BACKGROUND:Previous studies have shown that extracellular signal-regulated kinase 1/2(ERK1/2) and nitric oxide activation play a pivotal role in central sensitization and long-term neuronal plasticity induced by noxio...BACKGROUND:Previous studies have shown that extracellular signal-regulated kinase 1/2(ERK1/2) and nitric oxide activation play a pivotal role in central sensitization and long-term neuronal plasticity induced by noxious stimulation.However,their effects on compressive neuralg formation and maintenance remain poorly understood.OBJECTIVE:To investigate effects of the specific inhibitor of ERK1/2 signal pathway U0126 on neuronal nitric oxide synthase(nNOS) expression in the dorsal horn of the spinal cord in a compressive neuralgia rat model.DESIGN,TIME AND SETTING:A randomized,controlled experiment was performed at the Institu of Otolaryngology,Head and Neck Surgery,First Hospital of Jilin University from July 2008 to Marc 2009.MATERIALS:U0126(Bio-Mol,USA) was used in this study.METHODS:A total of 84 rats were randomly assigned to two groups.In the first part of the experiment,24 rats were used for behavioral testing,and they were randomly assigned to three sub-groups(n = 8):U0126,dimethyl sulfoxide(DMSO) and model control.In the second part of th experiment,60 rats were used for immunofluorescence and Western blot analysis,and they were randomly assigned to six sub-groups(n = 10):sham surgery,model control,U0126 post-injection 0.5,2,12 and 24 hours.Neuropathic pain was produced by chronic compression to the dorsal roo ganglion in rats from each sub-group.Rats in the U0126 group were administered a 5-μg U0126 intrathecal injection,and rats in the DMSO group were administered a 10-μL 5% DMSO intratheca injection.MAIN OUTCOME MEASURES:Changes in mechanical and thermal hyperalgesia were observed using von Frey filaments and thermalqia stimular.Thermal and mechanical hyperalgesia were stimulated at different time points following intrathecal injection of U0126.nNOS activation and expression in the spinal cord dorsal horn were determined by immunofluorescence and Western blot analysis.RESULTS:Intrathecal injection of U0126 significantly attenuated chronic compression of dorsal root ganglion-induced mechanical and thermal hyperalgesia.Immunofluorescence staining results demonstrated that,compared to the sham surgery group,the number of nNOS-positive neurons was significantly increased in the injured spinal dorsal horn in the model control group(P < 0.01).However,compared to the model control group,there were significantly decreasing numbers of nNOS-positive neurons in the U0126 post-injection groups at 0.5-hour,2-hour,and 12-hour(P < 0.05).Western blot analysis revealed similar results.CONCLUSION:Decreased activity in the ERK signal pathway resulted in down regulated nNOS expression in the dorsal horn of the spinal cord.These results suggested that ERK is involved in nitric oxide reaction to neuropathic pain.展开更多
BACKGROUND: Very few studies have addressed neuronal injury in cerebral vasospasm and subarachnoid hemorrhage (SAH), and the role of neurotransmitters in the regulation of extracellular signal-regulated kinase 1/2 (ER...BACKGROUND: Very few studies have addressed neuronal injury in cerebral vasospasm and subarachnoid hemorrhage (SAH), and the role of neurotransmitters in the regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) expression following SAH. OBJECTIVE: To analyze neurotransmitter regulation of ERK1/2 expression through the use of signal transduction, and to investigate cerebral injury mechanisms following SAH. DESIGN, TIME AND SETTING: A completely randomized grouping and controlled animal experiment was performed at the Experimental Center of Medical College of Xi'an Jiaotong University from March to December 2008. MATERIALS: Extracellular signal-regulated ERK1/2 polyclonal antibody and strepta-vidin-peroxidase method kits were purchased from Beijing Biosynthesis Biotechnology, China; DAB kit was purchased from Zhongshan Golden Bridge Biotechnology, China; TUNEL kit was purchased from Promega, USA. METHODS: A total of 114 male, Sprague Dawley rats, aged 55-63 days old, were randomly assigned to five groups: SAH (n = 30), saline control (n = 30), puncture control (n = 30), normal control (n = 6), and neurotransmitter-treated (n = 18). The SAH model was established by twice injecting blood through the cisterna magna. The neurotransmitters-treated group was subdivided into three groups according to drugs injected into the lateral cerebral ventricle: acetylcholine chloride, norepinephrine, and saline, with six animals in each group. MAIN OUTCOME MEASURES: Rats from the SAH, saline control, and puncture control groups were respectively sacrificed at 6, 12, and 24 hours, as well as 3 and 5 days, with six rats at each time point. The normal control group rats were sacrificed at 6 hours, and the neurotransmitter group rats were sacrificed 3 days following neurotransmitter injection. Morphological cellular changes were observed by hematoxylin and eosin staining. Immunohistochemical SP method was used to detect expression of ERK1/2 in the cortex, and cortical apoptosis was detected using the TUNEL method. RESULTS: Neural tissue edema, apoptosis, and necrosis occurred in the cortex of the SAH group. ERK1/2-positive cells were first observed at 6 hours, peaked at 12 hours following SAH in the cortex, and gradually decreased thereafter. Cellular apoptosis was observed in the cortex at 6 hours and peaked at 24 hours following SAH. ERK1/2 distribution in the brain overlapped apoptotic cells to a great degree. The number of ERK1/2-positive and apoptotic cells was significantly greater in the SAH group compared with the three control groups (P < 0.05). Compared to the number of ERK1/2-positive cells in the saline-treated group, acetylcholine chloride treatment resulted in decreased ERK1/2 expression and apoptosis (P < 0.05). Norepinephrine resulted in increased ERK1/2 expression, but there was no significance in apoptosis compared to the saline-treated group (P > 0.05). CONCLUSION: Apoptosis was observed early in the rat cortex following SAH. In addition, ERK1/2 was expressed earlier than apoptosis. Acetylcholine chloride treatment resulted in decreased numbers of apoptotic cells following SAH, possibly by down-regulating ERK1/2 expression.展开更多
Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WH...Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.展开更多
BACKGROUND: Prenatal stress has been shown to inhibit cell proliferation in the dentate gyrus and hippocampus, reduce hippocampal volume, and cause neuronal loss and oxidative damage in the hippocampus of offspring ra...BACKGROUND: Prenatal stress has been shown to inhibit cell proliferation in the dentate gyrus and hippocampus, reduce hippocampal volume, and cause neuronal loss and oxidative damage in the hippocampus of offspring rats, but the sexual difference of the effects on offsprings is seldom referred to. OBJECTIVE: To observe the effect of prenatal stress to adult pregnant rats on expression of extracellular signal-regulated kinases (ERK) in hippocampus of the offspring rats of different genders. DESIGN: A randomized and control animal experiment. SETTING: Department of Physiology and Pathophysiology, School of Medicine, Xi’an Jiaotong University. MATERIALS: The experiments were carried out in the Key Laboratory of Environment and Gene Related Diseases (Xi’an Jiaotong University), Ministry of Education between October 2005 and March 2006. Fifteen female and five male adult Sprague-Dawley rats were adopted. Female rats weighing 230-250 g and male rats weighing 280-350 g were used. METHODS: The virgin female rats were placed overnight with adult male rats (3:1) for mating. A total of twelve pregnant rats were randomly assigned to prenatal stress group (PNS group, n=6) and control group (n=6). The pregnant rats of the PNS group were exposed to restraint stress on days 14-20 of pregnancy three times a day, 45 minutes for each time[9,13]. The restraint device was a transparent plastic tube (6.8 cm in diameter) with air holes for breathing and closed end. The length could be adjusted to accommodate the size of the animals. To prevent habituation of animals to the daily procedure, restraint periods were randomly shifted within certain time periods (8:00-11:00, 11:00-14:00, and 16:00-19:00). After birth, offsprings of all groups were culled to 8-10 litters in each group and housed in the same animal room, and kept together with their biologic mothers. The pregnant rats of the control group were left undisturbed. On day 21, after all the offspring were weaned, male and female pups were separated and housed four in each cage respectively until test at 30 days of age. At the end of postnatal day 30, one male and female offspring rats from the same dam were selected with a random choice and a total of 24 animals from 12 different dams were used. The experimental rats were sacrificed by decapitation under anesthesia. Bilateral hippocampal tissues were isolated and homogenized in cold condition. Alkaline carbonate buffer (BCA) method was used to detect the concentration of extracellular signal-regulated kinases (ERK), then mixed with loading buffer, the constant voltage was 100 V. Finally, BCIP/NBT staining and electrophoresis were performed, the absorbance (A) value for the bands was detected with the Bandscan analytical software, and the expression of ERK in hippocampus of offspring rats of different genders in each group was quantitatively analyzed. MAIN OUTCOME MEASURES: The level of ERK expression in hippocampus of offspring rats of different genders in each group was observed. RESULTS: All the 24 offspring rats were involved in the analysis of results. ① The staining results of ERP activity in the extract of brain tissue detected with Western blotting technique and specific antibody analysis showed that the ERP in hippocampus of offspring rats had two subtypes of ERK-1 and ERK-2, and the latter was the main type. ② Standardized by the average A value in the control group, the quantitative data of the general A value of total ERK showed that the expression of ERK-2 in hippocampus of female offspring rats was obviously higher in the PNS group than in the control group (A value: 126±6.76,100±4.89,P < 0.01). ③ The expression of ERK-2 had no obvious difference between the female and male offspring rats in the control group. ④ The expression of ERK-2 in hippocampus of male offspring rats was a little higher in the PNS group than in the control group (A value: 104±6.27,102±5.48,P > 0.05). CONCLUSION: PNS significantly affects the increase of ERK expression in hippocampus of female offspring rats, but has no obvious influence on that of male ones.展开更多
BACKGROUND:Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However,there are few reports on the influence...BACKGROUND:Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However,there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE:To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Laboratory of Human Anatomy,Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS:Mouse anti-rat bromodeoxyuridine (BrdU) monoclonal antibody was provided by Sigma,USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase (ERK) specific inhibitor PD98059 were provided by Calbiochem,Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies,China. METHODS:A total of 126 rats were randomly assigned to four groups:model (n = 36),Du meridian (n = 36),Ren/Du meridian (n = 36),and Ren/Du meridian + PD98059 (n = 18). Rats in the Ren /Du meridian + PD98059 group were observed on days 7 (n = 6) and 14 (n = 12) after cerebral ischemia injury. Rats in the model,Du meridian,and Ren/Du meridian groups were observed on days 7,14,and 28 after cerebral ischemia injury,with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong (DU 26) and Baihui (DU 20) acupoints in the Du meridian group,as well as Chengjiang (RN 24),Guanyuan (RN 4),Renzhong,and Baihui acupoints in the Ren/Du meridian and Ren/Du meridian + PD98059 groups 2 days after model establishment. In addition,electro-acupuncture stimulation with disperse-dense waves was performed,with 30 Hz disperse wave,100 Hz dense wave,and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian + PD98059 group were treated with 0.2 μg PD98059 injection into the subventricular zone,2 μL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES:BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 (pERK1/2) expression in the subventricular zone. RESULTS:On days 14 and 28 after cerebral ischemia,there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren /Du meridian group compared with the Du meridian group (P < 0.05). PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the Ren and Du meridians (P < 0.05). On days 7,14,and 28 after treatment,pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group (P < 0.05). The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction (P < 0.05). However,PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression (P < 0.05). CONCLUSION:Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells,which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.展开更多
BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentia...BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type Ⅰ collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-β1 (TGF-β1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS: Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-β1 was examined using the reverse transcriptase-polymerase chain reaction. Type Ⅰ collagen in the culture medium was detected by enzyme- linked immunosorbent assay. RESULTS: 20, 50 and 100 μmol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1- phase arrest of acetaldehyde-induced HSCs in a dose- dependent manner. The secretion of type Ⅰ?collagen and the expression of cyclin D1, CDK4 and TGF-β1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 μmol/L PD98059, respectively.CONCLUSIONS: The ERK pathway regulates the cell proliferation, secretion of type Ⅰ collagen and the expression of TGF-β1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.展开更多
AIM: To investigate whether extracellular signal-regu- lated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was i...AIM: To investigate whether extracellular signal-regu- lated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were eval- uated by hematoxylin and eosin staining, and Masson’ s trichrome method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining. RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and peri- sinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vas- cular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK2 protein was up-regulated during the model course, and its level was the highest 4 wk after opera- tion, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days af- ter BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-SMAexpression (r = 0.958,P < 0.05).展开更多
AIM:To study the expression and phosphorylation of extracellular signal-regulated kinase(ERK)1 and ERK2 in multidrug resistant(MDR)hepatocellular carcinoma(HCC)cells.METHODS:MDR HCC cell lines,HepG2/adriamycin(ADM)and...AIM:To study the expression and phosphorylation of extracellular signal-regulated kinase(ERK)1 and ERK2 in multidrug resistant(MDR)hepatocellular carcinoma(HCC)cells.METHODS:MDR HCC cell lines,HepG2/adriamycin(ADM)and SMMC7721/ADM,were developed by exposing parental cells to stepwise increasing concentrations of ADM.MTT assay was used to determine drug sensitivity.Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein(P-gp)and multidrug resistant protein 1(MRP1)expression levels.ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR(QRTPCR).Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.SMMC7721/ADM were resistant not only to ADM,but also to multiple anticancer drugs.The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells(8.92%±0.22%vs 0.88%±0.05%,P<0.001)and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells(7.37%±0.26%vs 1.74%±0.25%,P<0.001).However,the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells.In addition,the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase.QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells.Compared with the expression of parental cells,ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells.However,ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells.Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION:ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells.ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.展开更多
AIM: To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase(ERK) signaling pathway in vitro.·METHODS: The expression levels of phos...AIM: To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase(ERK) signaling pathway in vitro.·METHODS: The expression levels of phosphorylated ERK(P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays,the differences in clone morphology and in K10, K19, PERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency,differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis.· RESULTS: The expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium.Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19.The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression.· CONCLUSION: We suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.展开更多
Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-reg...Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.展开更多
Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from inju...Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor(U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and Ku70 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and accelerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/ reperfusion.展开更多
In order to investigate the changes in the expression of extracellular signal regulated kinase (ERK1/ERK2) and angiotensin converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheuma...In order to investigate the changes in the expression of extracellular signal regulated kinase (ERK1/ERK2) and angiotensin converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheumatic heart diseases were examined. Nineteen patients had chronic persistent AF (AF≥6 months, CAF), 12 patients had paroxymal AF (PAF) and 21 patients had no history of AF. The ERK expression was detected at the mRNA level by reverse transcription polymerase chain reaction, at the protein level by Western blotting and at atrial tissue level by immunohistochemistry. ERK activating kinases (MEK1/2) and ACE were determined by Western blotting techniques. The expression of ERK2 mRNA was increased in the patients with CAF (74±19 U vs sinus rhythm: 32±24 U, P <0.05). Activated ERK1/ERK2 and MEK1/2 were increased to more than 150 % in the patients with AF compared to those with sinus rhythm. No significant difference between CAF and PAF was found. The expression of ACE was three fold increased in the patients with CAF compared to those with sinus rhythm. Patients with AF showed an increased expression of ERK1/ERK2 in atrial interstitial cells and marked atrial fibrosis. An ACE dependent increase in the amounts of activated ERK1/ERK2 in atrial interstitial cells may be one of molecular mechanisms for the development of atrial fibrosis in the patients with AF. These findings may have important impact on the treatment of AF.展开更多
Herb-partitioned moxibustion can effectively mitigate visceral pain, a major symptom in inflammatory bowel disease, but the analgesic mechanism is still unclear. Moreover, extracellular signal-regulated kinase, substa...Herb-partitioned moxibustion can effectively mitigate visceral pain, a major symptom in inflammatory bowel disease, but the analgesic mechanism is still unclear. Moreover, extracellular signal-regulated kinase, substance P, and neurokinin-1 are involved in formation of central hyperalgesia. Thus, we postulated that the analgesic effect of herb-partitioned moxibustion may be associated with these factors. Accordingly, in this study, we established an inflammatory bowel disease visceral pain model in rat by enema with a mixed solution of 5% trinitrobenzenesulfonic acid and 50% ethanol. Bilateral Tianshu(ST25) and Qihai(CV6) points were selected for herb-partitioned moxibustion. Our results showed that herb-partitioned moxibustion improved visceral pain and down-regulated extracellular signal-regulated kinase, substance P, and neurokinin-1 protein and m RNA expression in dorsal root ganglia. These results indicate that down-regulation of extracellular signal-regulated kinase, substance P, and neurokinin-1 protein and m RNA may be a central mechanism for the analgesic effect of herb-partitioned moxibustion.展开更多
BACKGROUND: The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and functi...BACKGROUND: The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE: To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN: Randomized controlled study. SETTING: Department of Anatomy, Sun Yat-Sen University. MATERIALS: A total of 60 healthy adult male Wistar rats weighing (250±10) g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS: The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups: normal group (n = 6), sham operation group (n = 18), model group (n = 18), and electroacupuncture group (n = 18). Middle cerebral artery occlusion (MCAO) was performed in the model group and electroacupuncture group. Zea Longa's grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between 1 and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery (CCA) was isolated, and the external carotid artery (ECA) was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture (People's Publishing House; 1997; First Edition). The Chengjiang, Qihai, and Guanyuan acupoints were labeled and connected to a G6805 electroacupuncture apparatus with sparse-dense waves (sparse waves were 30 Hz, dense waves were 100 Hz), with a frequency of 6-15 V. The duration was 20 minutes. Two days after surgery, the model and sham operation groups were placed with their backs on the operating table, but they received no acupuncture. However, the normal group received acupuncture. The experimental animals under anesthesia were sacrificed on days 7, 14, and 28 post-surgery. Western blot analysis was used to measure expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall. Expression was measured in the normal group at time points corresponding to the sham operation group. MAIN OUTCOME MEASURES: Expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall at different time points after intervention. RESULTS: All 60 rats were included in the final analysis, without any loss. Seven days after MCAO, there was no significant difference in extracellular signal-regulated kinases 1/2 expression in the electroacupuncture group compared to the model group (P > 0.05). However, extracellular signal-regulated kinases 1/2 expression significantly increased in the model group at 14 and 28 days after treatment (P < 0.05). CONCLUSION: Electroacupuncture at the Ren channel can enhance extracellular signal-regulated kinases1/2 expression in the inferior region of the lateral cerebral ventricle wall of rats with focal cerebral ischemia. However, this effect is not apparent until 14 days after electroacupuncture intervention.展开更多
BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC...BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.展开更多
Objectives To study extracellular signal-regulated kinase (ERK) activation in the endometrial carcinoma cell line Ishikawa with stimulation by 17-β-estradiol, and to elucidate the role of phosphatase and tensin homol...Objectives To study extracellular signal-regulated kinase (ERK) activation in the endometrial carcinoma cell line Ishikawa with stimulation by 17-β-estradiol, and to elucidate the role of phosphatase and tensin homologue (PTEN) and estrogen receptor (ER) subtype on the activation of ERKs. Methods Western blot was used to examine the expression of PTEN and PTEN (G129E) in Ishikawa cells after stable transfection as well as ERK activation in Ishikawa-EGFP, Ishikawa- PTEN and Ishikawa- PTEN (G129E) stimulated with various doses of 17-β-estradiol for different lengths of time. Western blot was also used for examining the expression of ERα and ERβ in NIH3T3 fibroblasts after transient transfection of pCXN2hERα and pCXN2hERβ. Then, ERK activation was examined after stimulation with 17-β-estradiol. Results 17-β-estradiol activated ERK cascades (mainly ERK2) in Ishikawa cells. The activation of ERK increased gradually as concentration of 17-β-estradiol also increased. The maximal activation of ERK2 took place 5 min after stimulation with 17-β-estradiol. The activation of ERK2 was inhibited markedly by PTEN, but not by PTEN (G129E). 17-β-estradiol activated ERK cascades in NIH3T3 fibroblasts after transient transfection of pCXN2hERα. Conclusions 17-β-estradiol activate ERK cascades in Ishikawa cells by integrating with ERα. Lipid phosphatase PTEN has an inhibitory role on the activation of ERK stimulated by 17-β-estradiol in Ishikawa cells.展开更多
The mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase 1/2(ERK1/2) signaling pathway is widely activated by a variety of extracellular stimuli, and its dysregulation is associated with the pr...The mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase 1/2(ERK1/2) signaling pathway is widely activated by a variety of extracellular stimuli, and its dysregulation is associated with the proliferation, invasion, and migration of cancer cells. ERK1/2 is located at the distal end of this pathway and rarely undergoes mutations, making it an attractive target for anticancer drug development. Currently, an increasing number of ERK1/2 inhibitors have been designed and synthesized for antitumor therapy, among which representative compounds have entered clinical trials. When ERK1/2 signal transduction is eliminated, ERK5 may provide a bypass route to rescue proliferation, and weaken the potency of ERK1/2 inhibitors. Therefore, drug research targeting ERK5 or based on the compensatory mechanism of ERK5 for ERK1/2 opens up a new way for oncotherapy. This review provides an overview of the physiological and biological functions of ERKs, focuses on the structure-activity relationships of small molecule inhibitors targeting ERKs, with a view to providing guidance for future drug design and optimization, and discusses the potential therapeutic strategies to overcome drug resistance.展开更多
Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2...Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin. Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4’,6’-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.展开更多
基金Science and Technology Development Project Program of Jilin Province,China,No. 200505195
文摘BACKGROUND:Previous studies have shown that extracellular signal-regulated kinase 1/2(ERK1/2) and nitric oxide activation play a pivotal role in central sensitization and long-term neuronal plasticity induced by noxious stimulation.However,their effects on compressive neuralg formation and maintenance remain poorly understood.OBJECTIVE:To investigate effects of the specific inhibitor of ERK1/2 signal pathway U0126 on neuronal nitric oxide synthase(nNOS) expression in the dorsal horn of the spinal cord in a compressive neuralgia rat model.DESIGN,TIME AND SETTING:A randomized,controlled experiment was performed at the Institu of Otolaryngology,Head and Neck Surgery,First Hospital of Jilin University from July 2008 to Marc 2009.MATERIALS:U0126(Bio-Mol,USA) was used in this study.METHODS:A total of 84 rats were randomly assigned to two groups.In the first part of the experiment,24 rats were used for behavioral testing,and they were randomly assigned to three sub-groups(n = 8):U0126,dimethyl sulfoxide(DMSO) and model control.In the second part of th experiment,60 rats were used for immunofluorescence and Western blot analysis,and they were randomly assigned to six sub-groups(n = 10):sham surgery,model control,U0126 post-injection 0.5,2,12 and 24 hours.Neuropathic pain was produced by chronic compression to the dorsal roo ganglion in rats from each sub-group.Rats in the U0126 group were administered a 5-μg U0126 intrathecal injection,and rats in the DMSO group were administered a 10-μL 5% DMSO intratheca injection.MAIN OUTCOME MEASURES:Changes in mechanical and thermal hyperalgesia were observed using von Frey filaments and thermalqia stimular.Thermal and mechanical hyperalgesia were stimulated at different time points following intrathecal injection of U0126.nNOS activation and expression in the spinal cord dorsal horn were determined by immunofluorescence and Western blot analysis.RESULTS:Intrathecal injection of U0126 significantly attenuated chronic compression of dorsal root ganglion-induced mechanical and thermal hyperalgesia.Immunofluorescence staining results demonstrated that,compared to the sham surgery group,the number of nNOS-positive neurons was significantly increased in the injured spinal dorsal horn in the model control group(P < 0.01).However,compared to the model control group,there were significantly decreasing numbers of nNOS-positive neurons in the U0126 post-injection groups at 0.5-hour,2-hour,and 12-hour(P < 0.05).Western blot analysis revealed similar results.CONCLUSION:Decreased activity in the ERK signal pathway resulted in down regulated nNOS expression in the dorsal horn of the spinal cord.These results suggested that ERK is involved in nitric oxide reaction to neuropathic pain.
基金the National Natural Science Foundation of China, No. 30870844 the National High Technology Research and Development Program of China (863 Program), No. 2006AA02Z4Z4the New Century Excellent Talents in University, Ministry of Education, No. NECT-05-0831
文摘BACKGROUND: Very few studies have addressed neuronal injury in cerebral vasospasm and subarachnoid hemorrhage (SAH), and the role of neurotransmitters in the regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) expression following SAH. OBJECTIVE: To analyze neurotransmitter regulation of ERK1/2 expression through the use of signal transduction, and to investigate cerebral injury mechanisms following SAH. DESIGN, TIME AND SETTING: A completely randomized grouping and controlled animal experiment was performed at the Experimental Center of Medical College of Xi'an Jiaotong University from March to December 2008. MATERIALS: Extracellular signal-regulated ERK1/2 polyclonal antibody and strepta-vidin-peroxidase method kits were purchased from Beijing Biosynthesis Biotechnology, China; DAB kit was purchased from Zhongshan Golden Bridge Biotechnology, China; TUNEL kit was purchased from Promega, USA. METHODS: A total of 114 male, Sprague Dawley rats, aged 55-63 days old, were randomly assigned to five groups: SAH (n = 30), saline control (n = 30), puncture control (n = 30), normal control (n = 6), and neurotransmitter-treated (n = 18). The SAH model was established by twice injecting blood through the cisterna magna. The neurotransmitters-treated group was subdivided into three groups according to drugs injected into the lateral cerebral ventricle: acetylcholine chloride, norepinephrine, and saline, with six animals in each group. MAIN OUTCOME MEASURES: Rats from the SAH, saline control, and puncture control groups were respectively sacrificed at 6, 12, and 24 hours, as well as 3 and 5 days, with six rats at each time point. The normal control group rats were sacrificed at 6 hours, and the neurotransmitter group rats were sacrificed 3 days following neurotransmitter injection. Morphological cellular changes were observed by hematoxylin and eosin staining. Immunohistochemical SP method was used to detect expression of ERK1/2 in the cortex, and cortical apoptosis was detected using the TUNEL method. RESULTS: Neural tissue edema, apoptosis, and necrosis occurred in the cortex of the SAH group. ERK1/2-positive cells were first observed at 6 hours, peaked at 12 hours following SAH in the cortex, and gradually decreased thereafter. Cellular apoptosis was observed in the cortex at 6 hours and peaked at 24 hours following SAH. ERK1/2 distribution in the brain overlapped apoptotic cells to a great degree. The number of ERK1/2-positive and apoptotic cells was significantly greater in the SAH group compared with the three control groups (P < 0.05). Compared to the number of ERK1/2-positive cells in the saline-treated group, acetylcholine chloride treatment resulted in decreased ERK1/2 expression and apoptosis (P < 0.05). Norepinephrine resulted in increased ERK1/2 expression, but there was no significance in apoptosis compared to the saline-treated group (P > 0.05). CONCLUSION: Apoptosis was observed early in the rat cortex following SAH. In addition, ERK1/2 was expressed earlier than apoptosis. Acetylcholine chloride treatment resulted in decreased numbers of apoptotic cells following SAH, possibly by down-regulating ERK1/2 expression.
文摘Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery.The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively.The control group were sham operated age-matched Wistar rats.Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00±33.00 mm Hg at the end of experiment, significantly higher than that in the control rats (P<0.01).Blood pressure in SHR4w (108.00±11.25 mm Hg) was similar to that in the controls.However, it rose to 122.25±21.75 mm Hg in SHR8w, and even up to 201.75±18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P<0.01).The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P<0.05).Hyaline degeneration of the afferent arterioles was found in WHR.In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w.Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2.The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09%±1.75%, 14.57%±4.58%, 29.44%±7.35%, and 13.63%±3.85%, respectively) than that of the controls(P<0.01).The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09%±1.40%, 24.17%±6.92%, 32.44%±4.05%, and 18.61%±3.35%, respectively) than that of the controls (P<0.01), too.The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P<0.01).Conclusion Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR.Phospho-ERK1/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.
基金the National Natural Science Foundation of China, No. 30270445
文摘BACKGROUND: Prenatal stress has been shown to inhibit cell proliferation in the dentate gyrus and hippocampus, reduce hippocampal volume, and cause neuronal loss and oxidative damage in the hippocampus of offspring rats, but the sexual difference of the effects on offsprings is seldom referred to. OBJECTIVE: To observe the effect of prenatal stress to adult pregnant rats on expression of extracellular signal-regulated kinases (ERK) in hippocampus of the offspring rats of different genders. DESIGN: A randomized and control animal experiment. SETTING: Department of Physiology and Pathophysiology, School of Medicine, Xi’an Jiaotong University. MATERIALS: The experiments were carried out in the Key Laboratory of Environment and Gene Related Diseases (Xi’an Jiaotong University), Ministry of Education between October 2005 and March 2006. Fifteen female and five male adult Sprague-Dawley rats were adopted. Female rats weighing 230-250 g and male rats weighing 280-350 g were used. METHODS: The virgin female rats were placed overnight with adult male rats (3:1) for mating. A total of twelve pregnant rats were randomly assigned to prenatal stress group (PNS group, n=6) and control group (n=6). The pregnant rats of the PNS group were exposed to restraint stress on days 14-20 of pregnancy three times a day, 45 minutes for each time[9,13]. The restraint device was a transparent plastic tube (6.8 cm in diameter) with air holes for breathing and closed end. The length could be adjusted to accommodate the size of the animals. To prevent habituation of animals to the daily procedure, restraint periods were randomly shifted within certain time periods (8:00-11:00, 11:00-14:00, and 16:00-19:00). After birth, offsprings of all groups were culled to 8-10 litters in each group and housed in the same animal room, and kept together with their biologic mothers. The pregnant rats of the control group were left undisturbed. On day 21, after all the offspring were weaned, male and female pups were separated and housed four in each cage respectively until test at 30 days of age. At the end of postnatal day 30, one male and female offspring rats from the same dam were selected with a random choice and a total of 24 animals from 12 different dams were used. The experimental rats were sacrificed by decapitation under anesthesia. Bilateral hippocampal tissues were isolated and homogenized in cold condition. Alkaline carbonate buffer (BCA) method was used to detect the concentration of extracellular signal-regulated kinases (ERK), then mixed with loading buffer, the constant voltage was 100 V. Finally, BCIP/NBT staining and electrophoresis were performed, the absorbance (A) value for the bands was detected with the Bandscan analytical software, and the expression of ERK in hippocampus of offspring rats of different genders in each group was quantitatively analyzed. MAIN OUTCOME MEASURES: The level of ERK expression in hippocampus of offspring rats of different genders in each group was observed. RESULTS: All the 24 offspring rats were involved in the analysis of results. ① The staining results of ERP activity in the extract of brain tissue detected with Western blotting technique and specific antibody analysis showed that the ERP in hippocampus of offspring rats had two subtypes of ERK-1 and ERK-2, and the latter was the main type. ② Standardized by the average A value in the control group, the quantitative data of the general A value of total ERK showed that the expression of ERK-2 in hippocampus of female offspring rats was obviously higher in the PNS group than in the control group (A value: 126±6.76,100±4.89,P < 0.01). ③ The expression of ERK-2 had no obvious difference between the female and male offspring rats in the control group. ④ The expression of ERK-2 in hippocampus of male offspring rats was a little higher in the PNS group than in the control group (A value: 104±6.27,102±5.48,P > 0.05). CONCLUSION: PNS significantly affects the increase of ERK expression in hippocampus of female offspring rats, but has no obvious influence on that of male ones.
基金the National Natural Science Foundation of China,No.30371808the Natural Science Foundation of Guangdong Province,No.5009688
文摘BACKGROUND:Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However,there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE:To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Laboratory of Human Anatomy,Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS:Mouse anti-rat bromodeoxyuridine (BrdU) monoclonal antibody was provided by Sigma,USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase (ERK) specific inhibitor PD98059 were provided by Calbiochem,Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies,China. METHODS:A total of 126 rats were randomly assigned to four groups:model (n = 36),Du meridian (n = 36),Ren/Du meridian (n = 36),and Ren/Du meridian + PD98059 (n = 18). Rats in the Ren /Du meridian + PD98059 group were observed on days 7 (n = 6) and 14 (n = 12) after cerebral ischemia injury. Rats in the model,Du meridian,and Ren/Du meridian groups were observed on days 7,14,and 28 after cerebral ischemia injury,with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong (DU 26) and Baihui (DU 20) acupoints in the Du meridian group,as well as Chengjiang (RN 24),Guanyuan (RN 4),Renzhong,and Baihui acupoints in the Ren/Du meridian and Ren/Du meridian + PD98059 groups 2 days after model establishment. In addition,electro-acupuncture stimulation with disperse-dense waves was performed,with 30 Hz disperse wave,100 Hz dense wave,and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian + PD98059 group were treated with 0.2 μg PD98059 injection into the subventricular zone,2 μL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES:BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 (pERK1/2) expression in the subventricular zone. RESULTS:On days 14 and 28 after cerebral ischemia,there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren /Du meridian group compared with the Du meridian group (P < 0.05). PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the Ren and Du meridians (P < 0.05). On days 7,14,and 28 after treatment,pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group (P < 0.05). The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction (P < 0.05). However,PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression (P < 0.05). CONCLUSION:Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells,which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.
文摘BACKGROUND: The pathogenesis of hepatic fibrosis and cirrhosis is still not fully understood. The extracellular signal-regulated kinase (ERK) pathway is involved in the regulation of cell proliferation and differentiation. The aim of this study was to investigate the effects of PD98059, a specific inhibitor of ERK, on the cell cycle, cell proliferation, secretion of type Ⅰ collagen and expression of cyclin D1 mRNA, CDK4 mRNA and transforming growth factor-β1 (TGF-β1) mRNA in rat hepatic stellate cells (HSCs) stimulated by acetaldehyde. METHODS: Rat HSCs stimulated by acetaldehyde were incubated with PD98059 at different concentrations. The cell cycle was analysed by flow cytometry. Cell proliferation was assessed by the methyl thiazolyl tetrazolium colorimetric assay. The mRNA expression of cyclin D1, CDK4 and TGF-β1 was examined using the reverse transcriptase-polymerase chain reaction. Type Ⅰ collagen in the culture medium was detected by enzyme- linked immunosorbent assay. RESULTS: 20, 50 and 100 μmol/L PD98059 significantly inhibited the proliferation and provoked a G0/G1- phase arrest of acetaldehyde-induced HSCs in a dose- dependent manner. The secretion of type Ⅰ?collagen and the expression of cyclin D1, CDK4 and TGF-β1 mRNA in acetaldehyde-induced HSCs were markedly inhibited by 50 and 100 μmol/L PD98059, respectively.CONCLUSIONS: The ERK pathway regulates the cell proliferation, secretion of type Ⅰ collagen and the expression of TGF-β1 mRNA in rat HSCs stimulated by acetaldehyde, which is likely related to its regulative effect on the cell cycle.
文摘AIM: To investigate whether extracellular signal-regu- lated kinase 1 (ERK1) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue. METHODS: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were eval- uated by hematoxylin and eosin staining, and Masson’ s trichrome method. ERK1 mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (α-SMA) staining. RESULTS: With the development of hepatic fibrosis, the positive staining cells of α-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and peri- sinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% ± 2.63%, 22.65% ± 2.16%, 27.45% ± 1.86%, 35.25% ± 2.34%, respectively) were significantly larger than those in the control group (5.88% ± 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vas- cular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK1 and ERK2 protein was up-regulated during the model course, and its level was the highest 4 wk after opera- tion, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK1 mRNA was expressed in normal rat livers as well, which was up-regulated two days af- ter BDL and reached the highest 4 wk after BDL. The expression of ERK1 was positively correlated with α-SMAexpression (r = 0.958,P < 0.05).
基金Supported by Innovation Fund of Fujian Province,No.2007-CXB-7Key Science and Technology Project of Xiamen,No.3502Z20077045
文摘AIM:To study the expression and phosphorylation of extracellular signal-regulated kinase(ERK)1 and ERK2 in multidrug resistant(MDR)hepatocellular carcinoma(HCC)cells.METHODS:MDR HCC cell lines,HepG2/adriamycin(ADM)and SMMC7721/ADM,were developed by exposing parental cells to stepwise increasing concentrations of ADM.MTT assay was used to determine drug sensitivity.Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein(P-gp)and multidrug resistant protein 1(MRP1)expression levels.ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR(QRTPCR).Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.SMMC7721/ADM were resistant not only to ADM,but also to multiple anticancer drugs.The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells(8.92%±0.22%vs 0.88%±0.05%,P<0.001)and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells(7.37%±0.26%vs 1.74%±0.25%,P<0.001).However,the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells.In addition,the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase.QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells.Compared with the expression of parental cells,ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells.However,ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells.Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION:ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells.ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.
基金Supported by National Natural Science Foundation of China (No.81100649)
文摘AIM: To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase(ERK) signaling pathway in vitro.·METHODS: The expression levels of phosphorylated ERK(P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays,the differences in clone morphology and in K10, K19, PERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency,differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis.· RESULTS: The expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium.Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19.The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression.· CONCLUSION: We suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.
基金Shanghai Medical Key Discipline Construction Foundation(05-Ⅲ-005-017).
文摘Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.
基金supported by the Hebei Province Natural Science Program,No.H2012401007a grant from the foundation Key Project of Hebei Province Education Ministry,No.ZD2010106
文摘Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor(U0126) was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and Ku70 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and accelerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/ reperfusion.
文摘In order to investigate the changes in the expression of extracellular signal regulated kinase (ERK1/ERK2) and angiotensin converting enzyme (ACE) in the patients with atrial fibrillation (AF), 52 patients with rheumatic heart diseases were examined. Nineteen patients had chronic persistent AF (AF≥6 months, CAF), 12 patients had paroxymal AF (PAF) and 21 patients had no history of AF. The ERK expression was detected at the mRNA level by reverse transcription polymerase chain reaction, at the protein level by Western blotting and at atrial tissue level by immunohistochemistry. ERK activating kinases (MEK1/2) and ACE were determined by Western blotting techniques. The expression of ERK2 mRNA was increased in the patients with CAF (74±19 U vs sinus rhythm: 32±24 U, P <0.05). Activated ERK1/ERK2 and MEK1/2 were increased to more than 150 % in the patients with AF compared to those with sinus rhythm. No significant difference between CAF and PAF was found. The expression of ACE was three fold increased in the patients with CAF compared to those with sinus rhythm. Patients with AF showed an increased expression of ERK1/ERK2 in atrial interstitial cells and marked atrial fibrosis. An ACE dependent increase in the amounts of activated ERK1/ERK2 in atrial interstitial cells may be one of molecular mechanisms for the development of atrial fibrosis in the patients with AF. These findings may have important impact on the treatment of AF.
基金supported by the National Natural Science Foundation of China,No.81273843,81674073a grant from the National Key Basic Research Program of China(973 Program)+1 种基金No.2015CB554501the Project Fund of Shanghai Municipal Commission of Health and Family Planning of China,No.20144Y0153,2017BR047
文摘Herb-partitioned moxibustion can effectively mitigate visceral pain, a major symptom in inflammatory bowel disease, but the analgesic mechanism is still unclear. Moreover, extracellular signal-regulated kinase, substance P, and neurokinin-1 are involved in formation of central hyperalgesia. Thus, we postulated that the analgesic effect of herb-partitioned moxibustion may be associated with these factors. Accordingly, in this study, we established an inflammatory bowel disease visceral pain model in rat by enema with a mixed solution of 5% trinitrobenzenesulfonic acid and 50% ethanol. Bilateral Tianshu(ST25) and Qihai(CV6) points were selected for herb-partitioned moxibustion. Our results showed that herb-partitioned moxibustion improved visceral pain and down-regulated extracellular signal-regulated kinase, substance P, and neurokinin-1 protein and m RNA expression in dorsal root ganglia. These results indicate that down-regulation of extracellular signal-regulated kinase, substance P, and neurokinin-1 protein and m RNA may be a central mechanism for the analgesic effect of herb-partitioned moxibustion.
基金the National Natural Science Foundation of China, No. 30371808Post-doctor's Project of Guangzhou University of Traditional Chinese Medicine, No.106B3YH0411
文摘BACKGROUND: The onset of focal cerebral ischemia activates extracellular signal-regulated kinases 1 and 2, regulates cell cycle, promotes cell proliferation and differentiation, and affects the normal stage and function of brain cells. OBJECTIVE: To observe the effects of electroacupuncture at the Ren channel on extracellular signal-regulated kinases 1/2 expression in the lateral cerebral ventricle wall of rats with focal cerebral ischemia. The effects were analyzed at different time points after intervention. DESIGN: Randomized controlled study. SETTING: Department of Anatomy, Sun Yat-Sen University. MATERIALS: A total of 60 healthy adult male Wistar rats weighing (250±10) g were provided by the Experimental Animal Center, Medical College of Sun Yat-Sen University. The animal experiment was conducted with confirmed consent by the local ethics committee. The GB6805-Ⅱ electric acupuncture apparatus was provided by Shanghai Medical Equipment High-techno Company. METHODS: The experiment was performed at the Laboratory of Anatomy, Sun Yat-Sen University, from February to July 2007. All experimental animals were randomly divided into the following groups: normal group (n = 6), sham operation group (n = 18), model group (n = 18), and electroacupuncture group (n = 18). Middle cerebral artery occlusion (MCAO) was performed in the model group and electroacupuncture group. Zea Longa's grading standard was used to assess neurological impairment after reperfusion; animals whose grades were between 1 and 4 were included in this study. The normal control group was not exposed to MCAO. In sham operation animals, the right common carotid artery (CCA) was isolated, and the external carotid artery (ECA) was damaged, but no embolism was induced. The electroacupuncture group was given acupuncture on the second day after surgery. The acupoint locations were chosen according to Experimental Acupuncture (People's Publishing House; 1997; First Edition). The Chengjiang, Qihai, and Guanyuan acupoints were labeled and connected to a G6805 electroacupuncture apparatus with sparse-dense waves (sparse waves were 30 Hz, dense waves were 100 Hz), with a frequency of 6-15 V. The duration was 20 minutes. Two days after surgery, the model and sham operation groups were placed with their backs on the operating table, but they received no acupuncture. However, the normal group received acupuncture. The experimental animals under anesthesia were sacrificed on days 7, 14, and 28 post-surgery. Western blot analysis was used to measure expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall. Expression was measured in the normal group at time points corresponding to the sham operation group. MAIN OUTCOME MEASURES: Expression of extracellular signal-regulated kinases 1/2 in the inferior region of the lateral cerebral ventricle wall at different time points after intervention. RESULTS: All 60 rats were included in the final analysis, without any loss. Seven days after MCAO, there was no significant difference in extracellular signal-regulated kinases 1/2 expression in the electroacupuncture group compared to the model group (P > 0.05). However, extracellular signal-regulated kinases 1/2 expression significantly increased in the model group at 14 and 28 days after treatment (P < 0.05). CONCLUSION: Electroacupuncture at the Ren channel can enhance extracellular signal-regulated kinases1/2 expression in the inferior region of the lateral cerebral ventricle wall of rats with focal cerebral ischemia. However, this effect is not apparent until 14 days after electroacupuncture intervention.
文摘BACKGROUND MicroRNAs(miRNAs)regulate gene expression and play a critical role in cancer physiology.However,there is still a limited understanding of the function and regulatory mechanism of miRNAs in gastric cancer(GC).AIM To investigate the role and molecular mechanism of miRNA-145-5p(miR145-5p)in the progression of GC.METHODS Real-time polymerase chain reaction(RT-PCR)was used to detect miRNA expression in human GC tissues and cells.The ability of cancer cells to migrate and invade was assessed using wound-healing and transwell assays,respectively.Cell proliferation was measured using cell counting kit-8 and colony formation assays,and apoptosis was evaluated using flow cytometry.Expression of the epithelial-mesenchymal transition(EMT)-associated protein was determined by Western blot.Targets of miR-145-5p were predicated using bioinformatics analysis and verified using a dual-luciferase reporter system.Serpin family E member 1(SERPINE1)expression in GC tissues and cells was evaluated using RT-PCR and immunohistochemical staining.The correlation between SERPINE1 expression and overall patient survival was determined using Kaplan-Meier plot analysis.The association between SERPINE1 and GC progression was also tested.A rescue experiment of SERPINE1 overexpression was conducted to verify the relationship between this protein and miR-145-5p.The mechanism by which miR-145-5p influences GC progression was further explored by assessing tumor formation in nude mice.RESULTS GC tissues and cells had reduced miR-145-5p expression and SERPINE1 was identified as a direct target of this miRNA.Overexpression of miR-145-5p was associated with decreased GC cell proliferation,invasion,migration,and EMT,and these effects were reversed by forcing SERPINE1 expression.Kaplan-Meier plot analysis revealed that patients with higher SERPINE1 expression had a shorter survival rate than those with lower SERPINE1 expression.Nude mouse tumorigenesis experiments confirmed that miR-145-5p targets SERPINE1 to regulate extracellular signal-regulated kinase-1/2(ERK1/2).CONCLUSION This study found that miR-145-5p inhibits tumor progression and is expressed in lower amounts in patients with GC.MiR-145-5p was found to affect GC cell proliferation,migration,and invasion by negatively regulating SERPINE1 levels and controlling the ERK1/2 pathway.
基金ThisstudywassupportedbyagrantfromtheNationalNaturalScienceFoundationofChina (No 3 0 0 0 0 178)
文摘Objectives To study extracellular signal-regulated kinase (ERK) activation in the endometrial carcinoma cell line Ishikawa with stimulation by 17-β-estradiol, and to elucidate the role of phosphatase and tensin homologue (PTEN) and estrogen receptor (ER) subtype on the activation of ERKs. Methods Western blot was used to examine the expression of PTEN and PTEN (G129E) in Ishikawa cells after stable transfection as well as ERK activation in Ishikawa-EGFP, Ishikawa- PTEN and Ishikawa- PTEN (G129E) stimulated with various doses of 17-β-estradiol for different lengths of time. Western blot was also used for examining the expression of ERα and ERβ in NIH3T3 fibroblasts after transient transfection of pCXN2hERα and pCXN2hERβ. Then, ERK activation was examined after stimulation with 17-β-estradiol. Results 17-β-estradiol activated ERK cascades (mainly ERK2) in Ishikawa cells. The activation of ERK increased gradually as concentration of 17-β-estradiol also increased. The maximal activation of ERK2 took place 5 min after stimulation with 17-β-estradiol. The activation of ERK2 was inhibited markedly by PTEN, but not by PTEN (G129E). 17-β-estradiol activated ERK cascades in NIH3T3 fibroblasts after transient transfection of pCXN2hERα. Conclusions 17-β-estradiol activate ERK cascades in Ishikawa cells by integrating with ERα. Lipid phosphatase PTEN has an inhibitory role on the activation of ERK stimulated by 17-β-estradiol in Ishikawa cells.
基金supported by grants from the National Natural Science Foundation of China (Grants 22177083,81922064,81874290,and 81803755)Sichuan Science and Technology Program (Grant No.2020JDRC0053,China)+1 种基金Fundamental Research Funds for the Central Universities (Grant No.2682020CX56,China)National Clinical Research Center for Geriatrics,West China Hospital,Sichuan University (Grant Z20201004,China)。
文摘The mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase 1/2(ERK1/2) signaling pathway is widely activated by a variety of extracellular stimuli, and its dysregulation is associated with the proliferation, invasion, and migration of cancer cells. ERK1/2 is located at the distal end of this pathway and rarely undergoes mutations, making it an attractive target for anticancer drug development. Currently, an increasing number of ERK1/2 inhibitors have been designed and synthesized for antitumor therapy, among which representative compounds have entered clinical trials. When ERK1/2 signal transduction is eliminated, ERK5 may provide a bypass route to rescue proliferation, and weaken the potency of ERK1/2 inhibitors. Therefore, drug research targeting ERK5 or based on the compensatory mechanism of ERK5 for ERK1/2 opens up a new way for oncotherapy. This review provides an overview of the physiological and biological functions of ERKs, focuses on the structure-activity relationships of small molecule inhibitors targeting ERKs, with a view to providing guidance for future drug design and optimization, and discusses the potential therapeutic strategies to overcome drug resistance.
文摘Background The role of extracellular signal-regulated kinase 1/2 (ERK1/2) in shikonin-induced HeLa cells apoptosis remains vague. This study was to investigate the activation of caspase pathways and the role of ERK1/2 in human cervical cancer cells, HeLa, by shikonin. Methods The inhibitory effect of shikonin on the growth of HeLa cells was measured by MTT assay. Fluorescent microscopic analysis of apoptotic cells stained with 4’,6’-oliiamiclino-2-phenylindole C (DAPI) and Hoechst 33258 was carried out. Caspase-3 and -8 activities were detected using caspase-3 substrate and caspase-8 substrate as substrates, respectively. The protein levels of ERK, p53 and p-ERK were determined by Western blot analysis.Results Shikonin inhibited cell growth in a time- and dose-dependent manner. Caspase-3 and caspase-8 were activated in the apoptotic process and caspase inhibitors effectively reversed shikonin-induced apoptosis. Phosphorylation of ERK resulted in up-regulation of p53 expression, which was blocked by mitogen-activated protein kinase (MEK), inhibitor PD 98059.Conclusion Shikonin induces HeLa cell apoptosis through the ERK, p53 and caspase pathways.
基金This study was supported by grants from National Natural Science Foundation of China （No. 30400195） and Dawn Program for Young Scientist of Wuhan （No. 20045006071-8）.
