AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to in...AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.展开更多
BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(...BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.展开更多
1-methyl-4-phenylpyridinium ion （MPP^＋） induces endoplasmic reticulum stress and activates caspase-12 in PC12 cells, leading to neuronal apoptosis. However, the underlying molecular mechanism remains unknown. The p...1-methyl-4-phenylpyridinium ion （MPP^＋） induces endoplasmic reticulum stress and activates caspase-12 in PC12 cells, leading to neuronal apoptosis. However, the underlying molecular mechanism remains unknown. The present study investigated the regulatory effects of nerve growth factor （Akt activator） and lithium chloride （glycogen synthase kinase-3β inhibitor） on the endoplasmic reticulum stress signaling pathway. The results revealed that MPP＋ induced expression of Bip and C/EBP homologous protein. The upregulation of Bip and C/EBP homologous protein, as well as the decreased pro-caspase-12 level induced by MPP^＋ were inhibited by pretreatment of the nerve growth factor or lithium chloride. These results suggest that the phosphatidylinositol 3 kinase-Aktglycogen synthase kinase-3β pathway is involved in MPP-induced endoplasmic reticulum stress.展开更多
Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum（ER） stress-mediated apoptotic and autophagic cell death i...Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum（ER） stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein（CHOP） and cleaved-caspase-3] and biomarkers of autophagy（Beclin-1 and light chain 3）, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1（IRE1）, tumor necrosis factor receptor-associated factor 2（TRAF2）, apoptosis signal-regulating kinase 1（ASK1）, c-Jun N-terminal kinase（JNK） and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.展开更多
Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeox...Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.展开更多
BACKGROUND： Endoplasmic reticulum （ER） stress impairs ER functions and leads to the accumulation of unfolded or misfolded proteins in the ER lumen. ER stress-induced cell death plays an important role in cerebral i...BACKGROUND： Endoplasmic reticulum （ER） stress impairs ER functions and leads to the accumulation of unfolded or misfolded proteins in the ER lumen. ER stress-induced cell death plays an important role in cerebral ischemia. Edaravon （3-methyl-1-phenyl-2-pyrazolin-5-one） is a potent and novel scavenger of free radicals that inhibit delayed neuronal death, as demonstrated by in vitro and animal studies. However, its effect on ER stress and induced neuronal apoptosis in a rat model of brief middle cerebral artery occlusion remains unclear. OBJECTIVE： To explore the effects of edaravone on the expression of ER stress-related factors and neuronal apoptosis, based on the hypothesis that edaravone influences ER stress in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of Department of Neurology, Xiangya Hospital and the Department of Laboratory Animals, Xiangya Medical College, Central South University in China from June 2005 to May 2006. MATERIALS： Edaravone was purchased from Simcere Pharmaceutical Group, China. METHODS： A total of 216 adult, male, Sprague Dawley rats were randomly assigned to sham-surgery, model and edaravone groups, with 72 rats in each group, Brief middle cerebral artery occlusion was established in the model and edaravone groups. In addition, the edaravone group rats were injected with 3 mg/kg edaravone through the tail vein. MAIN OUTCOME MEASURES： RNA-dependent protein kinase-like endoplasmic reticulum eukaryotic translation initiation factor 2a kinase （PERK） and C/EBP homology protein （CHOP） mRNA expression in the ischemic parietal cortex was determined by reverse transcriptionpolymerase chain reaction; phosphorylated PERK and CHOP protein expression was detected by immunohistochemistry; neuronal apoptosis was detected by TdT-mediated-dUTP nick end labeling. RESULTS： Neurological deficit scores were significantly reduced in the edaravone group compared to the model group at 12, 24, and 72 hours following reperfusion （P〈 0.05）. In addition, PERK and CHOP mRNA as well as phosphorylated PERK and CHOP protein expression were significantly reduced in the edaravone group compared to the model group at 1,3, and 6 hours following reperfusion （P 〈 0.05, P 〈 0.01）. CHOP mRNA expression was decreased in the edaravone group compared to the model group at 3, 6, 12, and 24 hours following reperfusion （P〈 0.01）, while CHOP protein expression was less than the model group at 6, 12, and 24 hours following reperfusion （P 〈 0.05）. CONCLUSION： Edaravone treatment resulted in decreased PERK and CHOP expression following ischemia/reperfusion, as well as reduced neuronal apoptosis. Edaravone exhibited a neuroprotective role by inhibiting endoplasmic reticulum stress.展开更多
Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize th...Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cerebral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the specific inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats intragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental findings indicate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 pathway.展开更多
The endoplasmic reticulum-nuclei-1 (ERN1) sensing and signaling enzyme mediates a set of complex intracellular signaling events known as the unfolded protein response. We have studied the effect of hypoxia and ischemi...The endoplasmic reticulum-nuclei-1 (ERN1) sensing and signaling enzyme mediates a set of complex intracellular signaling events known as the unfolded protein response. We have studied the effect of hypoxia and ischemic conditions (glucose or glutamine deprivation) on the expression of several casein kinase-1 and -2 genes in glioma U87 cells and its subline with suppressed function of ERN1. It was shown that blockade of ERN1, the key endoplasmic reticulum stress sensor, leads to an increase in the expression levels of casein kinase-1G2, -1E, -2B and NUCKS1 mRNA, but suppresses casein kinase-1A1, -1D and -2A1. Moreover, the expression levels of casein kinase-1A1, -1D and 1G3 as well as casein kinase-2A1 and -2A2 mRNAs are significantly increased under glutamine dep- rivation conditions both in control and ERN1- deficient glioma cells. At the same time, casein kinase-1E, -2B and NUCKS1 mRNA expression levels are also increased under this condition, but only in cells with suppressed function of ERN1. The expression level of NUCKS1 mRNA, however, is decreased both in control glioma cells and in genetically modified cells, but casein kinase-1G2—only in control U87 cells. Cell exposure to glucose deprivation conditions enhances the expression levels of casein kinase- 1D, 1G3, -1E and -2A1 in both types of glioma cells used, but casein kinase-2B expression levels increase only in cells with suppressed function of ERN1. Hypoxia induces or suppresses the expression of most of the studied genes mainly in ERN1-knockdown cells only. Results of this study show that hypoxia as well as glutamine and glucose deprivation conditions change the expression level most of casein kinase genes and that these effects are dependent on ERN1 signaling enzyme function.展开更多
Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the m...Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the mitochondrial inner membrane,and its role in Parkinson’s disease remains unclear.Protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK)is a factor that regulates cell fate during endoplasmic reticulum stress.Parkin is regulated by PERK and is a target of the unfolded protein response.It is unclear whether PERK regulates PHB2-mediated mitophagy thro ugh Parkin.In this study,we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson’s disease.We used adeno-associated virus to knockdown PHB2 expression.Our res ults showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson’s disease.Ove rexpression of PHB2 inhibited these abnormalities.We also established a 1-methyl-4-phenylpyridine(MPP+)-induced SH-SY5Y cell model of Parkinson’s disease.We found that ove rexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3,and promoted mitophagy.In addition,MPP+regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK.These findings suggest that PHB2 participates in the development of Parkinson’s disease by intera cting with endoplasmic reticulum stress and Parkin.展开更多
研究白藜芦醇(resveratrol,Res)对内质网应激途径细胞凋亡和糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)/Tau蛋白磷酸化作用的影响。体外原代培养神经元细胞,采用衣霉素(tunicamycin,TM)建立内质网应激模型,Western blo...研究白藜芦醇(resveratrol,Res)对内质网应激途径细胞凋亡和糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)/Tau蛋白磷酸化作用的影响。体外原代培养神经元细胞,采用衣霉素(tunicamycin,TM)建立内质网应激模型,Western blot法检测内质网分子伴侣蛋白葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、未折叠蛋白反应相关的肌醇需要酶1α(inositol-requiring enzyme 1α,IRE1α)的Ser724磷酸化、剪接的X盒结合蛋白1(spliced form of X-box binding protein 1s,XBP1s)表达、GSK-3β的Ser9和Tau蛋白的Ser396磷酸化水平。生物化学方法分析细胞质中半胱天冬酶-12(Caspase-12)和半胱天冬酶-3(Caspase-3)活性、细胞凋亡水平。结果显示,TM能够诱导内质网应激作用,导致神经元细胞GSK-3β的活化和Tau蛋白磷酸化水平升高(P<0.01)、神经元细胞经内质网途径凋亡(P<0.05)。与内质网应激抑制剂4-苯基丁酸结果相似,Res组显著降低了GRP78的表达(P<0.01)、降低了IRE1α-XBP1通路的活性(P<0.01)。Res可以减缓TM诱导的内质网途径细胞凋亡级联反应中Caspase-12和Caspase-3的活性(P<0.01)。Res抑制了TM诱导的GSK-3β的Ser9位点磷酸化水平和Tau蛋白Ser396位点的磷酸化水平(P<0.01)。结果表明,Res能够降低TM诱导的IRE1α-XBP1途径内质网应激作用、GSK-3β的活性及Tau蛋白发生磷酸化水平,减弱神经元细胞经内质网途径凋亡的级联反应作用。展开更多
基金Supported by the National Natural Science Foundation of China,No.81160067 and No.814600124
文摘AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.
基金Supported by the National Natural Science Foundation of China,No.81273952
文摘BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.
基金the National Natural Science Foundation of China, No. 30860085a grant from the Candidates of Young and Middle-Aged Academic Leaders of Yunnan Province, No. 2006PY01-07the Natural Science Foundation of Yunnan Province, No. 2007C177M
文摘1-methyl-4-phenylpyridinium ion （MPP^＋） induces endoplasmic reticulum stress and activates caspase-12 in PC12 cells, leading to neuronal apoptosis. However, the underlying molecular mechanism remains unknown. The present study investigated the regulatory effects of nerve growth factor （Akt activator） and lithium chloride （glycogen synthase kinase-3β inhibitor） on the endoplasmic reticulum stress signaling pathway. The results revealed that MPP＋ induced expression of Bip and C/EBP homologous protein. The upregulation of Bip and C/EBP homologous protein, as well as the decreased pro-caspase-12 level induced by MPP^＋ were inhibited by pretreatment of the nerve growth factor or lithium chloride. These results suggest that the phosphatidylinositol 3 kinase-Aktglycogen synthase kinase-3β pathway is involved in MPP-induced endoplasmic reticulum stress.
基金supported by the National Natural Science Foundation of China,No.81671159,No.81371298a grant from the Development of Science and Technology of Jilin Province of China,No.20160101099JC,No.20160101073JC+2 种基金a grant from the Youth Scientific Research of Health and Family Planning Commission in Jilin Province of China,No.2014Q022a grant from the Frontier Interdiscipline Program of Norman Bethune Health Science Center of Jilin University of China,No.2013107028a grant from the Young Scholars Program of Norman Bethune Health Science Center of Jilin University of China,No.2013207052
文摘Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum（ER） stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein（CHOP） and cleaved-caspase-3] and biomarkers of autophagy（Beclin-1 and light chain 3）, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1（IRE1）, tumor necrosis factor receptor-associated factor 2（TRAF2）, apoptosis signal-regulating kinase 1（ASK1）, c-Jun N-terminal kinase（JNK） and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.
基金supported by the National Natural Science Foundation of China,No.81170877the National Basic Research Program of China,No.2007CB512203
文摘Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.
文摘BACKGROUND： Endoplasmic reticulum （ER） stress impairs ER functions and leads to the accumulation of unfolded or misfolded proteins in the ER lumen. ER stress-induced cell death plays an important role in cerebral ischemia. Edaravon （3-methyl-1-phenyl-2-pyrazolin-5-one） is a potent and novel scavenger of free radicals that inhibit delayed neuronal death, as demonstrated by in vitro and animal studies. However, its effect on ER stress and induced neuronal apoptosis in a rat model of brief middle cerebral artery occlusion remains unclear. OBJECTIVE： To explore the effects of edaravone on the expression of ER stress-related factors and neuronal apoptosis, based on the hypothesis that edaravone influences ER stress in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of Department of Neurology, Xiangya Hospital and the Department of Laboratory Animals, Xiangya Medical College, Central South University in China from June 2005 to May 2006. MATERIALS： Edaravone was purchased from Simcere Pharmaceutical Group, China. METHODS： A total of 216 adult, male, Sprague Dawley rats were randomly assigned to sham-surgery, model and edaravone groups, with 72 rats in each group, Brief middle cerebral artery occlusion was established in the model and edaravone groups. In addition, the edaravone group rats were injected with 3 mg/kg edaravone through the tail vein. MAIN OUTCOME MEASURES： RNA-dependent protein kinase-like endoplasmic reticulum eukaryotic translation initiation factor 2a kinase （PERK） and C/EBP homology protein （CHOP） mRNA expression in the ischemic parietal cortex was determined by reverse transcriptionpolymerase chain reaction; phosphorylated PERK and CHOP protein expression was detected by immunohistochemistry; neuronal apoptosis was detected by TdT-mediated-dUTP nick end labeling. RESULTS： Neurological deficit scores were significantly reduced in the edaravone group compared to the model group at 12, 24, and 72 hours following reperfusion （P〈 0.05）. In addition, PERK and CHOP mRNA as well as phosphorylated PERK and CHOP protein expression were significantly reduced in the edaravone group compared to the model group at 1,3, and 6 hours following reperfusion （P 〈 0.05, P 〈 0.01）. CHOP mRNA expression was decreased in the edaravone group compared to the model group at 3, 6, 12, and 24 hours following reperfusion （P〈 0.01）, while CHOP protein expression was less than the model group at 6, 12, and 24 hours following reperfusion （P 〈 0.05）. CONCLUSION： Edaravone treatment resulted in decreased PERK and CHOP expression following ischemia/reperfusion, as well as reduced neuronal apoptosis. Edaravone exhibited a neuroprotective role by inhibiting endoplasmic reticulum stress.
文摘Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cerebral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the specific inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats intragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental findings indicate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 pathway.
文摘The endoplasmic reticulum-nuclei-1 (ERN1) sensing and signaling enzyme mediates a set of complex intracellular signaling events known as the unfolded protein response. We have studied the effect of hypoxia and ischemic conditions (glucose or glutamine deprivation) on the expression of several casein kinase-1 and -2 genes in glioma U87 cells and its subline with suppressed function of ERN1. It was shown that blockade of ERN1, the key endoplasmic reticulum stress sensor, leads to an increase in the expression levels of casein kinase-1G2, -1E, -2B and NUCKS1 mRNA, but suppresses casein kinase-1A1, -1D and -2A1. Moreover, the expression levels of casein kinase-1A1, -1D and 1G3 as well as casein kinase-2A1 and -2A2 mRNAs are significantly increased under glutamine dep- rivation conditions both in control and ERN1- deficient glioma cells. At the same time, casein kinase-1E, -2B and NUCKS1 mRNA expression levels are also increased under this condition, but only in cells with suppressed function of ERN1. The expression level of NUCKS1 mRNA, however, is decreased both in control glioma cells and in genetically modified cells, but casein kinase-1G2—only in control U87 cells. Cell exposure to glucose deprivation conditions enhances the expression levels of casein kinase- 1D, 1G3, -1E and -2A1 in both types of glioma cells used, but casein kinase-2B expression levels increase only in cells with suppressed function of ERN1. Hypoxia induces or suppresses the expression of most of the studied genes mainly in ERN1-knockdown cells only. Results of this study show that hypoxia as well as glutamine and glucose deprivation conditions change the expression level most of casein kinase genes and that these effects are dependent on ERN1 signaling enzyme function.
基金supported by the Key Science and Technology Research of Henan Province,No.222102310351(to JW)Luoyang 2022 Medical and Health Guiding Science and Technology Plan Project,No.2022057Y(to JY)Henan Medical Science and Technology Research Program Province-Ministry Co-sponsorship,No.SBGJ202002099(to JY)。
文摘Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the mitochondrial inner membrane,and its role in Parkinson’s disease remains unclear.Protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK)is a factor that regulates cell fate during endoplasmic reticulum stress.Parkin is regulated by PERK and is a target of the unfolded protein response.It is unclear whether PERK regulates PHB2-mediated mitophagy thro ugh Parkin.In this study,we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson’s disease.We used adeno-associated virus to knockdown PHB2 expression.Our res ults showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson’s disease.Ove rexpression of PHB2 inhibited these abnormalities.We also established a 1-methyl-4-phenylpyridine(MPP+)-induced SH-SY5Y cell model of Parkinson’s disease.We found that ove rexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3,and promoted mitophagy.In addition,MPP+regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK.These findings suggest that PHB2 participates in the development of Parkinson’s disease by intera cting with endoplasmic reticulum stress and Parkin.
文摘研究白藜芦醇(resveratrol,Res)对内质网应激途径细胞凋亡和糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)/Tau蛋白磷酸化作用的影响。体外原代培养神经元细胞,采用衣霉素(tunicamycin,TM)建立内质网应激模型,Western blot法检测内质网分子伴侣蛋白葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、未折叠蛋白反应相关的肌醇需要酶1α(inositol-requiring enzyme 1α,IRE1α)的Ser724磷酸化、剪接的X盒结合蛋白1(spliced form of X-box binding protein 1s,XBP1s)表达、GSK-3β的Ser9和Tau蛋白的Ser396磷酸化水平。生物化学方法分析细胞质中半胱天冬酶-12(Caspase-12)和半胱天冬酶-3(Caspase-3)活性、细胞凋亡水平。结果显示,TM能够诱导内质网应激作用,导致神经元细胞GSK-3β的活化和Tau蛋白磷酸化水平升高(P<0.01)、神经元细胞经内质网途径凋亡(P<0.05)。与内质网应激抑制剂4-苯基丁酸结果相似,Res组显著降低了GRP78的表达(P<0.01)、降低了IRE1α-XBP1通路的活性(P<0.01)。Res可以减缓TM诱导的内质网途径细胞凋亡级联反应中Caspase-12和Caspase-3的活性(P<0.01)。Res抑制了TM诱导的GSK-3β的Ser9位点磷酸化水平和Tau蛋白Ser396位点的磷酸化水平(P<0.01)。结果表明,Res能够降低TM诱导的IRE1α-XBP1途径内质网应激作用、GSK-3β的活性及Tau蛋白发生磷酸化水平,减弱神经元细胞经内质网途径凋亡的级联反应作用。
