While the Bushen Yizhi Formula can treat Alzheimer’s disease(AD),the yet to be ascertained specific mechanism of action was explored in this work.Methods:Different concentrations of the Bushen Yizhi Formula and amylo...While the Bushen Yizhi Formula can treat Alzheimer’s disease(AD),the yet to be ascertained specific mechanism of action was explored in this work.Methods:Different concentrations of the Bushen Yizhi Formula and amyloid-beta peptide(Aβ)were used to treat rat pheochromocytoma cells(P12)and human neuroblastoma cells(SH-SY5Y).Cell morphological changes were observed to determine the in vitro cell damage.Cell Counting Kit(CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle,respectively.Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmic reticulum stress(ERS)-related proteins(GRP78 and CHOP),p-IRE1α,IRE1α,ASK1,p-JNK,JNK,Bax,Bcl-2,XBP-1,and Bim.Fura 2-acetoxymethyl ester(Fura-2/AM)was used to determine the intracellular calcium(Ca^(2+))concentration.Also,an AD model was constructed by injecting Aβinto the CA1 area of the hippocampus in Sprague Dawley rats.AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutive days.The Morris water maze experiment was conducted to test the learning and memory of rats.Hematoxylin&Eosin(H&E)and Terminal-deoxynucleotidyl Transferase(TdT)-mediated dUTP Nick-End Labeling(TUNEL)staining were done to determine histopathological changes in the brain.Results:Bushen Yizhi Formula relieved the Aβ-induced effects including cell injury,decreased viability,increased apoptosis,G0/G1 phase cell cycle arrest,upregulation of GRP78,CHOP,p-IRE1α,p-JNK,Bax,XBP-1 and Bim,as well as down-regulation of Bcl-2.These results were also seen with IRE1αsilencing.While Aβsuppressed the learning and memory abilities of rats,the Bushen Yizhi Formula alleviated these effects of Aβ.Brain nerve cell injury induced by Aβcould also be treated with Bushen Yizhi Formula.Conclusion:Bushen Yizhi Formula could influence ERS through the IRE1αsignaling pathway to achieve its therapeutic effects on AD.展开更多
This study investigated the effects and possible targets of Fructus Broussonetiae extract, a traditional Chinese medicinal herb, on a model of Alzheimer＇s disease induced by beta-amyloid peptide 25 35 and D-galactose...This study investigated the effects and possible targets of Fructus Broussonetiae extract, a traditional Chinese medicinal herb, on a model of Alzheimer＇s disease induced by beta-amyloid peptide 25 35 and D-galactose. The results revealed that intragastric administration of Fructus Broussonetiae significantly increased the expression of immunoglobulin-binding protein, a key factor in the endoplasmic reticulum stress-signaling pathway in rat hippocampus. In contrast, the treatment significantly decreased expression levels of PKR-like endoplasmic reticulum kinase and C/EBP homologous protein, and substantially improved learning, memory and spatial recognition dysfunction in rats. This evidence indicates that Fructus Broussonetiae extract improves spatial learning and memory abilities in rats by affecting the regulation of hippocampal endoplasmic reticulum stress and activation of the apoptosis pathway.展开更多
In 1945,Porter et al.published an electon microscopy study of cultured chick fibroblasts in which they observed：＇a granular background and details of a darker lacelike reticulum which in places appears to be made up...In 1945,Porter et al.published an electon microscopy study of cultured chick fibroblasts in which they observed：＇a granular background and details of a darker lacelike reticulum which in places appears to be made up of chains of＂vesicles＂＇（Porter et al.,1945）.This constituted the first published observation of the endoplasmic reticulum（ER）and,while it was not evident at that time,this cytoplasmic system of interconnecting membrane-lined channels, comprising vesicles, tubules and cisternae, has numerous important functions.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
BACKGROUND： Studies have demonstrated that β-amyloid peptide （Aβ）, a characteristic pathological product of Alzheimer＇s disease （AD）, results in neuronal endoplasmic reticulum stress （ERS）. However, the mech...BACKGROUND： Studies have demonstrated that β-amyloid peptide （Aβ）, a characteristic pathological product of Alzheimer＇s disease （AD）, results in neuronal endoplasmic reticulum stress （ERS）. However, the mechanisms of traditional Chinese medicine against ERS in AD are poorly understood. OBJECTIVE： To measure expression levels of protective proteins （GRP78 and GRP94） of ER molecular partners and pro-apoptotic Caspase-12 ER membrane expression following application of traditional Chinese medicine natural cerebrolysin （NC） to treat Aβ1-40-induced ERS. DESIGN, TIME AND SETTING： A parallel-controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital of Southern Medical University between September 2006 and November 2008. MATERIALS： Sprague Dawley male rats, 6-8 weeks old, were used to harvest tibial and femoral bone marrow. Isolation and purification of mesenchymal stem cells （MSCs） were established from the whole bone marrow by removing non-adherent cells in primary and passage cultures. Aβ1-40 was provided by Sigma, USA. NC was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. NC was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yinxingye （Ginkgo Leaf） in a proportion of 1 ： 2： 2. Following conventional water extraction technology, an extract （1 ： 20） was prepared. Six adult, male, New Zealand rabbits underwent intragastric administration of NC extract （0.976 g/kg per day） for 1 month to prepare NC-positive serum, and the remaining 6 rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： A total of 500 nmol/L Aβ1-40 was used to establish ERS models of primary cultured MSCs. AD cell models were incubated with different doses of NC-positive serum （2.5%, 5%, and 10%）. MSCs treated with normal blank serum served as normal blank controls. MAIN OUTCOME MEASURES： Reverse transcription-polymerase chain reaction and fluorescent immunocytochemistry were respectively used to measure mRNA and protein expression levels of GRP78, GRP94, and Caspase-12 in MSCs. RESULTS： Following Aβ1-40 exposure, mRNA and protein expression levels of GRP78 and GRP94, as well as Caspase-12, significantly increased （P 〈 0.05）, suggesting successful establishment of ERS models. Following NC-positive serum application, mRNA and protein expression levels of GRP78 and GRP94 in MSCs significantly increased （P 〈 0.05 or P 〈 0.01）. However, mRNA and protein expression levels of Caspase-12 significantly decreased （P 〈 0.05, or P 〈 0.01） compared with the ERS model group. These effects were dose-dependent. CONCLUSION： NC downregulated Caspase-12 expression and upregulated GRP78 and GRP94 expression in MSCs in a dose-dependent manner under the state of Aβ1-40-induced ERS.展开更多
Objective： To explore the mechanism of endoplasmic reticulum stress（ERS） response and related apoptosis in dopaminergic neurons death. Methods： Nerve growth factor （NGF）-treatedPC12 cells were treated with 6-OHD...Objective： To explore the mechanism of endoplasmic reticulum stress（ERS） response and related apoptosis in dopaminergic neurons death. Methods： Nerve growth factor （NGF）-treatedPC12 cells were treated with 6-OHDA, MPP^＋ and rotenone. MTT assay and flow cytometry were used to measure the cell viability and the rate of celluar apoptosis induced by those neurotoxins. The expression of ERS-related gene XBP1, Grp78, CHOP, caspase-12 in drug-treated group and reserpine preincubafion group was determined with RT-polymerase chain reaction（RT-PCR） and immunohistochemistry. Results： After the exposure to different toxins, the viability of PC12 cells were decreased by 52%, 44%, 40% at 100μM6-OHDA, 75 μM MPP^＋, 20 nM rotenone for 24 h respectively. FCM assay confirmed time-dependent cell apoptosis （P 〈 0.01 ）. The gene and protein expression of XBP1, Grp78 in drug-treated group were significantly increased and reached their peaks 8 h after the treatment（P 〈 0.05）. The expression levels of CHOP and caspase-12 gene were increased 16-24 h after the treatment（P 〈 0.01 ）, but the expression level of caspase-12 was inhibited by reserpine preincubayion（P 〈 0.05）. Conclusion： The excessive ERS and relative activated cell apoptosis pathway may be associated with selective death of dopaminergic neurons.展开更多
Objective:To investigate the protective effects and its possible mechanism of Wuzi Yanzong Pill(WYP) on Parkinson’s disease(PD) model mice.Methods:Thirty-six C57BL/6 male mice were randomly assigned to 3 groups inclu...Objective:To investigate the protective effects and its possible mechanism of Wuzi Yanzong Pill(WYP) on Parkinson’s disease(PD) model mice.Methods:Thirty-six C57BL/6 male mice were randomly assigned to 3 groups including normal,PD,and PD+WYP groups,12 mice in each group.One week of intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) was used to establish the classical PD model in mice.Meanwhile,mice in the PD+WYP group were administrated with 16 g/kg WYP,twice daily by gavage.After 14 days of administration,gait test,open field test and pole test were measured to evaluate the movement function.Tyrosine hydroxylase(TH) neurons in substantia nigra of midbrain and binding immunoglobulin heavy chain protein(GRP78) in striatum and cortex were observed by immunohistochemistry.The levels of TH,GRP78,p-PERK,p-elF2α,ATF4,p-IRE1α,XBP1,ATF6,CHOP,ASK1,p-JNK,Caspase-12,-9 and-3 in brain were detected by Western blot.Results:Compared with the PD group,WYP treatment ameliorated gait balance ability in PD mice(P<0.05).Similarly,WYP increased the total distance and average speed(P<0.05or P<0.01),reduced rest time and pole time(P<0.05).Moreover,WYP significantly increased TH positive cells(P<0.01).Immunofluorescence showed WYP attenuated the levels of GRP78 in striatum and cortex.Meanwhile,WYP treatment significantly decreased the protein expressions of GRP78,p-PERK,p-elF2α,ATF4,p-IRE1α,XBP1,CHOP,Caspase-12 and Caspase-9(P<0.05or P<0.01).Conclusions:WYP ameliorated motor symptoms and pathological lesion of PD mice,which may be related to the regulation of unfolded protein response-mediated signaling pathway and inhibiting the endoplasmic reticulum stress-mediated neuronal apoptosis pathway.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
Background： Amyloid β （Aβ） deposits and the endoplasmic reticulum stress （ERS） are both well established in the development and progression of Alzheimer＇s disease （AD）. However, the mechanism and role of Aβ...Background： Amyloid β （Aβ） deposits and the endoplasmic reticulum stress （ERS） are both well established in the development and progression of Alzheimer＇s disease （AD）. However, the mechanism and role of Aβ-induced ERS in AD-associated pathological progression remain to be elucidated. Methods： The five familial AD （5×FAD） mice and wild-type （WT） mice aged 2, 7, and 12 months were used in the present study. Monis water maze test was used to evaluate their cognitive performance, lmmunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic （CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12） and anti-apoptotic factors （chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN 1]） in the ERS-associated unfolded protein response （UPR） pathway. Results： Compared with age-matched WT mice, 5 xFAD mice showed higher cleaved caspase-3, lower neuron-positive staining at the age of 12 months, but earlier cognitive deficit at the age of 7 months （all P 〈 0.05）. Interestingly, for 2-month-old 5×FAD mice, the related proteins involved in the ERS-associated UPR pathway, including CHOP, cleaved caspase-12, GRP 78, and SYVN 1, were significantly increased when compared with those in age-matched WT mice （all P 〈 0.05）. Moreover, ERS occurred mainly in neurons, not in astrocytes. Conclusions： These findings suggest that compared with those of age-matched WT mice, ERS-associated pro-apoptotic and anti-apoptotic proteins are upregulated in 2-month-old 5×FAD mice, consistent with intracellular Aβ aggregation in neurons.展开更多
Endoplasmic reticulum(ER)stress occurs when ER homeostasis is perturbed with accumulation of unfolded/misfolded protein or calcium depletion.The unfolded protein response(UPR),comprising of inositol-requiring enzyme 1...Endoplasmic reticulum(ER)stress occurs when ER homeostasis is perturbed with accumulation of unfolded/misfolded protein or calcium depletion.The unfolded protein response(UPR),comprising of inositol-requiring enzyme 1 a(IRE1 a),double-stranded RNA-dependent protein kinase(PKR)-like ER kinase(PERK)and activating transcription factor 6(ATF6)signaling pathways,is a protective cellular response activated by ER stress.However,UPR activation can also induce cell death upon persistent ER stress.The liver is susceptible to ER stress given its synthetic and other biological functions.Numerous studies from human liver samples and animal disease models have indicated a crucial role of ER stress and the UPR signaling pathways in the pathogenesis of liver diseases,including non-alcoholic fatty liver disease(NAFLD),alcoholic liver disease(ALD),alpha-1 antitrypsin(AAT)deficiency(AATD),cholestatic liver disease,drug-induced liver injury,ischemia/reperfusion(I/R)injury,viral hepatitis and hepatocel-lular carcinoma(HCC).Extensive investigations have demonstrated the potential underlying mechanisms of the induction of ER stress and the contribution of the UPR pathways during the development of the diseases.Moreover,ER stress and the UPR proteins and genes have become emerging therapeutic targets to treat liver diseases.展开更多
Objective： Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum （ER）. This ER function disorder is called ...Objective： Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum （ER）. This ER function disorder is called endoplasmic reticulum stress （ERS）. Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer＇s disease （AD）, Parkinson＇s disease and so on. Therefore, the protection effect of the traditional Chinese drug Tiantai No. 1 （天泰1号） on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug. Methods： Primarily cultured marrow mesenchymat stem cells （MSCs） of rats were treated by tunicamycin （TM） in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 （which would assist cells to resist cellular stress injury）, and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively. Results： Protein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs （P〈0.05）. All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group （P〈0.05 or P〈0.01） after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group （P〈0.05 or P〈0.01）. This effect showed a dose dependent manner. Conclusion： Tiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.展开更多
[Objectives]To study the effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on GRP78/ATF4 pathway in APP/PS1 mice.[Methods]Twelve 6-month-old female APP/PS1 mice were randomly divided into model group(MOD,...[Objectives]To study the effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on GRP78/ATF4 pathway in APP/PS1 mice.[Methods]Twelve 6-month-old female APP/PS1 mice were randomly divided into model group(MOD,n=6)and human umbilical cord mesenchymal stem cell treatment group(MSC,n=6);six 6-month-old C57BL/6N mice were used as control group(CON,n=6).The mice in each group were treated with the fourth generation of human umbilical cord mesenchymal stem cells through tail vein.Four weeks later,the mice in each group were killed.The expression of GFP78 and ATF4 in the cortex of mice in each group was detected by Western blotting and real-time fluorescence quantitative PCR.[Results]The results of immunoblotting and real-time fluorescence quantitative PCR showed that the expression of GRP78 in MOD group was lower than that in CON group and the expression of ATF4 increased.The expression of GRP78 protein in MSC group was higher than that in MOD group,but the expression of ATF4 protein was lower.The results of real-time fluorescence quantitative PCR showed that the mRNA level of GRP78 decreased and the mRNA level of ATF4 increased in MOD group compared with CON group.The mRNA level of GRP78 in MSC group was higher than that in MOD group,while the mRNA level of ATF4 in MSC group was lower than that in MOD group.[Conclusions]Human umbilical cord mesenchymal stem cells can regulate the expression of GRP78/ATF4 pathway in APP/PSI mice,which may be related to the stress level of endoplasmic reticulum in the brain of APP/PS1 mice mediated by human umbilical cord mesenchymal stem cells.展开更多
本文通过不同手段探讨了STCH(stress and chaperone)对多巴胺能神经元的作用。采用RT-PCR检测STCH的组织表达模式;采用免疫-激光捕获显微切割技术获得单一多巴胺能神经元,采用RT-PCR检测STCH在中脑不同亚区多巴胺能神经元的表达差异;分...本文通过不同手段探讨了STCH(stress and chaperone)对多巴胺能神经元的作用。采用RT-PCR检测STCH的组织表达模式;采用免疫-激光捕获显微切割技术获得单一多巴胺能神经元,采用RT-PCR检测STCH在中脑不同亚区多巴胺能神经元的表达差异;分别构建STCH过表达pEGFP-N2载体和pSuper-EGFP干扰载体,转染HEK293和SH-SY5Y细胞株,检测细胞对MPP+毒性的反应。结果显示:STCH在海马表达最低,肝脏最高;在中央灰质区的多巴胺能神经元可检测到表达,在黑质区检测不到;将STCH干扰片段转染至HEK293细胞,细胞死亡明显;转染至SH-SY5Y细胞,对细胞生长无明显影响,但细胞形态发生改变;与对照组相比过表达STCH的SH-SY5Y细胞对MPP+毒性有抵抗作用,并能抑制MPP+处理细胞中caspase-12的表达。这些结果提示:STCH对多巴胺能神经元具有潜在的保护作用,其机制可能是通过抑制内质网应激诱导的凋亡途径而实现。该结果为寻找Parkinson病的治疗靶向提供了有益的线索。展开更多
C/EBP homologous protein, an important transcription factor during endoplasmic reticulum stress, participates in cell apoptosis mediated by endoplasmic reticulum stress. Previous studies have shown that C/EBP homologo...C/EBP homologous protein, an important transcription factor during endoplasmic reticulum stress, participates in cell apoptosis mediated by endoplasmic reticulum stress. Previous studies have shown that C/EBP homologous protein mediates nerve injury during Alzheimer＇s disease, subarachnoid hemorrhage and spinal cord trauma. In this study, we introduced C/EBP homologous protein short hairpin RNA into the brains of ischemia/reperfusion rat models via injection of lentiviral vector through the left lateral ventricle. Silencing C/EBP homologous protein gene expression significantly reduced cerebral infarction volume, decreased water content and tumor necrosis factor-α and interleukin-1β mRNA expression in brain tissues following infarction, diminished the number of TUNEL-positive cells in the infarct region, decreased caspase-3 protein content and increased Bcl-2 protein content. These results suggest that silencing C/EBP homologous protein lessens cell apoptosis and inflammatory reactions, thereby protecting nerves.展开更多
基金supported by the National Natural Science Foundation of China[81904266,82004309].
文摘While the Bushen Yizhi Formula can treat Alzheimer’s disease(AD),the yet to be ascertained specific mechanism of action was explored in this work.Methods:Different concentrations of the Bushen Yizhi Formula and amyloid-beta peptide(Aβ)were used to treat rat pheochromocytoma cells(P12)and human neuroblastoma cells(SH-SY5Y).Cell morphological changes were observed to determine the in vitro cell damage.Cell Counting Kit(CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle,respectively.Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmic reticulum stress(ERS)-related proteins(GRP78 and CHOP),p-IRE1α,IRE1α,ASK1,p-JNK,JNK,Bax,Bcl-2,XBP-1,and Bim.Fura 2-acetoxymethyl ester(Fura-2/AM)was used to determine the intracellular calcium(Ca^(2+))concentration.Also,an AD model was constructed by injecting Aβinto the CA1 area of the hippocampus in Sprague Dawley rats.AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutive days.The Morris water maze experiment was conducted to test the learning and memory of rats.Hematoxylin&Eosin(H&E)and Terminal-deoxynucleotidyl Transferase(TdT)-mediated dUTP Nick-End Labeling(TUNEL)staining were done to determine histopathological changes in the brain.Results:Bushen Yizhi Formula relieved the Aβ-induced effects including cell injury,decreased viability,increased apoptosis,G0/G1 phase cell cycle arrest,upregulation of GRP78,CHOP,p-IRE1α,p-JNK,Bax,XBP-1 and Bim,as well as down-regulation of Bcl-2.These results were also seen with IRE1αsilencing.While Aβsuppressed the learning and memory abilities of rats,the Bushen Yizhi Formula alleviated these effects of Aβ.Brain nerve cell injury induced by Aβcould also be treated with Bushen Yizhi Formula.Conclusion:Bushen Yizhi Formula could influence ERS through the IRE1αsignaling pathway to achieve its therapeutic effects on AD.
基金the National Natural Science Foundation of China,No. 30973779
文摘This study investigated the effects and possible targets of Fructus Broussonetiae extract, a traditional Chinese medicinal herb, on a model of Alzheimer＇s disease induced by beta-amyloid peptide 25 35 and D-galactose. The results revealed that intragastric administration of Fructus Broussonetiae significantly increased the expression of immunoglobulin-binding protein, a key factor in the endoplasmic reticulum stress-signaling pathway in rat hippocampus. In contrast, the treatment significantly decreased expression levels of PKR-like endoplasmic reticulum kinase and C/EBP homologous protein, and substantially improved learning, memory and spatial recognition dysfunction in rats. This evidence indicates that Fructus Broussonetiae extract improves spatial learning and memory abilities in rats by affecting the regulation of hippocampal endoplasmic reticulum stress and activation of the apoptosis pathway.
文摘In 1945,Porter et al.published an electon microscopy study of cultured chick fibroblasts in which they observed：＇a granular background and details of a darker lacelike reticulum which in places appears to be made up of chains of＂vesicles＂＇（Porter et al.,1945）.This constituted the first published observation of the endoplasmic reticulum（ER）and,while it was not evident at that time,this cytoplasmic system of interconnecting membrane-lined channels, comprising vesicles, tubules and cisternae, has numerous important functions.
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the National Natural Science Foundation of China, No. 30973779the National Special Planning Project for Traditional Chinese Medicine of China, No.02-03LP41the Key Program of Scientific Planning of Guangdong Province, No. 2006B35630007
文摘BACKGROUND： Studies have demonstrated that β-amyloid peptide （Aβ）, a characteristic pathological product of Alzheimer＇s disease （AD）, results in neuronal endoplasmic reticulum stress （ERS）. However, the mechanisms of traditional Chinese medicine against ERS in AD are poorly understood. OBJECTIVE： To measure expression levels of protective proteins （GRP78 and GRP94） of ER molecular partners and pro-apoptotic Caspase-12 ER membrane expression following application of traditional Chinese medicine natural cerebrolysin （NC） to treat Aβ1-40-induced ERS. DESIGN, TIME AND SETTING： A parallel-controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital of Southern Medical University between September 2006 and November 2008. MATERIALS： Sprague Dawley male rats, 6-8 weeks old, were used to harvest tibial and femoral bone marrow. Isolation and purification of mesenchymal stem cells （MSCs） were established from the whole bone marrow by removing non-adherent cells in primary and passage cultures. Aβ1-40 was provided by Sigma, USA. NC was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. NC was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yinxingye （Ginkgo Leaf） in a proportion of 1 ： 2： 2. Following conventional water extraction technology, an extract （1 ： 20） was prepared. Six adult, male, New Zealand rabbits underwent intragastric administration of NC extract （0.976 g/kg per day） for 1 month to prepare NC-positive serum, and the remaining 6 rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： A total of 500 nmol/L Aβ1-40 was used to establish ERS models of primary cultured MSCs. AD cell models were incubated with different doses of NC-positive serum （2.5%, 5%, and 10%）. MSCs treated with normal blank serum served as normal blank controls. MAIN OUTCOME MEASURES： Reverse transcription-polymerase chain reaction and fluorescent immunocytochemistry were respectively used to measure mRNA and protein expression levels of GRP78, GRP94, and Caspase-12 in MSCs. RESULTS： Following Aβ1-40 exposure, mRNA and protein expression levels of GRP78 and GRP94, as well as Caspase-12, significantly increased （P 〈 0.05）, suggesting successful establishment of ERS models. Following NC-positive serum application, mRNA and protein expression levels of GRP78 and GRP94 in MSCs significantly increased （P 〈 0.05 or P 〈 0.01）. However, mRNA and protein expression levels of Caspase-12 significantly decreased （P 〈 0.05, or P 〈 0.01） compared with the ERS model group. These effects were dose-dependent. CONCLUSION： NC downregulated Caspase-12 expression and upregulated GRP78 and GRP94 expression in MSCs in a dose-dependent manner under the state of Aβ1-40-induced ERS.
基金National Natural Science Foundation of China(30570627)
文摘Objective： To explore the mechanism of endoplasmic reticulum stress（ERS） response and related apoptosis in dopaminergic neurons death. Methods： Nerve growth factor （NGF）-treatedPC12 cells were treated with 6-OHDA, MPP^＋ and rotenone. MTT assay and flow cytometry were used to measure the cell viability and the rate of celluar apoptosis induced by those neurotoxins. The expression of ERS-related gene XBP1, Grp78, CHOP, caspase-12 in drug-treated group and reserpine preincubafion group was determined with RT-polymerase chain reaction（RT-PCR） and immunohistochemistry. Results： After the exposure to different toxins, the viability of PC12 cells were decreased by 52%, 44%, 40% at 100μM6-OHDA, 75 μM MPP^＋, 20 nM rotenone for 24 h respectively. FCM assay confirmed time-dependent cell apoptosis （P 〈 0.01 ）. The gene and protein expression of XBP1, Grp78 in drug-treated group were significantly increased and reached their peaks 8 h after the treatment（P 〈 0.05）. The expression levels of CHOP and caspase-12 gene were increased 16-24 h after the treatment（P 〈 0.01 ）, but the expression level of caspase-12 was inhibited by reserpine preincubayion（P 〈 0.05）. Conclusion： The excessive ERS and relative activated cell apoptosis pathway may be associated with selective death of dopaminergic neurons.
基金Supported by the National Natural Science Foundation of China(Nos.81703978 and 81102552)the Special Fund for Science and Technology Innovation Team of Shanxi University of Chinese Medicine(No.2022TD1013)+5 种基金the Natural Science Foundation of Shanxi Province(No.201901D111334)the Returned Chinese Scholars Technology Activities Preferred Project,Shanxi Province of China(No.20200026)the Research Project supported by Shanxi Scholarship Council of China(No.2021-142)Shanxi University Science and Technology Innovation Project(No.2019L0724)the Key Science and technology R&D project of Jinzhong(No.Y213004)the Young Scientist Cultivation Program Project,Shanxi University of Chinese Medicine(No.2021PY-QN-03)。
文摘Objective:To investigate the protective effects and its possible mechanism of Wuzi Yanzong Pill(WYP) on Parkinson’s disease(PD) model mice.Methods:Thirty-six C57BL/6 male mice were randomly assigned to 3 groups including normal,PD,and PD+WYP groups,12 mice in each group.One week of intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) was used to establish the classical PD model in mice.Meanwhile,mice in the PD+WYP group were administrated with 16 g/kg WYP,twice daily by gavage.After 14 days of administration,gait test,open field test and pole test were measured to evaluate the movement function.Tyrosine hydroxylase(TH) neurons in substantia nigra of midbrain and binding immunoglobulin heavy chain protein(GRP78) in striatum and cortex were observed by immunohistochemistry.The levels of TH,GRP78,p-PERK,p-elF2α,ATF4,p-IRE1α,XBP1,ATF6,CHOP,ASK1,p-JNK,Caspase-12,-9 and-3 in brain were detected by Western blot.Results:Compared with the PD group,WYP treatment ameliorated gait balance ability in PD mice(P<0.05).Similarly,WYP increased the total distance and average speed(P<0.05or P<0.01),reduced rest time and pole time(P<0.05).Moreover,WYP significantly increased TH positive cells(P<0.01).Immunofluorescence showed WYP attenuated the levels of GRP78 in striatum and cortex.Meanwhile,WYP treatment significantly decreased the protein expressions of GRP78,p-PERK,p-elF2α,ATF4,p-IRE1α,XBP1,CHOP,Caspase-12 and Caspase-9(P<0.05or P<0.01).Conclusions:WYP ameliorated motor symptoms and pathological lesion of PD mice,which may be related to the regulation of unfolded protein response-mediated signaling pathway and inhibiting the endoplasmic reticulum stress-mediated neuronal apoptosis pathway.
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.
基金This work was supported by grants from the National Natural Science Foundation of China （No. 91232709, No. 811171216, and No. 81161120496 for Prof. Xiao-Chun Chen, and No. 81200991 for Prof. Xiao-Dong Pan） and the National and Fujian Province＇s Key Clinical Specialty Discipline Construction Programs.
文摘Background： Amyloid β （Aβ） deposits and the endoplasmic reticulum stress （ERS） are both well established in the development and progression of Alzheimer＇s disease （AD）. However, the mechanism and role of Aβ-induced ERS in AD-associated pathological progression remain to be elucidated. Methods： The five familial AD （5×FAD） mice and wild-type （WT） mice aged 2, 7, and 12 months were used in the present study. Monis water maze test was used to evaluate their cognitive performance, lmmunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic （CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12） and anti-apoptotic factors （chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN 1]） in the ERS-associated unfolded protein response （UPR） pathway. Results： Compared with age-matched WT mice, 5 xFAD mice showed higher cleaved caspase-3, lower neuron-positive staining at the age of 12 months, but earlier cognitive deficit at the age of 7 months （all P 〈 0.05）. Interestingly, for 2-month-old 5×FAD mice, the related proteins involved in the ERS-associated UPR pathway, including CHOP, cleaved caspase-12, GRP 78, and SYVN 1, were significantly increased when compared with those in age-matched WT mice （all P 〈 0.05）. Moreover, ERS occurred mainly in neurons, not in astrocytes. Conclusions： These findings suggest that compared with those of age-matched WT mice, ERS-associated pro-apoptotic and anti-apoptotic proteins are upregulated in 2-month-old 5×FAD mice, consistent with intracellular Aβ aggregation in neurons.
基金This work was supported by USA National Institute of Diabetes and Digestive and Kidney Diseases(NIDDK)R01 DK093807.
文摘Endoplasmic reticulum(ER)stress occurs when ER homeostasis is perturbed with accumulation of unfolded/misfolded protein or calcium depletion.The unfolded protein response(UPR),comprising of inositol-requiring enzyme 1 a(IRE1 a),double-stranded RNA-dependent protein kinase(PKR)-like ER kinase(PERK)and activating transcription factor 6(ATF6)signaling pathways,is a protective cellular response activated by ER stress.However,UPR activation can also induce cell death upon persistent ER stress.The liver is susceptible to ER stress given its synthetic and other biological functions.Numerous studies from human liver samples and animal disease models have indicated a crucial role of ER stress and the UPR signaling pathways in the pathogenesis of liver diseases,including non-alcoholic fatty liver disease(NAFLD),alcoholic liver disease(ALD),alpha-1 antitrypsin(AAT)deficiency(AATD),cholestatic liver disease,drug-induced liver injury,ischemia/reperfusion(I/R)injury,viral hepatitis and hepatocel-lular carcinoma(HCC).Extensive investigations have demonstrated the potential underlying mechanisms of the induction of ER stress and the contribution of the UPR pathways during the development of the diseases.Moreover,ER stress and the UPR proteins and genes have become emerging therapeutic targets to treat liver diseases.
基金Supported by the National Foundation of Traditional Chinese Medical Science and Technology(No.02-03LP41)Key Project of the Foundation of Science and Technology of Guangdong Province(No.2006B35630007)
文摘Objective： Changes of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum （ER）. This ER function disorder is called endoplasmic reticulum stress （ERS）. Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer＇s disease （AD）, Parkinson＇s disease and so on. Therefore, the protection effect of the traditional Chinese drug Tiantai No. 1 （天泰1号） on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug. Methods： Primarily cultured marrow mesenchymat stem cells （MSCs） of rats were treated by tunicamycin （TM） in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 （which would assist cells to resist cellular stress injury）, and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively. Results： Protein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs （P〈0.05）. All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group （P〈0.05 or P〈0.01） after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group （P〈0.05 or P〈0.01）. This effect showed a dose dependent manner. Conclusion： Tiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.
基金Supported by Major Project of Basic Scientific Research in Chengde Medical University(KY202217).
文摘[Objectives]To study the effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on GRP78/ATF4 pathway in APP/PS1 mice.[Methods]Twelve 6-month-old female APP/PS1 mice were randomly divided into model group(MOD,n=6)and human umbilical cord mesenchymal stem cell treatment group(MSC,n=6);six 6-month-old C57BL/6N mice were used as control group(CON,n=6).The mice in each group were treated with the fourth generation of human umbilical cord mesenchymal stem cells through tail vein.Four weeks later,the mice in each group were killed.The expression of GFP78 and ATF4 in the cortex of mice in each group was detected by Western blotting and real-time fluorescence quantitative PCR.[Results]The results of immunoblotting and real-time fluorescence quantitative PCR showed that the expression of GRP78 in MOD group was lower than that in CON group and the expression of ATF4 increased.The expression of GRP78 protein in MSC group was higher than that in MOD group,but the expression of ATF4 protein was lower.The results of real-time fluorescence quantitative PCR showed that the mRNA level of GRP78 decreased and the mRNA level of ATF4 increased in MOD group compared with CON group.The mRNA level of GRP78 in MSC group was higher than that in MOD group,while the mRNA level of ATF4 in MSC group was lower than that in MOD group.[Conclusions]Human umbilical cord mesenchymal stem cells can regulate the expression of GRP78/ATF4 pathway in APP/PSI mice,which may be related to the stress level of endoplasmic reticulum in the brain of APP/PS1 mice mediated by human umbilical cord mesenchymal stem cells.
文摘本文通过不同手段探讨了STCH(stress and chaperone)对多巴胺能神经元的作用。采用RT-PCR检测STCH的组织表达模式;采用免疫-激光捕获显微切割技术获得单一多巴胺能神经元,采用RT-PCR检测STCH在中脑不同亚区多巴胺能神经元的表达差异;分别构建STCH过表达pEGFP-N2载体和pSuper-EGFP干扰载体,转染HEK293和SH-SY5Y细胞株,检测细胞对MPP+毒性的反应。结果显示:STCH在海马表达最低,肝脏最高;在中央灰质区的多巴胺能神经元可检测到表达,在黑质区检测不到;将STCH干扰片段转染至HEK293细胞,细胞死亡明显;转染至SH-SY5Y细胞,对细胞生长无明显影响,但细胞形态发生改变;与对照组相比过表达STCH的SH-SY5Y细胞对MPP+毒性有抵抗作用,并能抑制MPP+处理细胞中caspase-12的表达。这些结果提示:STCH对多巴胺能神经元具有潜在的保护作用,其机制可能是通过抑制内质网应激诱导的凋亡途径而实现。该结果为寻找Parkinson病的治疗靶向提供了有益的线索。
文摘目的:探讨泛素-蛋白酶体系统(UPS)功能障碍诱发帕金森病(PD)细胞模型的作用机制,为深入研究PD的发病机制提供理论依据。方法:建立PSI诱导的PC12细胞模型,实验组、对照组分别加入PSI和DMSO(终浓度为10μmol.L-1)孵育36 h后提取蛋白,应用荧光差异凝胶电泳(DIGE)系统获得差异蛋白点,运用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF Pro MS)鉴定出差异蛋白。结果:实验组与对照组比较,在36 h可见细胞内嗜酸性类Lewy小体形成及细胞凋亡(细胞核呈固缩状或碎裂状),凋亡百分率为25.53%。实验组内质网蛋白ERp29与对照组比较表达量显著降低,差异有显著性(P<0.01)。结论:内质网应激(ERS)在UPS功能障碍引起PD的病理变化过程中起重要作用。
文摘C/EBP homologous protein, an important transcription factor during endoplasmic reticulum stress, participates in cell apoptosis mediated by endoplasmic reticulum stress. Previous studies have shown that C/EBP homologous protein mediates nerve injury during Alzheimer＇s disease, subarachnoid hemorrhage and spinal cord trauma. In this study, we introduced C/EBP homologous protein short hairpin RNA into the brains of ischemia/reperfusion rat models via injection of lentiviral vector through the left lateral ventricle. Silencing C/EBP homologous protein gene expression significantly reduced cerebral infarction volume, decreased water content and tumor necrosis factor-α and interleukin-1β mRNA expression in brain tissues following infarction, diminished the number of TUNEL-positive cells in the infarct region, decreased caspase-3 protein content and increased Bcl-2 protein content. These results suggest that silencing C/EBP homologous protein lessens cell apoptosis and inflammatory reactions, thereby protecting nerves.
