AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (D...AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.展开更多
Studies have shown that downregulation of nuclear-enriched autosomal transcript 1(Neat1)may adversely affect the recovery of nerve function and the increased loss of hippocampal neurons in mice.Whether Neat1 has prote...Studies have shown that downregulation of nuclear-enriched autosomal transcript 1(Neat1)may adversely affect the recovery of nerve function and the increased loss of hippocampal neurons in mice.Whether Neat1 has protective or inhibitory effects on neuronal cell apoptosis after secondary brain injury remains unclear.Therefore,the effects of Neat1 on neuronal apoptosis were observed.C57 BL/6 primary neurons were obtained from the cortices of newborn mice and cultured in vitro,and an oxygen and glucose deprivation cell model was established to simulate the secondary brain injury that occurs after traumatic brain injury in vitro.The level of Neat1 expression in neuronal cells was regulated by constructing a recombinant adenovirus to infect neurons,and the effects of Neat1 expression on neuronal apoptosis after oxygen and glucose deprivation were observed.The experiment was divided into four groups:the control group,without any treatment,received normal culture;the oxygen and glucose deprivation group were subjected to the oxygen and glucose deprivation model protocol;the Neat1 overexpression and Neat1 downregulation groups were treated with Neat1 expression intervention techniques and were subjected to the in oxygen and glucose deprivation protocol.The protein expression levels of neurons p53-induced death domain protein 1(PIDD1,a pro-apoptotic protein),caspase-2(an apoptotic priming protein),cytochrome C(a pro-apoptotic protein),and cleaved caspase-3(an apoptotic executive protein)were measured in each group using the western blot assay.To observe changes in the intracellular distribution of cytochrome C,the expression levels of cytochrome C in the cytoplasm and mitochondria of neurons from each group were detected by western blot assay.Differences in the cell viability and apoptosis rate between groups were detected by cell-counting kit 8 assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay,respectively.The results showed that the apoptosis rate,PIDD1,caspase-2,and cleaved caspase-3 expression levels significantly decreased,and cell viability significantly improved in the Neat1 overexpression group compared with the oxygen and glucose deprivation group;however,Neat1 downregulation reversed these changes.Compared with the Neat1 downregulation group,the cytosolic cytochrome C level in the Neat1 overexpression group significantly decreased,and the mitochondrial cytochrome C level significantly increased.These data indicate that Neat1 upregulation can reduce the release of cytochrome C from the mitochondria to the cytoplasm by inhibiting the PIDD1-caspase-2 pathway,reducing the activation of caspase-3,and preventing neuronal apoptosis after oxygen and glucose deprivation,which might reduce secondary brain injury after traumatic brain injury.All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University,China,on December 19,2020(approval No.2020-895).展开更多
基金National Natural Science Foundation of China,No.39770300,30070873the Overseas Chinese Affairs Office of the State Council Foundation,No.98-33
文摘AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.
基金supported by the Youth Scienceof the National Natural Science Foundation of China,No.81701226(to LJ)。
文摘Studies have shown that downregulation of nuclear-enriched autosomal transcript 1(Neat1)may adversely affect the recovery of nerve function and the increased loss of hippocampal neurons in mice.Whether Neat1 has protective or inhibitory effects on neuronal cell apoptosis after secondary brain injury remains unclear.Therefore,the effects of Neat1 on neuronal apoptosis were observed.C57 BL/6 primary neurons were obtained from the cortices of newborn mice and cultured in vitro,and an oxygen and glucose deprivation cell model was established to simulate the secondary brain injury that occurs after traumatic brain injury in vitro.The level of Neat1 expression in neuronal cells was regulated by constructing a recombinant adenovirus to infect neurons,and the effects of Neat1 expression on neuronal apoptosis after oxygen and glucose deprivation were observed.The experiment was divided into four groups:the control group,without any treatment,received normal culture;the oxygen and glucose deprivation group were subjected to the oxygen and glucose deprivation model protocol;the Neat1 overexpression and Neat1 downregulation groups were treated with Neat1 expression intervention techniques and were subjected to the in oxygen and glucose deprivation protocol.The protein expression levels of neurons p53-induced death domain protein 1(PIDD1,a pro-apoptotic protein),caspase-2(an apoptotic priming protein),cytochrome C(a pro-apoptotic protein),and cleaved caspase-3(an apoptotic executive protein)were measured in each group using the western blot assay.To observe changes in the intracellular distribution of cytochrome C,the expression levels of cytochrome C in the cytoplasm and mitochondria of neurons from each group were detected by western blot assay.Differences in the cell viability and apoptosis rate between groups were detected by cell-counting kit 8 assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay,respectively.The results showed that the apoptosis rate,PIDD1,caspase-2,and cleaved caspase-3 expression levels significantly decreased,and cell viability significantly improved in the Neat1 overexpression group compared with the oxygen and glucose deprivation group;however,Neat1 downregulation reversed these changes.Compared with the Neat1 downregulation group,the cytosolic cytochrome C level in the Neat1 overexpression group significantly decreased,and the mitochondrial cytochrome C level significantly increased.These data indicate that Neat1 upregulation can reduce the release of cytochrome C from the mitochondria to the cytoplasm by inhibiting the PIDD1-caspase-2 pathway,reducing the activation of caspase-3,and preventing neuronal apoptosis after oxygen and glucose deprivation,which might reduce secondary brain injury after traumatic brain injury.All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University,China,on December 19,2020(approval No.2020-895).
