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Objective:To evaluate the role of Rac1 in cerebral ischemia-reperfusion (I/R) injury and the relationship with mitopaghy in diabetic rats.Methods:SPF healthy adult male Sprague-Dawley rats, aged 8 weeks, weighing 250-280 g, in which diabetes mellitus was induced by intraperitoneal streptozotocin, were used in this study.Forty-eight rats with diabetes mellitus were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (sham group), cerebral I/R group (I/R group), I/R plus lentivirus inhibiting Rac1 group (I/R+ shRac1 group), and I/R plus lentivirus-negative control group (I/R+ NC group). Cerebral I/R was induced by 90-min middle cerebral artery occlusion followed by 24-h reperfusion.In I/R+ shRac1 and I/R+ NC groups, Rac1 shRNA lentivirus vector and lentivirus negative control vector 10 μl were injected via the right lateral cerebral ventricle at 7 days before establishing the model, respectively.Rats were sacrificed at 24 h of reperfusion, and brains were removed for determination of cerebral infarct size, expression of BNIP3, P62, LC3Ⅰ and LC3Ⅱ (by Western blot). The LC3Ⅱ/LC3 Ⅰ ratio was calculated. Results:Compared with sham group, the cerebral infarct size was significantly increased in the other three groups ( P<0.05). Compared with I/R group, the cerebral infarct size was significantly decreased, LC3Ⅱ/LC3Ⅰratio was increased, the expression of BNIP3 was up-regulated, and the expression of P62 was down-regulated in group I/R+ shRac1 ( P<0.05 or 0.01), and no significant change was found in each index in group I/R+ NC ( P>0.05). Compared with I/R+ NC group, the cerebral infarct size was significantly decreased, LC3Ⅱ/LC3Ⅰratio was increased, the expression of BNIP3 was up-regulated, and the expression of P62 was down-regulated in group I/R+ shRac1 ( P<0.05 or 0.01). Conclusion:The mechanism by which Rac1 reduces cerebral I/R injury is related to enhancing mitophagy in diabetic rats.
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Objective To evaluate the role of long non-coding RNA maternally expressed gene 3(MEG3)in hyperglycose-induced neurocyte damage and the relationship with mitochondrion-dependent ap-optosis in rats.Methods Normally cultured PC12 cells were divided into 5 groups(n＝18 each)using a random number table method: normal concentration of glucose control group(C group),normal concentra-tion of glucose plus MEG3 group(C+MEG3 group),high-concentration glucose group(HG group),high-concentration glucose plus MEG3 group(HG+MEG3 group),and high-concentration glucose plus negative lentiviral vector(LV-NC)group(HG+NC group).PC12 cells were cultured in DMEM medium with 25 mmol/L glucose in group C.PC12 cells were cultured in DMEM medium with 25 mmol/L glucose after being transfected with MEG3 lentiviral vector(LV-MEG3)in C+MEG3 group.PC12 cells were cultured in DMEM medium with 250 mmol/L glucose in HG group.PC12 cells were incubated in DMEM medium con-taining 250 mmol/L glucose after being transfected with LV-MEG3 in HG+MEG3 group.PC12 cells were in-cubated in DMEM medium containing 250 mmol/L glucose after being transfected with LV-NC in HG+NC group.After the cells were cultured or incubated for 1 day,the cell viability was measured by CCK8 assay,the apoptosis rate and level of reactive oxygen species(ROS)were determined by flow cytometry,and the amount of lactic dehydrogenase(LDH)released was measured by DCFH-DA,the expression of Cyt c,caspase-3,caspase-9,Bcl-2,Bax and Apaf-1 was determined by Western blot,and the opening of mito-chondrial permeability transition pore(mPTP)was determined by fluorescent method.Blc-2/Bax ratio was calculated.Results Compared with group C,the cell viability was significantly decreased,the amount of LDH released,ROS level and apoptosis rate were increased,the opening of mPTP was increased,and the expression of caspase-3,caspase-9,Cyt c,Bax,Bcl-2 and Apaf-1 was up-regulated in HG,HG+MEG3 and HG+NC groups,and Bcl-2/Bax ratio was increased in HG+MEG3 group and decreased in HG and HG+NC groups(P＜0.05).Compared with HG group and HG+NC group,the cell activity was significantly in-creased,the amount of LDH released,ROS level and apoptosis rate were decreased,the opening of mPTP was decreased,the expression of caspase-3,caspase-9,Cyt c,Bax,and Apaf-1 was down-regulated,the expression of Bcl-2 was up-regulated,and Bcl-2/Bax ratio was increased in HG+MEG3 group(P＜0.01).Conclusion MEG3 may be involved in the endogenous protective mechanism during hyperglycose-induced neurocyte damage by inhibiting mitochondrion-dependent apoptosis in rats.
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Objective@#To evaluate the role of long non-coding RNA maternally expressed gene 3 (MEG3) in hyperglycose-induced neurocyte damage and the relationship with mitochondrion-dependent apoptosis in rats.@*Methods@#Normally cultured PC12 cells were divided into 5 groups (n=18 each) using a random number table method: normal concentration of glucose control group (C group), normal concentration of glucose plus MEG3 group (C+ MEG3 group), high-concentration glucose group (HG group), high-concentration glucose plus MEG3 group(HG+ MEG3 group), and high-concentration glucose plus negative lentiviral vector (LV-NC) group (HG+ NC group). PC12 cells were cultured in DMEM medium with 25 mmol/L glucose in group C. PC12 cells were cultured in DMEM medium with 25 mmol/L glucose after being transfected with MEG3 lentiviral vector (LV-MEG3) in C+ MEG3 group.PC12 cells were cultured in DMEM medium with 250 mmol/L glucose in HG group.PC12 cells were incubated in DMEM medium containing 250 mmol/L glucose after being transfected with LV-MEG3 in HG+ MEG3 group.PC12 cells were incubated in DMEM medium containing 250 mmol/L glucose after being transfected with LV-NC in HG+ NC group.After the cells were cultured or incubated for 1 day, the cell viability was measured by CCK8 assay, the apoptosis rate and level of reactive oxygen species (ROS) were determined by flow cytometry, and the amount of lactic dehydrogenase (LDH) released was measured by DCFH-DA, the expression of Cyt c, caspase-3, caspase-9, Bcl-2, Bax and Apaf-1 was determined by Western blot, and the opening of mitochondrial permeability transition pore (mPTP) was determined by fluorescent method.Blc-2/Bax ratio was calculated.@*Results@#Compared with group C, the cell viability was significantly decreased, the amount of LDH released, ROS level and apoptosis rate were increased, the opening of mPTP was increased, and the expression of caspase-3, caspase-9, Cyt c, Bax, Bcl-2 and Apaf-1 was up-regulated in HG, HG+ MEG3 and HG+ NC groups, and Bcl-2/Bax ratio was increased in HG+ MEG3 group and decreased in HG and HG+ NC groups (P<0.05). Compared with HG group and HG+ NC group, the cell activity was significantly increased, the amount of LDH released, ROS level and apoptosis rate were decreased, the opening of mPTP was decreased, the expression of caspase-3, caspase-9, Cyt c, Bax, and Apaf-1 was down-regulated, the expression of Bcl-2 was up-regulated, and Bcl-2/Bax ratio was increased in HG+ MEG3 group (P<0.01).@*Conclusion@#MEG3 may be involved in the endogenous protective mechanism during hyperglycose-induced neurocyte damage by inhibiting mitochondrion-dependent apoptosis in rats.
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Objective To investigate the effects of remote ischemic postconditioning (RIPoC) on global cerebral ischemia-reperfusion (I/R) injury in rats.Methods One hundred and twenty-eight male adult SD rats weighing 200-250 g were randomly divided into 4 groups ( n =32 each):sham operation group (group S),group I/R,group I/R + RIPoC and remote I/R group (group RI/R ).Global cerebral I/R was induced by four-vessel occlusion.Group I/R + RIPoC received 3 cycles of 15 min reperfusion followed by 15 min ischemia in bilateral femoral arteries at the beginning of cerebral reperfusion.The rats were sacrificed at 24 and 48 h of cerebral reperfusion,and brains were removed for determination of neuronal apoptosis (by TUNEL method) in hippocampal CA1 region and the parietal cortex,Bcl-2 and Bax expression (by Western blot) in hippocampal CA1 region.The superoxide dismutase (SOD) and catalase (CAT) activity and malondialdehyde (MDA) content in hippocampal CA1 region and the parietal cortex were also measured at 48 h of cerebral reperfusion.Morris water maze task was used to test the learning and memory function at 4 d of cerebral reperfusion,and the rats were sacrificed at 7 d of cerebral reperfusion,and brains were removed for determination of neuronal density in hippocampal CAl region and the parietal cortex.Results Cerebral I/R significantly increased the number of apoptotic neurons and MDA content,upregulated Bcl-2 and Bax expression,decreased neuronal density,SOD and CAT activity and learning and memory function in group I/R as compared with group S.RIPoC significantly attenuated these cerebral I/R-induced changes.Conclusion RIPoC could protect brain against global cerebral I/R-induced injury,and the mechanism may be related to inhibiting lipid peroxidation,regulating the balance between Bcl-2 and Bax and inhibiting apoptosis.