| Aims: We sought to establish a low temperature oxygen and glucose deprivation/reperfusion(OGD/R)HT22 cells model to mimic the DHCA-induced brain cell injury,thus to explore the regulatory effect of hydrogen on miR-29 family in injuried brain cells.In addition,we would further study the effects of miR-29 family on low temperature OGD/R-associated brain cell injury and involved mechanisms.Methods: HT22 cells were randomly divided into 10 groups:(1)control group;(2)low temperature OGD/R group;(3)low temperature OGD/R+hydrogen group;(4)miR-NC group;(5)agomiR-29a/b/c groups(5-7);(6)antamiR-29a/b/c groups(8-10).A low temperature OGD/R model was established using an airtight container and an anaeropack.The expression of miR-29 family in HT22 cells of different groups was detected by quantitative PCR.The cell viability assay was conducted by CCK-8 kit and the expressions of KEAP1,NRF2,BAX and PUMA protein were measured by western blot.The levels of reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)in HT22 cells were determined by JC-1 and H2 DCFDA kits,respectively.Dual luciferase reporter assay was used to validate the downstream target of miR-29 family.Results: We found that hydrogen can significantly increase the expression of miR-29 family in OGD/R-induced HT22 cells.After transfection of miR-29 family analogs into damaged brain cells,the brain cell injuries were significantly reduced: the number of living cells was increased,the content of ROS was decreased,the reduction of MMP was suppressed,the expression of oxidative stress-related protein KEAP1 and apoptosis-related protein PUMA and BAX were decreased and the level of antioxidant stress-related protein NRF2 was increased.The results of dual luciferase reporter assay also showed that miR-29 family could directly target pro-apoptotic protein PUMA and inhibit its expression.Conclusions: The protective effect of hydrogen on brain cells is closely related to the upregulation of miR-29 family.miR-29 family can protect DHCA-induced neuronal injury by acting on a member of the pro-apoptotic BCL-2 family-PUMA.Aims: Investigate the regulatory effect of HRS on the expression of miR-29 family inhippocampus of DHCA-associated rats,and further study the effects of miR-29 family analogs on brain injury of DHCA-associated rats and the involved mechanisms.Methods: The model of DHCA was established by modified Pulsineli four-vessel occlusion method.The miR-29 family synthetics were injected into the hippocampus of rats by using a microinjector and a stereotaxic apparatus.Quantitative PCR was used to detect the expression of miR-29 family in the hippocampus.The morphological features of DHCA-related hippocampal neurons were observed by histopathology.The contents of inflammatory and oxidative factors in the hippocampus and serum were determined by specific kits.Western blot was used to quantize the expression of Caspase-3,BAX,PUMA,KEAP1 and NRF2 proteins.Results: HRS could significantly upregulate the expression of miR-29 family in hippocampus of DHCA-treated rats.After the hippocampal injection of miR-29 analogs,the brain injury of DHCA-treated rats was significantly reduced: apoptosis of hippocampal cells was decreased,histopathological features of hippocampal CA1 cells was improved,MDA content and NOS activity in the hippocampus and serum were decreased and SOD activity was increased,inflammatory factors TNF-? and IL-1? were reduced,anti-inflammatory factor IL-10 was increased.The expression of oxidative stress-related protein KEAP1 and apoptosis-related proteins Caspase-3,PUMA and BAX were decreased and the expression of antioxidant stress-related protein NRF2 was increased.Conclusions: The brain protective effect of HRS on DHCA rats is related to the upregulation of miR-29 family expression,which exerts its protective effect through anti-apoptotic,anti-inflammatory and antioxidant pathways. |
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