RAC2通过调控Bcl-2/Bax平衡与PARP-1/caspase-3通路促进脑缺血再灌注神经元凋亡OA

Objective To elucidate the specific role of Ras-related C3 botulinum toxin substrate 2(RAC2)in neuronal apoptosis induced by cerebral ischemia-reperfusion(CIR),and investigate the molecular mechanisms underlying its regulation of apoptosis.Methods ① Cell culture and grouping(n=5):mouse hippocampal neuronal cell line HT22 was divided into normal control(NC)and oxygen-glucose deprivation/reoxygenation(OGD/R)groups.The NC group was maintained under standard culture conditions,while the OGD/R group was subjected to 8 hours'OGD/R.Cell proliferation was assessed using CCK-8 assay and EdU staining,and cell apoptosis was evaluated by flow cytometry.Western blotting was performed to measure the protein levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cleaved caspase-3,cleaved poly(ADP-ribose)polymerase-1(PARP-1),RAC2,and extracellular signal-regulated kinase(ERK)/p-ERK.Quantitative real-time PCR(qRT-PCR)was applied to detect the mRNA level of RAC2.② Gene knockdown:HT22 cells were transfected with sh-RAC2 or non-targeting control shRNA(sh-NC)before OGD/R treatment.Subsequently,the cells were divided into OGD/R:sh-NC and OGD/R:sh-RAC2 groups(n=5).Apoptosis-related proteins were detected by Western blotting.③ Pathway validation:The ERK inhibitor FR180204 was employed to establish 4 groups(n=5):NC,OGD/R,FR180204 alone,and FR180204+OGD/R(pretreated with FR180204 followed by OGD/R treatment).Expression of cleaved caspase-3,RAC2,and p-ERK was analyzed with Western blotting.Results ①Compared with the NC group,OGD/R treatment significantly inhibited HT22 cell proliferation and increased apoptotic rates(P<0.05).OGD/R upregulated pro-apoptotic proteins(Bax,cleaved PARP-1,and cleaved caspase-3),downregulated the anti-apoptotic protein Bcl-2,and markedly elevated RAC2 expression at both mRNA and protein levels(P<0.05).② Specific knockdown of RAC2(sh-RAC2 group)partially restored Bcl-2 expression and attenuated OGD/R-induced upregulation of Bax and cleaved PARP-1 compared with the OGD/R group(P<0.05).③ OGD/R treatment activated the ERK signaling pathway(elevated p-ERK/ERK ratio),upregulated RAC2 expression,and promoted caspase-3 cleavage compared with the NC group(P<0.05).Conversely,pretreatment with FR180204 effectively blocked OGD/R-induced RAC2 upregulation and caspase-3 activation compared with the OGD/R group(P<0.05).Conclusion RAC2 plays a key role in CIR-induced neuronal apoptosis by modulating the Bcl-2/Bax balance and PARP-1 cleavage through the ERK signaling pathway.