基于TLR4信号通路探讨银杏二萜内酯对海马神经元缺氧/复氧损伤的影响OA

Objective To investigate the effects of ginkgo diterpene lactones(GDL)on hypoxia/reoxygenation injury in hippocampal neurons based on the Toll-like receptor 4(TLR4)signaling pathway.Methods Hippocampal neurons were isolated from embryonic day 18 fetal rats under sterile conditions.After 7 days of culture,the neurons were randomly divided into normal group,model group,GDL group,GDL+TAK242 group(TLR4 inhibitor),and GDL+lipopolysaccharide(LPS)group(TLR4 agonist).Except for the normal group,hippocampal neurons in all other groups were subjected to 4 h of hypoxia followed by 24 h of reoxygenation to establish the hypoxia/reoxygenation injury model.After modeling,the normal and model groups were replaced with fresh Neurobasal medium,while the other groups were treated with drug-containing medium.Following 24 h of incubation,the viability of hippocampal neurons was assessed by MTT assay,apoptosis rate was assessed by flow cytometry,reactive oxygen species(ROS)level in neurons was assessed by DCFH-DA assay,oxidative stress indicators[malondialdehyde(MDA)content and superoxide dismutase(SOD)and catalase(CAT)activities]were assessed by spectrophotometry,inflammatory cytokine levels were assessed by ELISA,TLR4 and nuclear factor-κB(NF-κB)p65 mRNA expression levels were assessed by RT-PCR,and expressions of TLR4,NF-κB p65,apoptosis-related proteins were assessed by Western blot.Results Significant differences were observed among the 5 groups in terms of viability,apoptosis rate,the level of ROS,the content of MDA,the activities of SOD and CAT,the levels of inflammatory cytokines[interleukin-1β(IL-1β),IL-6,and tumor necrosis factor-α(TNF-α)],the mRNA and protein expression levels of TLR4 and NF-κB p65,and the expression ratios of B-cell lymphoma 2 associated X protein/B-cell lymphoma 2(Bax/Bcl-2)and Cleaved Caspase-3/Caspase-3(F=74.954,147.688,71.609,79.970,40.886,19.880,65.847,41.434,88.121,43.226,40.420,30.743,43.397,118.710,60.193,respectively;all P<0.05).Compared with the normal group,the model group showed significantly decreased neuronal viability,increased apoptosis rate(both P<0.05),elevated levels of ROS,MDA,IL-1β,IL-6,TNF-α,TLR-4 and NF-κB p65 mRNA and protein expressions,and increased Bax/Bcl-2 and Cleaved Caspase3/Caspase3 ratios,along with decreased SOD and CAT activities,the differences were statistically significant(all P<0.05).Compared with the model group,the GDL group and GDL+TAK242 group showed significantly increased neuronal viability,decreased apoptosis rate(P<0.05),reduced levels of ROS,MDA,IL-1β,IL-6,TNF-α,TLR-4 and NF-κB p65 mRNA and protein expressions,and decreased Bax/Bcl-2 and Cleaved Caspase3/Caspase3 ratios,along with increased SOD and CAT activities,the differences were statistically significant(all P<0.05).Compared with the GDL group,the regulatory effects on all measured parameters in hypoxia/reoxygenation-injured hippocampal neurons were significantly enhanced in the GDL+TAK242 group,whereas the GDL+LPS group significantly reversed the regulatory effects of GDL;the differences were statistically significant(P<0.05).Conclusion By inhibiting the TLR4 signaling pathway,GDL may reduce the downstream inflammatory response,oxidative stress,and neuronal apoptosis mediated by this pathway,thereby alleviating hypoxia/reoxygenation injury in hippocampal neurons.