二甲双胍经SIRT1/HMGB1通路改善LPS诱导的小鼠小胶质细胞炎症反应OA

Objective To investigate the effect of metformin(MET)on the inflammatory response induced by lipopolysaccharide(LPS)in mouse microglia(BV2 cells)and explore its underlying mechanism.Methods BV2 cells were cultured in vitro and divided into six groups:CON group,LPS group,L-MET group,M-MET group,H-MET group and H-MET+EX527(a specific SIRT1 inhibitor)group.Cells in CON group were not treated.Cells in LPS group were exposed to LPS at a final concentration of 1 μg/mL for 24 h.Cells in MET groups were incubated with metformin at 0.25,1 and 4 μmol/L for 12 h,respectively,and then stimulated with LPS for 24 h.Cells in H-MET+EX527 group were pretreated with 50 μmol/L EX527 for 1 h,then incubated with 4 μmol/L MET for 12 h,and then stimulated with LPS for 24 h.CCK-8 method was used to assess cell viability,Griess method was used to measure nitric oxide(NO)release,and RT-qPCR and Western blot were employed to detect the mRNA and protein expression levels of SIRT1,HMGB1,TLR4 and NF-κB.Simultaneously,a co-culture system of BV2 cell-conditioned medium and mouse neuronal cells(HT22 cells)was used as an experimental model,and the viability of HT22 cells was measured to evaluate the indirect neuroprotective effect of MET.Results Compared with CON group,NO release increased in LPS group(P<0.01),SIRT1 expression was down-regulated(P<0.05),and expressions of HMGB1,TLR4,and NF-κB were up-regulated(P<0.05),and viability of HT22 cells decreased under co-culture condi-tions(P<0.05).Compared with the LPS group,SIRT1 expression was significantly increased in all MET groups(P<0.05),whereas the expressions of HMGB1,TLR4 and NF-κB,as well as NO release,were significantly decreased(P<0.05),with the effects becoming more pronounced at higher MET concentrations.Meanwhile,the viability of HT22 neurons was significantly enhanced(P<0.05).Compared with the H-MET group,the H-MET+EX527 group showed decreased SIRT1 expression(P<0.05),elevated expressions of HMGB1,TLR4 and NF-κB,and increased NO release(P<0.05),along with a decline in HT22 cell viability back to lower levels(P<0.05).Conclusion Metformin may inhibit LPS-induced BV2 microglial activation and significantly alleviate neuroinflammatory responses through regulation of the SIRT1/HMGB1 signaling pathway.