Effects Of HMGB1/TLR4/NF-κB Signaling Pathway On Microglia And The Intervention Effect Of Calycosin Under Oxygen-glucose Deprivation (OGD) Environment

Objective:To explore the protective effect of calycosin on inflammatory injury in microglia after oxygen-glucose deprivation/reoxygenation（OGD/R）,and to clarify whether its protective effect is related to the HMGB1/TLR4/NF-κB signaling pathway.Methods:In this study,rodent microglia BV2 and HAPI were used for research,and the cells were divided into control group,OGD/R group,calycosin group,TAK-242 group and combined treatment group,ODG/R was performed in all groups except the control group.In calycosin group,BV2and HAPI were pretreated with different concentrations of calycosin(1×10^-6mol/L,2×10^-6mol/L,4×10^-6mol/L)for 24 hours.In TAK-242 group,BV2 and HAPI were pretreated with TAK-242(10×10^-9mol/L)for 0.5 hours.In combined treatment group,the two drugs,calycosin(4×10^-6mol/L)and TAK-242(10×10^-9 mol/L),were used together,and the pretreatment time was the same as calycosin groups and TAK-242 group.In this experiment,OGD/R model was established in vitro to simulate cerebral ischemia-reperfusion injury.After drug pretreatment,BV2 cells were OGD for 4 hours and HAPI for 2 hours.After 24 hours of reoxygenation,cck8 was used to detect the activity of cells,and western blot was used to detect the protein expression levels of TLR4,NF-κB,p-NF-κB,IκBα,and p-IκBα.Elisa detected the expression levels of HMGB1,IL-1β,IL-6 and TNF-αin the supernatant of cells.The m RNA expression levels of HMGB1 and TLR4 were detected by q RT-PCR.Results:After OGD/R,the cells viability decreased,and the expression of HMGB1,TLR4 and NF-κB increased,and the release of inflammatory factors IL-6,IL-1and TNF increased.Pretreatment with calycosin can ameliorate this phenomenon,increase cell viability,reduce HMGB1,TLR4 and NF-κB expression,and reduce inflammatory cytokine production.In addition,the effect of calycosin is similar to that of TAK-242,the combined treatment further enhanced the protective effect of OGD/R inflammatory injury.Conclusion:HMGB1/TLR4/NF-κB signaling pathway is activated during OGD/R and it exacerbates the inflammatory damage of microglia.Calycosin may play a protective role against cerebral ischemia-reperfusion injury by inhibiting HMGB1/TLR4/NF-κB signaling pathway and reducing the inflammatory injury of microglia after OGD/R in a dose-dependent manner.