Sophoridine Regulates the Antiendotoxin Inflammatory Effects of Macrophages through Autophagy
Objective To investigate how sophoridine(SRI)regulates the inflammatory response induced by lipopolysaccharide(LPS)in macrophages through the autophagy pathway.Methods RAW264.7 cells were treated with LPS to establish an inflammatory response model.The experiment was divided into two parts.In the first part of the experiment,cells were treated with SRI and autophagy inhibitor 3-methyladenine(3-MA).The experimental groups were blank control group,SRI group,LPS group,SRI+LPS group,3-MA+LPS group.In the second part of the experiment,cells were pretreated with toll-like receptor 4(TLR4)inhibitor resatorvid(TAK-242).The experiment consisted of five groups,TAK-242 group,TAK-242+SRI group,TAK-242+LPS group,TAK-242+SRI+LPS group,and TAK-242+3-MA+LPS group.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in cell supernatant were measured using ELISA.The mRNA expression levels of TNF-α,IL-6andIL-1β were determined using RT-qPCR.Protein expressionlevels of TLR4,NF-κB,Beclin-1 and ATG5 were analyzed by Western blot.Fluorescence microscopy was used to observe the expression of reactive oxygen species(ROS)and autophagy protein LC3B.Results Pretreatment of LPS-induced RAW264.7 cells with SRI or inhibitors such as TAK-242,3-MA or their combinations significantly reduced secretion levels of TNF-α,IL-6,IL-1 β compared to control groups(P all＜0.05).Reduced ROS production was observed in treated cells(P＜0.05).Moreover,the protein expression levels of TLR4 and phosphorylated NF-κB decreased(P all＜0.05).Additionally,the expressionlevels of Beclin-1 and ATG5 proteins involved in autophagy regulation also decreased(P all＜0.05).Decreased fluorescence intensity for LC3B protein indicated a decrease in cellular autophagic activity(P＜0.05),with combined interventions showing better effects than single-inhibitor treatmen.Conclusion SRI exerted an anti-inflammatory and antioxidative effects by modulating macrophage autophagy pathway through TLR4/NF-κB axis,and antagonize the inflammatory response of endotoxin.
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