竹节参总皂苷通过HMGB1/TLR4/NF-κB信号通路改善游离脂肪酸诱导肝细胞脂肪变性的实验研究

Effect of total saponins from panax japonicus on on free fatty acid-induced hepatocyte steatosis via HMGB1/TLR4/NF-κB signaling pathway

目的:通过HMGB1/TLR4/NF-κB信号通路探讨竹节参总皂苷(TSPJ)改善游离脂肪酸(FFA)诱导的肝癌细胞HepG2脂肪变性的作用机制。方法:体外培养HepG2细胞,随机分成对照组、FFA组、FFA+高迁移率族蛋白1(HMGB1) p-NC组、FFA+HMGB1 siRNA组、FFA+TSPJ组、FFA+pcDNA+TSPJ组、FFA+HMGB1 pcDNA+TSPJ组、FFA+HMGB1 p-NC+TSPJ组、FFA+HMGB1 siRNA+TSPJ组。RT-qPCR和Western blot检测HMGB1 siRNA干扰效率;油红O染色观察TSPJ对细胞脂质蓄积的影响;检测TSPJ对甘油三酯(TG)的作用。RT-qPCR和Western blot检测各组细胞HMGB1、蛋白Toll样受体4(TLR4)和核因子κB(NF-κB)表达水平。结果:HepG2细胞经0.5 mmol/L FFA处理后,脂质水平及TG含量升高(P<0.05);TSPJ干预使肝细胞脂质水平及TG含量降低(P<0.05);干扰HMGB1表达后,肝细胞脂质水平及TG含量降低(P<0.05);TSPJ处理的同时过表达HMGB1,FFA诱导的脂质水平和TG含量恢复(P<0.05)。FFA诱导使肝细胞HMGB1、TLR4和NF-κB表达升高(P<0.05);TSPJ处理使肝细胞HMGB1、TLR4和NF-κB表达降低(P<0.05);干扰HMGB1表达后TLR4和NF-κB p65进一步降低(P<0.05)。结论:竹节参总皂苷通过HMGB1/TLR4/NF-κB信号通路改善FFA诱导肝细胞脂肪变性。

Objective:To investigate the mechanism of total saponins from Panax japonicum(TSPJ)in improving free fatty acid(FFA)-induced steatosis of HepG2 cells through HMGB1/TLR4/NF-κB signaling pathway.Methods:HepG2 cells were cultured in vitro and randomly separated into control group,FFA group,FFA+high mobility group box 1(HMGB1)p-NC group,FFA+HMGB1 siRNA group,FFA+TSPJ group,FFA+pcDNA+TSPJ group,FFA+HMGB1 pcDNA+TSPJ group,FFA+HMGB1 p-NC+TSPJ group,and FFA+HMGB1 siRNA+TSPJ group.RT-qPCR and Western blot were performed to detect the interference efficiency of HMGB1 siRNA;oil red O staining was performed to observe the effect of TSPJ on cell lipid accumulation.The effect of TSPJ on triglyceride(TG)was determined.The expression levels of HMGB1,Toll-like receptor 4(TLR4),and nuclear factorκB(NF-κB)were detected by RT-qPCR and Western blot.Results:The lipid level and TG content of HepG2 cells were increased after 0.5 mmol/L FFA treatment(P<0.05);TSPJ intervention reduced the lipid level and TG content of hepatocytes(P<0.05);after the interference of HMGB1 expression,the lipid level and TG content of hepatocytes were decreased(P<0.05);simultaneous administration of TSPJ and overexpression of HMGB1 restored FFA-induced lipid levels and TG content.After FFA induction,the expression of HMGB1,TLR4,and NF-κB in hepatocytes increased(P<0.05);the expressions of HMGB1,TLR4 and NF-κB in hepatocytes were decreased after TSPJ treatment(P<0.05);after HMGB1 expression was disrupted,TLR4,and NF-κB p65 were further decreased(P<0.05).Conclusion:Total saponins from Panax japonicum ameliorate FFA-induced hepatocyte steatosis through HMGB1/TLR4/NF-κB signaling pathway.