参苓白术散调控TLR4/NF-κB通路抑制LPS诱导心肌细胞炎性损伤的机制

Mechanism of Shenling Baizhu Powder in inhibiting LPS-induced inflammatory injury of myocardial cells by regulating TLR4/NF-κB signaling pathway

目的基于Toll样受体4(Toll-like receptor 4,TLR4)/核因子κB(nuclear factor-κB,NF-κB)信号通路探讨参苓白术散含药血清对脂多糖(lipopolysaccharide,LPS)诱导H9C2心肌细胞炎性损伤的调控机制。方法将分化完全的H9C2心肌细胞分为空白血清组、模型组、参苓白术散含药血清组、TLR4抑制剂(TAK-242)组,每组6个复孔。除空白血清组外,其余各组以10μg·mL^(-1) LPS处理细胞,构建心肌细胞炎症损伤体外模型。空白血清组、模型组及TAK-242组以10%空白血清进行培养,参苓白术散含药血清组用10%含药血清培养,均培养24 h。采用CCK-8法测定各组细胞增殖情况;采用ELISA法及免疫荧光法检测细胞白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量及蛋白表达水平;采用Western blot法检测TLR4、NF-κB、NF-κB抑制蛋白α(degradation of proteinκB-α,IκBα)蛋白表达情况。结果CCK-8法结果发现,10%参苓白术散含药血清对细胞增殖无抑制作用。与空白血清组比较,模型组细胞的TNF-α、IL-1β含量及荧光表达量明显增高(P<0.01),TLR4、NF-κB、Iκ-Bα蛋白表达明显上调(P<0.01)。与模型组比较,参苓白术散含药血清组IL-1β、TNF-α含量显著降低(P<0.01),TLR4、NF-κB、IκBα蛋白表达明显下调(P<0.01)。结论参苓白术散含药血清能抑制LPS诱导的心肌细胞炎性反应,其作用机制可能与调控TLR4/NF-κB通路,下调细胞炎症因子TNF-α、IL-1β表达有关。

Objective To investigate the regulatory mechanism of medicated serum containing Shenling Baizhu Powder(SLBZP)on lipopolysaccharide(LPS)-induced inflammatory injury of H9C2 myocardial cells based on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB)signaling pathway.Methods The fully differentiated H9C2 cells were divided into blank serum group,model group,medicated serum containing SLBZP group,and TLR4 inhibitor(TAK-242)group,with six replicates in each group.Except for the blank serum group,cells of the other groups were treated with LPS 10μg·mL^(-1) to establish an in vitro model of myocardial cell inflammatory injury.Blank serum group,model group,and TAK-242 group were cultured with 10%blank serum,and medicated serum containing SLBZP group with 10%medicated serum.All groups had been cultured for 24 h.CCK-8 assay was used to test the cell proliferation of each group;ELISA and immunofluorescence were used to determine the content of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)and their protein expression levels in cells,respectively;Western blot was used to check the protein expression levels of TLR4,NF-κB,and degradation of proteinκB-α(IκBα).Results The results of CCK-8 assay showed that 10%SLBZP-medicated serum had no inhibitory effects on cell proliferation.Compared with the blank serum group,the content of TNF-αand IL-1βand their fluorescent expression levels in cells of model group significantly increased(P<0.01),and the protein expression levels of TLR4,NF-κB,and IκBαwere significantly up-regulated(P<0.01).Compared with the model group,medicated serum containing SLBZP group showed significantly reduced content of IL-1βand TNF-α(P<0.01)and significantly down-regulated protein expression levels of TLR4,NF-κB,and IκBα(P<0.01).Conclusion Medicated serum containing SLBZP can inhibit the LPS-induced inflammatory response in H9C2 cells.Its mechanism may be related to regulating TLR4/NF-κB pathway and down-regulating the expression of inflammatory factors of TNF-αand IL-1β.