基于PI3K/Akt信号通路探讨“赤芍-附片”对肝细胞炎症损伤的影响

Effects of the couplet medicines of"peony root and prepared common monkshood branched root"on the inflammatory damage of hepatocytes based on the PI3K/Akt signaling pathway

目的基于磷脂酰激醇3激酶(phosphati-dylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB又称Akt)信号通路,探讨“赤芍-附片”对肝细胞炎症损伤的保护作用。方法体外培育L02细胞株,设立正常组、模型组、空白血浆组、“赤芍-附片”组、Copanlisib(PI3K抑制剂)组,采用D-半乳糖联合脂多糖干预构建肝细胞炎症损伤模型。通过CCK-8法筛选含药血浆最佳干预浓度及时间;透射电镜观察细胞超微结构;ELISA法检测白介素-1β(interleukin-1β,IL-1β)、白介素-6(interleukin-6,IL-6)及肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)表达水平;实时荧光定量PCR(quantitative real-time PCR,RT-q PCR)、蛋白免疫印迹法(Western blot)测定PI3K/Akt通路m RNA及蛋白表达水平。结果较之正常组,模型组受损细胞器及自噬体数量增加,IL-1β、IL-6、TNF-α含量显著升高(P<0.01),PI3K、Akt mRNA及磷酸化蛋白表达水平明显降低(P<0.01)。较之模型组,“赤芍-附片”组细胞形态相对正常且自噬体数量减少,IL-1β、IL-6、TNF-α含量显著下降(P<0.01),PI3K、Akt mRNA及磷酸化蛋白表达上调(P<0.01或P<0.05)。结论“赤芍-附片”药对可能通过调控PI3K/Akt信号通路,减少IL-1β、IL-6、TNF-α分泌,从而减轻D-半乳糖联合脂多糖诱导的肝细胞损伤。

Objective To explore the protective effect of the couplet medicines of"peony root and prepared common monkshood branched root"on the inflammatory damage of hepatocytes based on the phosphati-dylinositol 3-kinase(PI3K)/protein kinase B(PKB,also known as Akt)signaling pathway.Methods L02 cell strains were cultured in vitro,and a normal group,a model group,a blank plasma group,a medicine group(peony root--prepared common monkshood branched root)and a Copanlisib(PI3K inhibitor)group were established.D-galactose combined with lipopolysaccharide were used to intervene in the establishment of a hepatocyte inflammatory injury model,and corresponding interventions were given respectively.The optimal intervention concentration and time of drugs were screened through the CCK-8 method.The ultrastructure of cells was observed by transmission electron microscope.The expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were detected by ELISA.The mRNA and protein expression levels of the PI3K/Akt signaling pathway were determined by quantitative real time PCR(RT-q PCR)and Western blot.Results Compared with the normal group,the number of damaged organelles and autophagosomes in the model group was increased,and the contents of IL-1β,IL-6 and TNF-αwere significantly increased(P<0.01),while the mRNA and phosphorylated protein expression levels of PI3K and Akt were significantly decreased(P<0.01).Compared with the model group,the cell morphology was relatively normal in the medicine group,and the number of autophagosomes was reduced,the contents of IL-1β,IL-6 and TNF-αwere significantly decreased(P<0.01),while the mRNA and phosphorylated protein expression levels of PI3K and Akt were up-regulated(P<0.01 or P<0.05).Conclusion The couplet medicines of"peony root and prepared common monkshood branched root"may reduce the secretion of IL-1β,IL-6 and TNF-αby regulating the PI3K/Akt signaling pathway,so as to alleviate the liver cell injury induced by D-galactose combined with lipopolysaccharide.