化滞柔肝颗粒通过诱导自噬缓解非酒精性脂肪肝病细胞模型脂肪变性

Huazhi Rougan Granules attenuates steatosis in cell model of nonalcoholic fatty liver disease by inducing autophagy

探讨化滞柔肝颗粒对混合脂肪酸诱导的非酒精性脂肪肝病L02细胞模型自噬的影响及其缓解脂肪变性的可能机制。棕榈酸(palmitic acid,PA)和油酸(oleic acid,OA)以1∶2比例混合制备成游离脂肪酸(free fatty acid,FFA)混合液,诱导L02细胞24 h发生脂肪变性,建立非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFLD)体外模型。CCK-8法测定各组的细胞增殖活力;油红O染色法观察肝细胞脂肪变性的程度;酶联免疫吸附法(enzyme-linked immunosorbnent assay,ELISA)测定肝细胞中甘油三酯(triglyceride,TG)含量;透射电镜(transmission electron microscopy,TEM)观察细胞内自噬体的形成;LysoBrite Red荧光探针检测溶酶体内pH变化;mRFP-GFP-LC3腺病毒转染检测对细胞内自噬流的影响;免疫印迹法(Western blot)检测自噬标记物LC3B-Ⅰ/LC3B-Ⅱ、自噬底物p62、沉默信息调节因子1(silent information regulator 1,SIRT1)/腺苷酸激活蛋白激酶[adenosine 5′-monophosphate(AMP)-activated protein kinase,AMPK]信号通路蛋白表达水平。FFA在0.2 mmol·L-1PA和0.4 mmol·L-1OA条件下成功诱导NAFLD细胞模型;与模型组相比,化滞柔肝颗粒不同剂量均显著降低细胞内TG含量(P<0.05,P<0.01);油红O染色显示各给药组脂滴分布较模型组减少;透射电镜观察到细胞内自噬体形成增多;mRFP-GFP-LC3腺病毒转染结果显示化滞柔肝颗粒促进自噬溶酶体形成,从而促进自噬流的形成;LysoBrite Red染色结果显示化滞柔肝颗粒通过调控溶酶体内pH影响溶酶体功能;Western blot结果显示化滞柔肝颗粒显著升高LC3B-Ⅱ/LC3B-Ⅰ、SIRT1、p-AMPK和磷酸化蛋白激酶A(phospho-protein kinase A,p-PKA)蛋白的表达水平(P<0.05,P<0.01),显著降低p62蛋白的表达水平(P<0.01)。化滞柔肝颗粒可以有效地防治混合脂肪酸诱导的NAFLD细胞模型脂肪变性,其作用机制可能与促进细胞自噬和调控SIRT1/AMPK信号通路有关。

To investigate the effect of Huazhi Rougan Granules(HZRG)on autophagy in a steatotic hepatocyte model of free fatty acid(FFA)-induced nonalcoholic fatty liver disease(NAFLD)and explore the possible mechanism.FFA solution prepared by mixing palmitic acid(PA)and oleic acid(OA)at the ratio of 1∶2 was used to induce hepatic steatosis in L02 cells after 24 h treatment,and an in vitro NAFLD cell model was established.After termination of incubation,cell counting kit-8(CCK-8)assay was performed to detect the cell viability;Oil red O staining was employed to detect the intracellular lipid accumulation;enzyme-linked immunosorbnent assay(ELISA)was performed to measure the level of triglyceride(TG);to monitor autophagy in L02 cells,transmission electron microscopy(TEM)was used to observe the autophagosomes;LysoBrite Red was used to detect the pH change in lysosome;transfection with mRFP-GFP-LC3 adenovirus was conducted to observe the autophagic flux;Western blot was performed to determine the expression of autophagy marker LC3B-Ⅰ/LC3B-Ⅱ,autophagy substrate p62 and silent information regulator 1(SIRT1)/adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)signaling pathway.NAFLD cell model was successfully induced by FFA at 0.2 mmol·L-1 PA and 0.4 mmol·L-1 OA.HZRG reduced the TG level(P<0.05,P<0.01)and the lipid accumulation of FFAinduced L02 cells,while elevated the number of autophagosomes and autophagolysosomes to generate autophagic flux.It also affected the functions of lysosomes by regulating their pH.Additionally,HZRG up-regulated the expression of LC3B-Ⅱ/LC3B-Ⅰ,SIRT1,p-AMPK and phospho-protein kinase A(p-PKA)(P<0.05,P<0.01),while down-regulated the expression of p62(P<0.01).Furthermore,3-methyladenine(3-MA)or chloroquine(CQ)treatment obviously inhibited the above effects of HZRG.HZRG prevented FFA-induced steatosis in L02 cells,and its mechanism might be related to promoting autophagy and regulating SIRT1/AMPK signaling pathway.