Shi Ya-Min, Fu Zhi-Hui, Zhu Chun-Sheng, Li Xiao-Ping
the First Affiliated Hospital of Zhengzhou University Zhengzhou 450052, China.
Zhongguo Zhong Yao Za Zhi. 2023 Apr;48(7):1770-1778. doi: 10.19540/j.cnki.cjcmm.20221111.401.
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 FFA-induced 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.
探讨化脂柔肝颗粒（HZRG）对游离脂肪酸（FFA）诱导的非酒精性脂肪性肝病（NAFLD）脂肪变性肝细胞模型自噬的影响，并探究其可能机制。将棕榈酸（PA）和油酸（OA）按1∶2比例混合配制的FFA溶液用于处理L02细胞24小时以诱导肝脂肪变性，从而建立体外NAFLD细胞模型。孵育结束后，采用细胞计数试剂盒-8（CCK-8）法检测细胞活力；用油红O染色检测细胞内脂质蓄积；采用酶联免疫吸附测定（ELISA）法测定甘油三酯（TG）水平；为监测L02细胞中的自噬，使用透射电子显微镜（TEM）观察自噬体；用溶酶体荧光探针LysoBrite Red检测溶酶体pH变化；进行mRFP-GFP-LC3腺病毒转染以观察自噬通量；采用蛋白质免疫印迹法检测自噬标志物LC3B-Ⅰ/LC3B-Ⅱ、自噬底物p62以及沉默信息调节因子1（SIRT1）/5'-单磷酸腺苷（AMP）激活的蛋白激酶（AMPK）信号通路的表达。0.2 mmol·L⁻¹ PA和0.4 mmol·L⁻¹ OA的FFA成功诱导了NAFLD细胞模型。HZRG降低了TG水平（P<0.05，P<0.01）以及FFA诱导的L02细胞的脂质蓄积，同时增加了自噬体和自噬溶酶体的数量以产生自噬通量。它还通过调节溶酶体pH来影响溶酶体功能。此外，HZRG上调了LC3B-Ⅱ/LC3B-Ⅰ、SIRT1、磷酸化AMPK（p-AMPK）和磷酸化蛋白激酶A（p-PKA）的表达（P<0.05，P<0.01），而下调了p62的表达（P<0.01）。此外，3-甲基腺嘌呤（3-MA）或氯喹（CQ）处理明显抑制了HZRG的上述作用。HZRG可预防FFA诱导的L02细胞脂肪变性，其机制可能与促进自噬和调节SIRT1/AMPK信号通路有关。
