小檗碱通过抑制肝星状细胞自噬改善小鼠肝纤维化

Berberine Relieves Liver Fibrosis in Mice by Inhibiting Autophagy of Hepatic Stellate Cells

目的 研究小檗碱(BBR)对肝星状细胞(HSC)自噬与活化的调控及其抗肝纤维化作用。方法分别采用不同质量浓度BBR(0、2、5、10、20、50μmol/L)干预血小板衍生生长因子-BB(PDGF-BB,20μg/L)处理的LX-2细胞24 h,使用CCK-8法检测细胞活力;采用不同质量浓度BBR(0、2、5、10μmol/L)干预PDGF-BB(20μg/L)处理的LX-2细胞24 h或48 h,使用5-乙炔基-2’脱氧尿嘧啶核苷(EdU)方法检测细胞增殖率,细胞划痕实验检测细胞迁移率。实时荧光定量聚合酶链式反应、免疫印迹法检测小檗碱(5μmol/L)对LX-2细胞α-SMA,COL1A1 mRNA及蛋白表达的影响;检测Atg5、 Becn1、 Atg7、 LC3、p62的表达。动物实验共18只小鼠,随机分为3组,对照组[橄榄油(Oil)+磷酸盐缓冲液(PBS)]、模型组[四氯化碳(CCl4)+PBS]和药物干预组(CCl4+BBR),每组6只小鼠,造模5周,2次/周。第3周开始给与BBR干预,2次/周,连续给药3周。造模5周后处死小鼠,检测各组小鼠肝脏组织中的羟脯胺酸水平,苏木素-伊红(HE)和天狼星红染色观察肝脏组织炎性细胞浸润及胶原沉积情况。结果 BBR作用LX-2细胞24 h的半数抑制浓度(IC50)为(31.51±2.66)μmol/L;BBR可呈剂量依赖性地抑制LX-2细胞的增殖及迁移(P<0.05)。BBR(5μmol/L)下调LX-2细胞的α-SMA、COL1A1 mRNA和蛋白表达水平,抑制细胞活化(P<0.05)。BBR(5μmol/L)下调LX-2细胞的Atg5 mRNA和蛋白表达水平,上调p62蛋白表达水平及减少LC3-Ⅱ/LC3-Ⅰ比值,抑制细胞自噬(P<0.05)。CCl4+BBR与CCl4+PBS组小鼠肝脏比较,BBR干预后,肝细胞脂肪变性坏死和炎性细胞浸润明显减轻,胶原纤维沉积减少,羟脯胺酸含量减少(P<0.05)。结论 BBR在小鼠体内具有抗肝纤维化的作用,在体外可抑制LX-2细胞的增殖、迁移及活化,这可能与BBR抑制HSC自噬水平有关。

Objective To study the regulation of berberine(BBR) on autophagy and activation of hepatic stellate cells(HSC) and its anti-fibrosis effect.Methods LX-2 cells pretreated with platelet-derived growth factor-BB(PDGF-BB,20μg/L) were treated with different concentrations of BBR(0,2,5,10,20,50μmol/L) for 24 h,and the cell viability was detected by cell counting kit-8(CCK-8).LX-2 cells pretreated with PDGF-BB(20μg/L) were treated with different concentrations of BBR(0,2,5,10μmol/L) for 24 h or 48 h,and the cell proliferation rate was detected by 5-acetyne-2‘deoxyuracil nucleoside(Ed U) method and the cell migration rate was detected by scratch test.The effects of BBR(5μmol/L) on mRNA and protein expressions of α-SMA and COL1A1 in LX-2 cells were detected by real-time fluorescence quantitative polymerase chain reaction(qPCR) and western blotting.The expressions of Atg5,Becn1,Atg7,LC3 and p62 were detected.Eighteen male C57BL/6 mice were randomly divided into 3 groups:control group[n=6,Oil+phosphate buffer solution(PBS)],model group[n=6,carbon tetrachloride(CCl4)+PBS],and drug intervention group(n=6,CCl4+BBR),modeled for 5 weeks,twice a week.BBR intervention was started at week 3,twice a week for 3 weeks.After 5 weeks of modeling,the mice were sacrificed.The hydroxyproline content in the liver tissues of each group was detected.The inflammatory cell infiltration and collagen deposition in liver tissues were observed by hematoxylin-eosin(HE) and Sirius red staining.Results The half maximal inhibitory concentration(IC50) of BBR on LX-2 cells for 24 h was(31.51±2.66)μmol/L.BBR inhibited the proliferation and migration of LX-2 cells in a dose-dependent manner(P<0.05).BBR(5μmol/L) downregulated mRNA and protein levels of α-SMA and COL1A1 in LX-2 cells,and inhibited cell activation(P<0.05).BBR(5μmol/L) down-regulated mRNA and protein expression of Atg5 in LX-2 cells,up-regulated p62 protein level,reduced LC3-II/LC3-I ratio,and inhibited autophagy(P<0.05).Comparison of liver between CCl4+BBR group and CCl4+PBS group found that after BBR intervention,steatosis,necrosis and inflammatory cell infiltration of hepatocytes were significantly alleviated,collagen fiber deposition was reduced,hydroxyprolinate content was decreased(P<0.05).Conclusion For mice,BBR has an anti-fibrosis effect in vivo and can inhibit the proliferation,migration and activation of LX-2 cells in vitro,which may be related to its inhibition of HSC autophagy level.