石蒜碱通过PI3K/AKT信号通路调节自噬抑制HBV诱发肝纤维化  

Lycoline regulates autophagy through PI3K/AKT signaling pathway and inhibits HBV-induced liver fibrosis

作　　者：张福华[1] 楚胜[1] 李传俊[1] 李先佳[1] ZHANG Fu-hua;CHU Sheng;LI Chuan-jun;LI Xian-jia(Luohe Medical College,Luohe,Henan 462002,China)

机构地区：[1]漯河医学高等专科学校,河南漯河462002

出　　处：《现代预防医学》2022年第4期720-726,共7页Modern Preventive Medicine

基　　金：河南省高等学校重点科研项目(18A310018)。

摘　　要：目的观察石蒜碱抑制乙型肝炎病毒(HBV)诱发肝纤维化的机制。方法50只雄性C57BL/6小鼠随机分为5组,每组10只:正常对照组、模型组、石蒜碱低、高剂量组(50、100 mg/kg)、阳性对照组(秋水仙碱0.12 mg/kg)。除了正常对照组,其余各组采用HBV诱发建立肝纤维化动物模型,治疗组小鼠腹腔注射给药,正常对照组小鼠腹腔注射等体积生理盐水,1次/d,持续8周。并采用细胞间非接触式共培养体系培养HSC-T6,实时荧光定量逆转录聚合酶链反应(q RT-PCR)法检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(collagen I)、转化生长因子-β(TGF-β1)m RNA水平,蛋白免疫印迹(western blot)检测细胞纤维化、自噬、磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路相关蛋白表达水平,运用PI3K-si RNA、自噬抑制剂(3-MA)探讨石蒜碱通过自噬对肝纤维化的影响。结果体内实验显示,石蒜碱能减少肝小叶胶原纤维,降低小鼠体内谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、羟脯氨酸、α-SMA、collagen I、TGF-β1水平,下调p-AKT/AKT、p-PI3K/PI3K水平(t=20.937,P=0.001;t=23.505,P=0.001;t=15.778,P=0.007;t=26.279,P=0.001)。增加Beclin-1、LC3 II/LC3 I水平(t=8.050,P=0.008;t=12.593,P=0.003;t=5.095,P=0.008;t=7.202,P=0.004)。体外实验显示,0.313~20μmol/L的石蒜碱对HSC-T6细胞增殖具有抑制作用(P<0.05);与对照组相比,2.5、10μmol/L石蒜碱能降低α-SMA、collagen I、TGF-β1,下调p-AKT/AKT、pPI3K/PI3K水平(t=8.229,P=0.001;t=11.325,P=0.003),增加LC3 II/LC3 I水平(t=3.798,P=0.019;t=5.735,P=0.005);与石蒜碱组比较,石蒜碱+PI3K-si RNA组collagen I、p-PI3K/PI3K水平降低(t=6.877,P=0.004;t=7.206,P=0.002),LC3 II/LC3 I水平增加(t=4.461,P=0.011),而石蒜碱+3-MA组collagen I、p-PI3K/PI3K水平上调(t=4.073,P=0.015;t=3.105,P=0.036),LC3 II/LC3 I水平下调(t=3.454,P=0.026)。结论石蒜碱能促进肝细胞自噬,下调细胞纤维化水平,其机制可能与调控PI3K/AKT信号通路有关Objective To observe the mechanism of lycoline in inhibiting HBV-induced liver fibrosis.Methods Fifty male C57 BL/6 mice were randomly divided into five groups,with 10 in each group:normal control group,model group,low dose group of lycorine(50 mg/kg),high dose group of lycorine(100 mg/kg),positive control group(colchicine,0.12 mg/kg).Except for the normal group,other groups established animal models of liver fibrosis induced by HBV.The mice in the treatment group were injected intraperitoneally,and the mice in the normal control group were injected intraperitoneally with an equal volume of normal saline,once a day for 8 weeks.HSC-T6 with a non-contact co-culture system between cells was cultured.The method of qRT-PCR was used to detectα-SMA,collagen I,and TGF-β1 mRNA levels.Western blot was used to detect related protein expression levels of cell fibrosis,autophagy,PI3 K/AKT signaling pathway.PI3 K-siRNA and autophagy inhibitor(3-MA)were used to explore the effect of lycoline on liver fibrosis through autophagy.Results In vivo experiments showed that sophoracarpine could reduce liver lobular collagen fibers,and reduce ALT,AST,ALP,hydroxyproline,α-SMA,collagen I,TGF-β1,decrease p-AKT/AKT,p-PI3 K/PI3 K levels(t=20.937,P=0.001;t=23.505,P=0.001;t=15.778,P=0.007;t=26.279,P=0.001),and increase Beclin-1,LC3 II/LC3 I levels(t=8.050,P=0.008;t=12.593,P=0.003;t=5.095,P=0.008;t=7.202,P=0.004).In vitro experiments showed that 0.313 to 20μmol/L lycoline could inhibit the proliferation of HSC-T6 cells(P<0.05).Compared with the control group,2.5 and 10μmol/L lycoline could reduceα-SMA and collagen I,TGF-β1,decrease p-AKT/AKT,p-PI3 K/PI3 K levels(t=8.229,P=0.001;t=11.325,P=0.003),and increase LC3 II/LC3 I levels(t=3.798,P=0.019;t=5.735,P=0.005).Compared with lycoline group,collagen I,-PI3 K/PI3 K levels in lycoline+PI3 K-siRNA group significantly decreased(t=6.877,P=0.004;t=7.206,P=0.002),LC3 II/LC3 I levels were significantly increased(t=4.461,P=0.011),and collagen I,p-PI3 K/PI3 K levels significantly increased(t=4.073,

关 键 词：石蒜碱 PI3K/AKT通路 自噬 HBV 肝纤维化 

分 类 号：R512.62[医药卫生—内科学] R575.2[医药卫生—临床医学]