虎杖苷对高脂喂养的中年LDLr-/-小鼠非酒精性脂肪肝炎的作用及机制研究

Effect of polydatin on age-and diet-associated non-alcoholic steatohepatitis in LDL receptor knockout mice and its possible mechanisms

目的探讨虎杖苷对高脂喂养的中年低密度脂蛋白受体基因敲除(low-density lipoprotein receptor knockout,LDLr-/-)小鼠非酒精性脂肪肝炎的保护作用及机制。方法12月龄雄性LDLr-/-小鼠随机分为对照组、模型组、白藜芦醇(200 mg/kg)组和虎杖苷低、高剂量(100、200 mg/kg)组,对照组给予常规饲料喂养,其余各组给予高脂饲料喂养;同时给予虎杖苷和白藜芦醇干预12周后,检测各组小鼠血清代谢参数;采用油红O染色、苏木素-伊红(HE)染色和Masson染色考察各组小鼠肝脏组织病理变化;采用试剂盒检测各组小鼠肝脏中三酰甘油(triglycerides,TG)、总胆固醇(total cholesterol,TC)、游离脂肪酸(free fatty acid,FFA)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin 6,IL-6)、丙二醛(malonaldehyde,MDA)、羟脯氨酸(hydroxyproline,HYP)水平及超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathion peroxidase,GSH)活性;采用免疫共沉淀法检测各组小鼠肝脏组织中乙酰化肝激酶B1(liver kinase Bl,LKB1)表达情况;采用Western blotting法检测各组小鼠肝脏组织中氧化应激、纤维化及sirtuin l(SIRT1)信号通路相关蛋白表达情况。结果虎杖苷显著改善高脂喂养的中年LDLr-/小鼠代谢基础特征(P<0.05),抑制肝脏脂肪变性和肝纤维化;显著降低肝脏组织中TC、TG、TNF-α、IL-6、MDA和HYP水平(P<0.05);显著升高SOD、GSH和CAT活性(P<0.05);显著降低肝脏中脂肪酸合成酶(fatty acid synthase,FASN)、硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase 1,SCD1)、转化生长因子-β(transforming growth factor-β,TGF-β)和α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)蛋白表达水平(P<0.05),显著升高过氧化物酶体增殖激活受体α(peroxisome proliferator-activated receptorα,PPARα)和肉毒碱棕榈酰基转移酶1α(carnitine palmitoyltransferasela,CPT1α)蛋白表达水平(P<0.05);显著增加核因子E2相关因子(nuclear factor E2-:related factor2,Nrf2)入核表达(P<0.05);显著升高SIRT1蛋白表达水平(P<0.05),降低SIRT1靶蛋白肝激酶B1(liver kinase B1,LKB1乙酰化水平(P<0.05);显著升高p-LKB1/LKB1和磷酸化腺苷酸活化蛋白激酶(phosphorylated AMP-activated protein kinase,p-AMPK)/AMPK蛋白表达水平(P<0.05),显著降低p65乙酰化水平和p65入核表达(P<0.05)。结论虎杖苷对高脂喂养的中年LDLr-/-小鼠非酒精性脂肪肝炎具有保护作用,且与SIRT1的激活有关。

Objective To explore the protective effect and mechanism of polydatin on high-fat-fed middle-aged low-density lipoprotein receptor knockout(LDLr-/-)mice with non-alcoholic steatohepatitis.Methods Male LDLr-/-mice(12-month-old)were randomly divided into control group,model group,resveratrol(200 mg/kg)group,low-and high-dose polydatin(100,200 mg/kg)group,rats in control group were fed with regular feed,and the others were fed with high-fat feed;After 12 weeks of intervention with polydatin and resveratrol,mice in each group were tested for serum metabolic parameters;Oil red O staining,hematoxylin-eosin(HE)staining and Masson staining were used to investigate pathological changes of liver in mice of each group;Kits were used to detect levels of triglycerides(TG),total cholesterol(TC),free fatty acid(FFA),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),malonaldehyde(MDA),hydroxyproline(HYP)and activities of superoxide dismutase(SOD),catalase(CAT),glutathion peroxidase(GSH)in liver of mice in each group.Immunoprecipitation method was used to detect expression of acetylated liver kinase B1(LKB1)in liver of mice;Western blotting was used to detect expressions of oxidative stress,fibrosis and sirtuin 1(SIRT1)signaling pathway related proteins in liver of mice in each group.Results Polydatin significantly improved the basic metabolic characteristics of middle-aged LDLr-/-mice induced by fed high-fat diet(P<0.05),inhibited liver steatosis and liver fibrosis,reduced levels of TC,TG,TNF-α,IL-6,MDA and HYP in liver(P<0.05),increased activities of SOD,GSH and CAT(P<0.05),reduced expressions of fatty acid synthase(FASN),stearoyl-CoA desaturase 1(SCD1),transforming growth factor-β(TGF-β)andα-smooth muscle actin(α-SMA)in liver(P<0.05),increased expressions of peroxisome proliferator-activated receptorα(PPARα)and carnitine palmitoyltransferase 1α(CPT1α)in liver(P<0.05),increased expression of nuclear factor E2-related factor 2(Nrf2)into nucleus(P<0.05),increased SIRT1 expression(P<0.05),reduced SIRT1 target protein liver kinase B1(LKB1)acetylation level(P<0.05),increased expressions of p-LKB1/LKB1 and phosphorylated AMP-activated protein kinase(p-AMPK)/AMPK(P<0.05),reduced p65 acetylation level and expression of p65 into nucleus(P<0.05).Conclusion Polydatin has a protective effect on non-alcoholic steatohepatitis in middle-aged LDLr-/-mice fed with high fat,which is related to the activation of SIRT1.