丹参泽泻汤对代谢相关脂肪性肝病大鼠AMPK/Nrf2信号通路的调节作用

Regulation of Danshen Zexie Decoction on AMPK/Nrf2 Signaling Pathway in Rats with Metabolic-associated Fatty Liver Disease

目的:探讨丹参泽泻汤对代谢相关脂肪性肝病(MAFLD)大鼠的防治作用及其作用机制。方法:60只SD大鼠随机分为正常组、模型组、易善复组(0.144 g·kg^(-1))和丹参泽泻汤低、中、高剂量组(1.16、2.32、4.64 g·kg^(-1)),给予高脂饲料建立MAFLD模型。造模同时给予药物治疗,连续8周。检测血清中总胆固醇(TC)、甘油三酯(TG)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)及肝组织中TC、TG、游离脂肪酸(FFA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、丙二醛(MDA)的含量或活性变化;苏木素-伊红(HE)染色和油红O染色观察肝组织病理形态学变化,并计算脂肪变性评分和油红染色面积;免疫荧光法检测肝组织中活性氧(ROS)含量;蛋白免疫印迹法(Western blot)检测肝组织中腺苷酸活化蛋白激酶(AMPK)、磷酸化(p)-AMPK、核因子E2相关因子(Nrf2)、谷胱氨酸连接酶催化亚基(GCLC)、谷胱氨酸连接酶调节亚基(GCLM)和醌氧化还原酶1(NQO1)的蛋白表达情况。结果:与正常组比较,模型组大鼠血清中TC、TG、ALT、AST及肝组织中TC、TG、FFA、MDA、ROS含量或活性均显著升高(P<0.01),肝组织中SOD活性、GSH含量及p-AMPK、Nrf2、GCLC、GCLM和NQO1蛋白表达水平均明显降低(P<0.05,P<0.01),肝细胞内出现大量脂滴空泡和较多的气球样病变,脂肪变性评分和油红染色面积均显著升高(P<0.01)。与模型组比较,丹参泽泻汤各剂量组均能显著降低大鼠血清中TC、TG、ALT、AST及肝组织中TC、TG、FFA、MDA、ROS含量或活性(P<0.01),升高肝组织中SOD活性、GSH含量及p-AMPK、Nrf2、GCLC、GCLM和NQO1蛋白表达水平(P<0.05,P<0.01),明显改善肝组织脂肪变性(P<0.05,P<0.01)。结论:丹参泽泻汤可能通过激活AMPK/Nrf2信号通路,抑制氧化应激,发挥防治MAFLD的作用。

Objective:To explore the efficacy and mechanism of Danshen Zexie decoction in treating the rats with metabolic-associated fatty liver disease(MAFLD).Method:Sixty male SD rats were randomized into control group,model group,essentiale(polyene phosphatidylcholine capsules,0.144 g·kg^(-1))group,and low-,medium-,and high-dose Danshen Zexie decoction(1.16,2.32,and 4.64 g·kg^(-1),respectively)groups.The rat model of MAFLD was established with high fat diet,and 8-week therapy started at the induction of MAFLD.The serum levels of total cholesterol(TC),triglyceride(TG),alanine aminotransferase(ALT),and aspartate aminotransferase(AST),as well as the liver levels of TC,TG,free fatty acid(FFA),superoxide dismutase(SOD),glutathione(GSH),and malondialdehyde(MDA),were measured.The pathological changes of liver were observed by hematoxylin-eosin(HE)staining and oil red O staining,and the steatosis score and oil red O staining area were calculated.The level of reactive oxygen species(ROS)in the liver was detected by immunofluorescence.The protein levels of adenosine monophosphate-activated protein kinase(AMPK),p-AMPK,nuclear factor E2-related factor 2(Nrf2),glutamate-cysteine ligase catalytic subunit(GCLC),glutamate-cysteine ligase modifier subunit(GCLM),and nicotinamide adenine dinucleotide phosphate:quinine oxidoreductase(NQO1)in the liver were determined by Western blot.Result:Compared with control group,the modeling of MAFLD elevated the levels of TC,TG,ALT,and AST in the serum and TC,TG,FFA,MDA,and ROS in the liver(P<0.01),lowered the levels of SOD and GSH and down-regulated the protein levels of phosphorylation(p)-AMPK,Nrf2,GCLC,GCLM,and NQO1 in the liver(P<0.05,P<0.01).Further,a large number of lipid droplet vacuoles and balloon-like lesions appeared in the hepatocytes,and the steatosis score and oil red O staining area increased in the model group(P<0.01).Compared with model group,Danshen Zexie decoction lowered the levels of TC,TG,ALT,and AST in the serum and TC,TG,FFA,MDA,and ROS in the liver(P<0.01),increased the levels of SOD and GSH and up-regulated the protein levels of p-AMPK,Nrf2,GCLC,GCLM,and NQO1 in the liver(P<0.05,P<0.01).Moreover,the decoction alleviated the degree of liver steatosis(P<0.05,P<0.01).Conclusion:Danshen Zexie decoction protects against MAFLD by activating AMPK/Nrf2 signaling pathway and suppressing oxidative stress.