黄芪总皂苷配伍荷叶总生物碱对高脂血症大鼠肝脏AMPK/SREBP-1c/ACC通路的影响

Effects of Total Saponins of Astragalus Radix Combined with Total Alkaloids of Nelumbinis Folium on the Hepatic AMPK/SREBP-1c/ACC Pathway in Hyperlipidemic Rats

目的 探讨黄芪总皂苷配伍荷叶总生物碱对高脂血症大鼠的防治作用及机制。方法 将60只SD雄性大鼠随机分为正常组、模型组、阳性药(辛伐他汀,2.1 mg·kg^(-1))组和黄芪总皂苷+荷叶总生物碱低(17+40 mg·kg^(-1))、中(34+80 mg·kg^(-1))、高(68+160 mg·kg^(-1))剂量组;采用高脂饲料喂养复制高脂血症大鼠模型,同时给予药物灌胃治疗,每日1次,连续4周。检测大鼠血清总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)、天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)水平;采用HE染色法观察肝脏组织病理形态学变化;qRT-PCR法检测肝组织中腺苷酸活化蛋白激酶(AMPK)、固醇调节元件结合蛋白1c(SREBP-1c)、乙酰辅酶A羧化酶(ACC)、肉碱棕榈酰转移酶1(CPT-1)m RNA表达情况;免疫荧光法检测肝组织中p-AMPK、SREBP-1c、ACC、CPT-1蛋白表达情况;Western Blot法检测肝组织中AMPK、p-AMPK、SREBP-1c、ACC、p-ACC、CPT-1蛋白表达情况。结果 与正常组比较,模型组大鼠血清TG、TC、FFA、LDL-C、AST、ALT水平显著升高(P<0.01),HDL-C水平显著下降(P<0.01);大鼠肝细胞内有大量的脂滴聚集,胞浆疏松,出现了大量脂滴空泡和气球样变,细胞核偏移;大鼠肝组织AMPK、CPT-1 mRNA表达显著下调(P<0.01),ACC、SREBP-1c mRNA表达显著上调(P<0.01);大鼠肝脏中p-AMPK、CPT-1蛋白阳性表达明显减少(P<0.05,P<0.01),SREBP-1c、ACC蛋白阳性表达显著增加(P<0.01);大鼠肝组织中AMPK蛋白表达未见明显变化(P>0.05),p-AMPK、p-ACC、CPT-1蛋白表达水平明显下降(P<0.01),SREBP-1c、ACC蛋白表达水平显著升高(P<0.01)。与模型组比较,各给药组大鼠血清TG、TC、FFA、LDL-C、AST、ALT水平明显下降(P<0.05,P<0.01),HDL-C水平明显升高(P<0.01);大鼠肝细胞内的脂肪沉积均有明显减轻,仅见少量的脂滴空泡;大鼠肝组织AMPK、CPT-1mRNA的表达明显上调(P<0.05,P<0.01),SREBP-1c、ACC mRNA表达明显下调(P<0.05,P<0.01);大鼠肝脏中p-AMPK、CPT-1蛋白阳性表达明显增加(P<0.05,P<0.01),SREBP-1c、ACC蛋白阳性表达显著减少(P<0.01);大鼠肝组织中AMPK蛋白表达未见明显变化(P>0.05),p-AMPK、p-ACC、CPT-1蛋白表达水平均明显升高(P<0.05,P<0.01),SREBP-1c、ACC蛋白表达水平显著降低(P<0.01)。结论 黄芪总皂苷配伍荷叶总生物碱可能通过调节AMPK/SREBP-1c/ACC信号通路,抑制脂肪合成,促进脂肪酸氧化分解来防治高脂血症。

Objective To investigate the effect and mechanism of the combination of total saponins of Astragalus Radix and total alkaloids of Nelumbinis Folium on the prevention and treatment of hyperlipidaemia in rats.Methods Sixty SD male rats were randomly divided into the normal group,the model group,the positive drug(Simvastatin,2.1 mg·kg^(-1))group and total saponins of Astragalus Radix + total alkaloids of Nelumbinis Folium low-(17+40 mg·kg^(-1)),medium-(34+80 mg·kg^(-1))and high-(68+160 mg·kg^(-1))dose groups;a replicated hyperlipidaemic rat model was fed high-fat diet and given concomitant drug treatment(once daily)for consecutive 4 weeks. Serum total cholesterol(TC), triglyceride(TG), free fatty acid(FFA), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were measured;the histomorphological changes of liver were observed by HE staining;mRNA expressions of adenosine monophosphate activated protein kinase(AMPK),sterol regulatory element binding protein 1c(SREBP-1c),acetyl coenzyme A carboxylase(ACC)and carnitine palmityl transferase 1(CPT-1)in liver tissue were detected by qRTPCR;protein expressions of p-AMPK, SREBP-1c, ACC and CPT-1 in liver tissue were detected by immunofluorescence;Western Blot was used to detect the protein expressions of AMPK,p-AMPK,SREBP-1c,ACC,p-ACC,CPT-1 in liver tissue.Results Compared with the normal group,serum TG,TC,FFA,LDL-C,AST and ALT levels were significantly increased(P<0.01)and HDL-C level was significantly decreased(P<0.01)in the model group;there was a large amount of lipid droplet aggregation,cytoplasmic laxity,a large number of lipid droplet vacuoles and ballooning degeneration, and nuclei shift in rat liver cells;the mRNA expressions of AMPK and CPT-1 in rat liver tissue were significantly down-regulated(P<0.01),mRNA expressions of ACC and SREBP-1c were significantly up-regulated(P<0.01);protein positive expressions of p-AMPK and CPT-1 in rat liver were significantly reduced(P<0.05, P<0.01), positive expressions of protein SREBP-1c and ACC were significantly increased(P<0.01);protein expression of AMPK in rat liver tissue did not show significant changes(P>0.05),protein expression levels of p-AMPK,p-ACC and CPT-1 were significantly decreased(P<0.01),protein expression levels of SREBP-1c and ACC were significantly increased(P<0.01). Compared with the model group,levels of serum TG,TC,FFA,LDL-C,AST and ALT were significantly decreased(P<0.05,P<0.01)and HDL-C level was significantly increased(P<0.01)in each administration group;fat deposition in rat hepatocytes was significantly reduced, and only a small amount of lipid droplet vacuoles were seen;the mRNA expressions of AMPK and CPT-1 in rat liver tissue were significantly up-regulated(P<0.05,P<0.01)and mRNA expressions of SREBP-1c and ACC were significantly down-regulated(P<0.05,P<0.01);the positive expressions of protein p-AMPK and CPT-1 in rat liver were significantly increased(P<0.05, P<0.01)and the positive expressions of protein SREBP-1c and ACC were significantly decreased(P<0.01);there were no significant changes were seen in protein expression of AMPK in rat liver tissue(P>0.05), protein expression levels of pAMPK, p-ACC and CPT-1 were significantly increased(P<0.05, P<0.01), and protein expression levels of SREBP-1c and ACC were significantly decreased(P<0.01).Conclusion The combination of total saponins of Astragalus Radix and total alkaloids of Nelumbinis Folium may prevent hyperlipidemia by regulating the AMPK/SREBP-1c/ACC signaling pathway,inhibiting lipid synthesis and promoting the oxidative breakdown of fatty acids.