苓桂术甘汤对非酒精性脂肪肝相关肝细胞癌模型小鼠肝脂肪酸代谢重编程的影响

Effectof Linggui Zhugan Decoctionn(苓桂术甘汤)on Reprogramming of Liver Fatty Acid Metabolismin Mice Model of Hepatocellular Carcinoma Associated with Non-alcoholic Fatty Liver

目的从肝脂肪酸代谢重编程角度探讨苓桂术甘汤治疗非酒精性脂肪肝相关肝细胞癌(NAFLD-HCC)的可能作用机制。方法将60只C57BL/6小鼠随机分为正常组、脂肪肝组、脂肪肝相关肝癌组以及苓桂术甘汤低、中、高剂量组,每组10只。脂肪肝相关肝癌组及苓桂术甘汤低、中、高剂量组小鼠采用腹腔注射二乙基亚硝胺和高脂饲料喂养相结合方式建立NAFLD-HCC模型;脂肪肝组小鼠采用高脂饲料喂养建立非酒精性脂肪性肝病模型;正常组予普通饲料喂养。造模成功后,苓桂术甘汤低、中、高剂量组小鼠分别给予苓桂术甘汤4.875、9.750、19.500 g/(kg·d)灌胃,正常组、脂肪肝组、脂肪肝相关肝癌组小鼠每天给予10 ml/kg生理盐水灌胃,各组均连续灌胃30天。检测各组小鼠血脂含量[包括甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)]及肝脏游离脂肪酸(FFA)含量,HE及油红O染色观察小鼠肝脏组织病理学改变;检测各组小鼠血清甲胎蛋白(AFP)、癌胚抗原(CEA)含量及肝脏肿瘤增殖抗原Ki-67蛋白表达,肝脏脂蛋白脂肪酶(LPL)、白细胞分化抗原36(CD36)、肉毒碱棕榈酰基转移酶1α(CPT1α)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达,LPL、CD36、CPT1αmRNA表达。结果与正常组比较,脂肪肝相关肝癌组小鼠肝脏炎性细胞浸润,脂肪变性明显,部分细胞出现小细胞性增生的病理改变,血清TC、TG、LDL-C、AFP、CEA含量升高,HDL-C含量下降(P<0.05或P<0.01),肝脏Ki-67蛋白、Bcl-2蛋白表达均升高,LPL、CD36、CPT1αmRNA及蛋白表达升高(P<0.05或P<0.01);脂肪肝组仅血清TC、TG和LDL-C含量升高,病理显示肝脏组织仅有脂肪变性和炎细胞浸润,AFP、CEA含量及肝脏Ki-67蛋白表达差异无统计学意义(P>0.05)。与脂肪肝相关肝癌组比较,苓桂术甘汤高剂量组可显著改善肝脏病理形态,显著降低肝脏脂质沉积面积,降低血清TC、TG、LDL-C、AFP、CEA含量,升高HDL-C含量,降低肝脏Ki-67、Bcl-2蛋白表达和LPL、CD36、CPT1α蛋白及mRNA表达(P<0.05或P<0.01);苓桂术甘汤中、低剂量组上述各指标改善效果不及高剂量组。结论苓桂术甘汤可能通过影响LPL/CD36/CPT1信号轴调控脂肪酸代谢重编程,从而抑制NAFLD-HCC的发生发展。

Objective To observe the potential mechanism of Linggui Zhugan Decoction(苓桂术甘汤,LZD)in the treatment of nonalcoholic fatty liver disease-associated hepatocellular carcinoma(NAFLD-HCC)from fatty acid metabolism reprogramming.Methods Sixty C57BL/6 mice were randomly divided into normal group,NAFLD group,NAFLD-HCC group,low-,medium-and high-dose LZD groups,with 10 mice in each group.NAFLD-HCC mouse models were established by intraperitoneal injection of diethylnitrosamine(DEN)combined with a high-fat diet(HFD)in the NAFLD-HCC group,low-,medium-and high-dose LZD groups,while NAFLD models were made through HFD in the NAFLD group;the mice in the normal group were fed with normal diet.After the model was suc-cessfully established,low-,medium-and high-dose LZD groups were intragastrically administered with 4.875,9.750 and 19.500 g/(kg·d)of LZD solutions,respectively,while the other groups were intragastrically administered with 10ml/kg of normal saline,all for 30 days.The levels of blood lipid indicators including triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)as well as free fatty acid(FFA)content in liver tissue were measured;pathological changes in liver tissue were observed by hematoxylin and eosin(HE)and Oil Red O staining;serum alpha-fetoprotein(AFP)and carcinoembryonic antigen(CEA)levels were measured,and Ki-67 protein expression in liver was examined;liver lipoprotein lipase(LPL),leukocyte differentiation antigen 36(CD36),carnitine palmitoyltransferase lα(CPT1α),B lymphocytoma-2 gene(Bcl,),and Bcl-2 related X protein(Bax)protein expression as well as LPL,CD36 and CPT1αmRNA expression were detected.Results Compared to those in the normal group,the mice in the NAFLD-HCC group had liver inflammatory cell infiltration,obvious fatty degeneration,and small cell hyperplasia in certain cells;the contents of TC,TG,LDL-C,AFP and CEA significantly increased,and the content of HDL-C significantly decreased(P<O.05 or P<0.01);the expression of Ki-67 protein and Bcl,protein increased,and the expression of LPL,CD36 and CPT1αmRNA and protein increased as well(P<0.05 or P<0.01).In the NAFLD group,only serum TC,TG and LDL-C content significantly increased compared to those in the normal group,and only steatosis and inflammatory cell infiltration in the liver tissue were found through pathological observation;there was no significant difference in AFP and CEA content and liver Ki-67 protein expression between the two groups(P>0.05).Compared to those in the NAFLD-HCC group,the pathological morphology of the liver was significantly improved in the high-dose LZD group,and the area of lipid deposition was reduced;the serum TC,TG,LDL-C,AFP and CEA content decreased,while the HDL-C content increased;Ki-67 and Bcl,protein expression decreased,as well as LPL,CD36,CPTlαmRNA and protein expression(P<0.05 or P<0.01);and the above indexes in high-dose LZD group were superior to those in the lowand medium-dose LZD group.Conclusion LZD may inhibit the occurrence and development of NAFLD-HCC by regulating fatty acid metabolism reprogramming through LPL-CD36-CPT1 metabolic axis.