下瘀血汤及其组分对蛋氨酸-胆碱缺乏饮食诱导的小鼠非酒精性脂肪性肝炎的干预作用

Effects of Xiayuxue Decoction and its components on nonalcoholic steatohepatitis induced by methionine-choline deficient diet in mice

目的:探讨下瘀血汤及其组分对蛋氨酸-胆碱缺乏(MCD)饮食诱导的小鼠非酒精性脂肪性肝炎(NASH)的治疗作用及潜在机制。方法:40只C57/BL6J雄性小鼠随机分为对照组、模型组、下瘀血汤全方组(3.03 g/kg)、下瘀血汤组分组(乙酸乙酯部位萃取液,0.65 g/kg)及吡格列酮组(10 mg/kg),每组8只。对照组小鼠给予蛋氨酸-胆碱充分(MCS)饲料,其他组小鼠给予MCD饲料诱导NASH模型,连续6周。造模第4周起,各药物组灌胃给予相应药液,连续3周。末次给药后,收集肝组织,分离血清。试剂盒检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平以及肝组织三酰甘油(TG)含量。HE染色和油红O染色分别观察肝组织形态学变化及脂滴沉积。PCR检测肝组织脂质代谢[二酰甘油酰基转移酶(Dgat)1、Dgat2、脂滴包被蛋白2(PLIN2)、诱导细胞凋亡的DFF45样效应因子C(CIDEC)、白细胞分化抗原簇36(CD36)、脂肪酸转运蛋白3(FATP3)、脂酰辅酶A氧化酶(ACO)、长链脂酰辅酶A脱氢酶(LCAD)]、炎症[F4/80、CD11b、CD11c、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)]、血管新生[血管内皮生长因子(VEGF)、血管内皮生长因子受体2(VEGFR2)、血管性血友病因子(vWF)、血小板/内皮细胞黏附分子(PECAM-1/CD31)、CD34、内皮糖蛋白(ENG/CD105)、E-选择素(E-sele)、血管细胞黏附分子(VCAM)]相关因子的mRNA表达水平。结果:(1)与对照组相比,模型组小鼠血清ALT、AST水平及肝脏TG含量显著增加(P<0.05,P<0.01);HE染色发现,模型组小鼠肝组织可见肝细胞脂肪变,大量脂肪空泡,肝细胞气球样变以及炎症细胞浸润,且NAS积分>4分;油红O染色发现,模型组小鼠肝细胞内有大量红色脂滴沉积。与模型组相比,下瘀血汤组分组小鼠血清ALT水平显著下降(P<0.05),下瘀血汤全方组及组分组小鼠血清AST水平及肝组织TG含量均显著降低(P<0.05,P<0.01),两组肝组织脂肪变、炎症浸润以及脂滴沉积明显改善,且NAS积分和油红O染色阳性表达面积占比均显著降低(P<0.01)。组分组ALT和AST水平、全方组AST水平较吡格列酮组明显降低(P<0.05,P<0.01)。(2)与对照组相比,模型组小鼠肝组织脂质代谢相关基因Dgat1、PLIN2、CIDEC、CD36、FATP3的mRNA表达水平明显升高(P<0.01),而ACO与LCAD的mRNA表达水平显著降低(P<0.05,P<0.01);炎症相关基因F4/80、CD11b、CD11c、TNF-α及TGF-β的mRNA表达,以及血管新生相关基因VEGF、VEGFR2、vWF、CD31、CD34、E-sele、VCAM的mRNA表达均显著上调(P<0.05,P<0.01)。与模型组相比,脂质代谢方面,下瘀血汤全方组与组分组Dgat1、Dgat2、PLIN2、CIDEC、CD36及FATP3的mRNA表达显著降低(P<0.05,P<0.01),LCAD的mRNA表达显著上调(P<0.01);下瘀血汤全方组ACO mRNA表达亦明显升高(P<0.01);炎症方面,下瘀血汤全方组及组分组F4/80、CD11b、CD11c及TNF-α的mRNA表达显著降低(P<0.05,P<0.01),组分组TGF-β mRNA表达亦明显下调(P<0.01);血管新生方面,下瘀血汤全方组及组分组VEGF、VEGFR2、vWF、CD31、CD34、E-sele、VCAM、CD105的mRNA表达均显著降低(P<0.05,P<0.01)。结论:下瘀血汤及其乙酸乙酯部位组分可有效治疗MCD诱导的小鼠NASH,其作用机制可能与改善肝脏脂质代谢、炎症反应及血管新生有关。

Objective: To investigate the therapeutic effects and potential mechanisms of Xiayuxue Decoction and its components on methionine-choline deficient(MCD) diet-induced nonalcoholic steatohepatitis(NASH) in mice. Methods: Forty male C57/BL6 J mice were randomly divided into the control group, model group, Xiayuxue Decoction group(3.03 g/kg), Xiayuxue Decoction fraction group(ethyl acetate extract, 0.65 g/kg) and pioglitazone group(10 mg/kg), 8 mice in each group. The mice in the control group were fed with methionine-choline sufficient(MCS) diet, and the mice in the other groups were fed with MCD diet for 6 weeks to induce NASH model. From the fourth week of modeling, the drug-treatment groups were treated with the corresponding drugs by intragastric administration for 3 weeks. After last administration, the liver tissue was collected and the serum was separated. The serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) and the content of triglyceride(TG) in liver tissue were detected by the kits. The morphological changes and lipid deposition in liver tissues were observed by HE and oil red O staining, respectively. The mRNA expression levels of factors associated with lipid metabolism, inflammation and angiogenesis were detected by PCR. The lipid metabolism-related genes included diacylglycerol acyltransferase(Dgat) 1, Dgat2, perilipin 2(PLIN2), cell death-inducing DFFA-like effector C(CIDEC), cluster of differentiation 36(CD36), fatty acid transporter protein 3(FATP3), acyl-CoA oxidase(ACO) and long-chain acyl-CoA dehydrogenase(LCAD). The inflammation-related genes included F4/80, CD11 b, CD11 c, tumor necrosis factor-α(TNF-α) and transforming growth factor-β(TGF-β). The angiogenesis-related genes included vascular endothelial growth factor(VEGF), vascular endothelial growth factor receptor 2(VEGFR2), von Willebrand factor(vWF), platelet/endothelial cell adhesion molecule(PECAM-1/CD31), CD34, endothelial glycoprotein(ENG/CD105), E-selectin(E-sele) and vascular cell adhesion molecule(VCAM). Results:(1)Compared with the control group, the serum levels of ALT and AST and the hepatic TG content in the model group were significantly increased(P<0.05, P<0.01). HE staining showed that hepatic steatosis, lots of fat vacuoles, hepatocyte ballooning and inflammatory cell infiltration were found in the model group, and the NAS score was more than 4. Oil red O staining showed that a large number of red-stained lipid droplets were deposited in the liver of the model mice. Compared with the model group, the serum level of ALT was significantly decreased in the Xiayuxue Decoction fraction group(P<0.05), the serum level of AST and the hepatic TG content were significantly decreased in the Xiayuxue Decoction group and Xiayuxue Decoction fraction group(P<0.05, P<0.01), the hepatic steatosis, inflammatory infiltration and lipid deposition were significantly improved in these two groups, and the NAS score and positive expression area proportion in oil red O staining were decreased significantly(P<0.01). The ALT and AST levels in the Xiayuxue Decoction fraction group and the AST level in the Xiayuxue Decoction group were lower than those in the pioglitazone group(P<0.05, P<0.01).(2)Compared with the control group, the mRNA expression levels of lipid metabolism-related genes, including Dgat1, PLIN2, CIDEC, CD36 and FATP3, were significantly increased in the model group(P<0.01), while the mRNA expression levels of ACO and LCAD were significantly decreased(P<0.05, P<0.01);the mRNA expression levels of inflammation-related genes F4/80, CD11 b, CD11 c, TNF-α and TGF-β as well as angiogenesis-related genes VEGF, VEGFR2, vWF, CD31, CD34, E-sele and VCAM were dramatically up-regulated(P<0.05, P<0.01). Compared with the model group, for the lipid metabolism-related genes, the mRNA expressions of Dgat1, Dgat2, PLIN2, CIDEC, CD36 and FATP3 were significantly decreased in the Xiayuxue Decoction group and its fraction group(P<0.05, P<0.01), while the mRNA expression of LCAD was significantly up-regulated(P<0.01);the expression of ACO mRNA was also obviously increased in the Xiayuxue Decoction group(P<0.01). Compared with the model group, for the inflammation-related genes, the mRNA expressions of F4/80, CD11 b, CD11 c and TNF-α were significantly decreased in the Xiayuxue Decoction group and its fraction group(P<0.05, P<0.01), the expression of TGF-β mRNA was also obviously decreased in the Xiayuxue Decoction fraction group( P<0.01). Compared with the model group,for the angiogenesis-related genes,the mRNA expressions of VEGF,VEGFR2,v WF,CD31,CD34,E-sele,VCAM and CD105 were significantly decreased in the Xiayuxue Decoction group and its fraction group( P<0.05,P<0.01). Conclusion: Xiayuxue Decoction and its ethyl acetate fraction components can effectively treat MCDinduced NASH in mice,and its mechanism may be related to the improvement of liver lipid metabolism,inflammation and angiogenesis.