调肝祛脂方通过miR-34a/SIRT1/PPARα通路对非酒精性脂肪肝大鼠的保护作用研究

Study on mechanism of protective effect in nonalcoholic fatty liver rats by TiaoganQuzhi Formula based on miR-34a/SIRT1/PPARαsignaling pathway

目的观察调肝祛脂方通过微小RNA-34a(miR-34a)/沉默调节蛋白1(SIRT1)/过氧化物酶体增殖物激活受体α(PPARα)通路对非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)大鼠的保护作用。方法选择SD大鼠40只,按随机数字表法分为对照组、模型组、调肝祛脂方低剂量组、调肝祛脂方高剂量组,每组10只。对照组给予正常饲料喂养,其余各组以高脂饲料建立NAFLD模型。各组成模后,调肝祛脂方低、高剂量组分别按9.045、36.18 g/kg给药,灌胃体积均为1 mL/100 g,对照组和模型组予等体积蒸馏水灌胃。各组均每日灌胃1次,连续给药8周。比较各组肝脏HE染色病理改变,血脂及肝功能指标,RT-PCR法检测肝组织miR-34a、SIRT1、PPARα基因表达。结果造模后对照组大鼠精神良好、反应灵敏、一般状况较好,其余各组出现精神萎靡、活动减少等表现;干预后调肝祛脂方低、高剂量组大鼠一般状态均有改善。对照组肉眼观察肝脏光泽红润、无明显充血水肿,HE显示肝细胞排列规律、肝索清晰;模型组肉眼可见肝组织颜色苍白、充血水肿,HE染色显示肝细胞排列紊乱、有脂肪空泡变性;治疗后调肝祛脂方低、高剂量组大鼠肝脏肉眼及HE观察均有好转。与对照组相比,模型组总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、谷丙转氨酶(ALT)、谷草转氨酶(AST)水平均升高(P<0.01),高密度脂蛋白胆固醇(HDL-C)水平降低(P<0.01);与模型组相比,调肝祛脂方低、高剂量组TC、TG、LDL-C、ALT、AST水平均降低,HDL-C水平高,其中调肝祛脂方高剂量组上述指标与模型组比较,差异有统计学意义(P<0.05)。与对照组相比,模型组miR-34a表达高(P<0.05),SIRT1、PPARα基因表达低(P<0.05);与模型组相比,调肝祛脂方低、高剂量组miR-34a表达低,SIRT1、PPARα基因表达高,且调肝祛脂方高剂量组上述指标与模型组比较差异有统计学意义(P<0.05)。结论调肝祛脂方可通过降低miR-34a表达,提高SIRT1/PPARα表达,调节血脂及肝功,改善NAFLD大鼠肝细胞变性。

Objective To explore the protective effect of TiaoganQuzhi Formula(regulatingliver andreducing fat)on nonalcoholic fatty liver(NAFLD)by microRNA-34a(miR-34a)/silent mating type information regulation 2 homolog-1(SIRT1)/peroxisome proliferator-activated receptor(PPARα)signaling pathway.Methods Forty SD rats were selected and randomly divided into a control group,a model group,a low-dose TiaoganQuzhi group,and a high-dose TiaoganQuzhi group,with ten rats in each group.The control group was fed with normal feed,while other groups were fed with high-fat feed to establish a nonalcoholic fatty liver model.The low and high dose TiaoganQuzhi groups were given 9.045 g/kg and 36.18 g/kg drugs respectively after modeling,with the volume of 1 mL/100 g,and the normal group and model group were given same volume of distilled water.Rats were gavaged once a day for 8 weeks.The pathological changes of liver hematoxylin-eosin(HE),blood lipids and liver function indicators in each group were compared,and miR-34a,SIRT1 and PPARαgene expression in liver tissueweredetected by reverse transcriptionpolymerase chain reaction(RT-PCR)method.Results After modeling,the control group showed good mental health,sensitive response and generally good condition.Other groups showed symptoms such as mental exhaustion and decreased activity.After intervention,the general state of treatment group rats were improved.Liver of the control group was shiny and ruddy,without obvious congestion or edema by visual observation.HE showed regular arrangement of liver cells,and liver cord was clear.The model group showed liver tissue pale with congestion and edema in visible,and liver HE showed disordered arrangement of liver cells with fatty vacuolar degeneration.The treatment group showed improvement in liver observation.Compared with the control group,the model group had higher levels of total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C)(P<0.01),while lower levels of high-density lipoprotein cholesterol(HDL-C)(P<0.01).Compared with the model group,the levels of TC,TG,LDL-C in each TiaoganQuzhi dose group of were low,and the levels of HDL-C were high.The above indicators in TiaoganQuzhi high-dose group of were statistically significant compared with the model group(P<0.05).Compared with the control group,the levels of alanine transaminase(ALT)and aspartate transaminase(AST)in model group were higher(P<0.01).Compared with the model group,the levels of ALT and AST in each TiaoganQuzhi dose group were lower,and the above indicators in the TiaoganQuzhi high-dose group had statistical significance compared with the model group(P<0.05).Compared with the control group,the model group showed higher expression of miR-34a(P<0.05)and lower expression of SIRT1 and PPARαgenes(P<0.05).Compared with the model group,the expression of miR-34a was lower in each TiaoganQuzhi dose group,while the expression of SIRT1 and PPARαgenes was higher.Moreover,there was a statistically significant difference in the above indicators between the TiaoganQuzhi high-dose group and the model group(P<0.05).Conclusion TiaoganQuzhi Formula can reduce miR-34a expression,increase SIRT1/PPARαexpression,regulate blood lipids and liver function,and improve liver cell degeneration in NAFLD rats.