金丝桃苷对心力衰竭大鼠肝纤维化的影响及其分子机制

Effect and mechanism of hyperin on liver fibrosis in rats with heart failure

目的探讨金丝桃苷对心力衰竭(以下简称心衰)大鼠肝纤维化的影响,并从TGF-β1/Smad通路和TIMP/MMP轴的角度探讨其分子机制。方法取雄性Wistar大鼠24只,随机分为Sham组、心衰组、金丝桃苷100 mg/kg组和金丝桃苷200 mg/kg组各6只。心衰组和金丝桃苷组采用腹主动脉—腔静脉分流术制作心衰模型,Sham组行假手术充分暴露腹主动脉段后直接将腹腔内器官复位。造模完成后,100 mg/kg金丝桃苷组和200 mg/kg金丝桃苷组分别给予100、200 mg/kg金丝桃苷灌胃,Sham组和心衰组给予等体积溶剂灌胃,每天1次,连续4周。给药完成后,使用小动物超声系统测量大鼠心输出量(CO)、左心室末期舒张压(LVEDP)、左心室收缩末压(LVESP)、左心室内压最大上升速率(+dp/dtmax)和最大下降速率(-dp/dtmax)、左心室舒张末期容积(LVEDV)、左心室收缩末期容积(LVESV),计算左心室射血分数(LVEF);ELISA法检测血清肝功能指标[丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)];称取大鼠体质量(BW)、肝脏质量(LW),计算肝脏系数;HE染色观察肝脏病理形态学表现,Masson染色测算胶原形成面积;分别采用碱水解法、比色法、硫代巴比妥酸法、羟胺法检测肝组织氧化应激指标羟脯胺酸(Hyp)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、总超氧化物歧化酶(T-SOD);Real-time PCR法检测TGF-β1/Smad通路相关基因[转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)]和TIMP/MMP轴相关基因[金属蛋白酶组织抑制因子1(TIMP1)、基质金属蛋白酶1(MMP1)、基质金属蛋白酶2(MMP2)、Ⅲ型胶原(collagenⅢ)]mRNA表达;Western blotting法检测TGF-β1、Smad2/3、p-Smad2/3、CTGF及TIMP1、MMP1、MMP2、collagenⅢ蛋白表达。结果与Sham组相比,心衰组CO、LVEF、+dp/dtmax及-dp/dtmax降低,LVESP、LVEDP、LVESV升高(P均<0.01);与心衰组相比,金丝桃苷100 mg/kg组LVESP、LVEDP、LVESV降低(P均<0.01);与心衰组相比,金丝桃苷200 mg/kg组LVESP、LVEDP、LVESV降低(P均<0.01),CO、LVEF、+dp/dtmax及-dp/dtmax升高(P<0.05或<0.01)。与Sham组相比,心衰组血清ALT、AST水平升高(P均<0.01);与心衰组相比,100 mg/kg金丝桃苷组血清AST水平下降,200 mg/kg金丝桃苷组血清ALT、AST水平均下降(P<0.05或<0.01)。四组肝脏系数比较差异无统计学意义(P均>0.05)。与Sham组相比,心衰组肝组织弥漫性的炎症浸润及脂肪变性,肝细胞变大,形状不均,肝小叶结构消失,肝板破坏及中央静脉坍塌,胶原形成面积增加(P<0.05);与心衰组相比,金丝桃苷100、200 mg/kg组肝脏脂肪变性、炎细胞浸润、纤维化程度均有不同程度的减轻,胶原形成面积减少(P<0.05)。与Sham组相比,心衰组肝组织Hyp、MDA水平升高,GSH-Px、TSOD水平降低(P均<0.05);与心衰组相比,金丝桃苷100 mg/kg组肝组织Hyp、MDA水平降低,T-SOD水平升高,金丝桃苷200 mg/kg组Hyp、MDA水平降低,GSH-Px、T-SOD水平升高(P均<0.05)。与Sham组相比,心衰组肝组织TGF-β1、CTGF、TIMP1、MMP2、collagenⅢmRNA及蛋白水平升高,p-Smad2/3蛋白水平升高,MMP1 mRNA及蛋白水平下降(P均<0.05);与心衰组相比,金丝桃苷100 mg/kg组肝组织TGF-β1、CTGF、TIMP1、MMP2、collagenⅢmRNA及蛋白水平下降,p-Smad2/3蛋白水平下降,MMP1蛋白水平升高(P均<0.05);与心衰组相比,金丝桃苷200 mg/kg组肝组织TGF-β1、CTGF、TIMP1、MMP2、collagenⅢmRNA及蛋白水平下降,p-Smad2/3蛋白水平下降,MMP1 mRNA及蛋白水平升高(P均<0.05)。结论心衰可导致大鼠发生肝纤维化;金丝桃苷对肝纤维化具有抑制作用,其机制可能与调节TGF-β1/Smad通路的活化及TIMP/MMP轴的平衡有关。

Objective To investigate the effect of hyperin on liver fibrosis in rats with heart failure,and to explore its molecular mechanism from the perspective of TGF-β1/Smad pathway and TIMP/MMP axis.Methods Twenty-four male Wistar rats were randomly divided into the Sham group,heart failure group,100 mg/kg hyperin group,and 200 mg/kg hyperin group.Heart failure models were made by abdominal aorta-vena cava shunt in the heart failure group and hyperoside group,and abdominal organs were directly reset after Sham operation.Rats in the 100 mg/kg hyperin group and 200 mg/kg hyperin group were given 100 and 200 mg/kg hyperin,respectively.Rats in the Sham group and heart failure group were given the same volume of solvent,once a day,for 4 weeks.After administration,cardiac output(CO)and left ventricular ejection fraction(LVEF)were measured by small animal ultrasound system.Serum liver function indexes[alanine aminotransferase(ALT)and aspartate aminotransferase(AST)]were detected by ELISA.The body weight(BW)and liver weight(LW)of rats were weighed and the liver coefficient was calculated.Liver pathomorphology was observed by HE staining,and collagen formation area was measured by Masson staining.Oxidative stress indicators[hydroxyprolide(Hyp),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),and total superoxide dismutase(T-SOD)]of liver tissues were detected by alkaline hydrolysis,colorimetry,thiobarbituric acid and hydroxylamine methods,respectively.Transforming growth factor-β1(TGF-β1)/Smad pathway-related genes[transforming growth factorβ1(TGF-β1),and connective tissue growth factor(CTGF)mRNA]and TIMP/MMP axis-related genes[TIMP1,matrix metalloproteinase 1(MMP1),MMP2,and collagenⅢ]were detected by real-time PCR;the expression levels of TGF-β1,Smad2/3,p-Smad2/3,CTGF,TIMP1,MMP1,MMP2 and collagenⅢwere detected by Western blotting.Results Compared with the Sham group,CO,LVEF,+dp/dtmax and-dp/dtmax decreased,LVESP,LVEDP,and LVESV increased in the heart failure group(all P<0.01).Compared with the heart failure group,LVESP,LVEDP,and LVESV in the 100 mg/kg hyperin group decreased(all P<0.01).Compared with the heart failure group,LVESP,LVEDP,and LVESV in 200 mg/kg hyperin group decreased(all P<0.01),while CO,LVEF,+dp/dtmax,and-dp/dtmax increased(P<0.05 or P<0.01).Compared with the Sham group,the serum ALT and AST levels in the heart failure group were higher(all P<0.01).Compared with the heart failure group,the serum AST level decreased in the 100 mg/kg hyperin group,and the serum ALT and AST levels decreased in the 200 mg/kg hyperin group(P<0.05 or P<0.01).There was no significant difference in liver coefficient among the four groups(P>0.05).Compared with the Sham group,the liver tissue in heart failure group had diffuse inflammatory infiltration and steatosis,the liver cells became larger and more uneven in shape,the liver lobule structure disappeared,the liver plate was destroyed and the central vein collapsed,and the collagen formation area increased(all P<0.05).Compared with the heart failure group,hepatic steatosis,inflammatory cell infiltration and fibrosis in 100 mg/kg and 200 mg/kg hyperin groups were reduced in different degrees,and collagen formation area was reduced(all P<0.05).Compared with the Sham group,the levels of Hyp and MDA in the liver tissues of the heart failure group increased,while the levels of GSH-Px and T-SOD decreased(all P<0.05).Compared with the heart failure group,the levels of Hyp and MDA decreased and T-SOD increased in the 100 mg/kg hyperin group;the levels of Hyp and MDA decreased,and the levels of GSH-Px and T-SOD increased in the 200 mg/kg hyperin group(all P<0.05).Compared with the Sham group,the mRNA and protein levels of TGF-β1,CTGF,TIMP1,MMP2 and collagenⅢin the liver tissues of the heart failure group increased,while the protein level of p-Smad2/3 increased and MMP1 mRNA and protein decreased(all P<0.05).Compared with the heart failure group,the mRNA and protein levels of TGF-β1,CTGF,TIMP1,MMP2 and collagenⅢin the liver tissues of 100 mg/kg hyperin group decreased,while the protein level of p-Smad2/3 decreased and MMP1 protein increased(all P<0.05).Compared with the heart failure group,the mRNA and protein levels of TGF-β1,CTGF,TIMP1,MMP2,and collagenⅢdecreased,the protein level of p-Smad2/3 decreased and the mRNA and protein levels of MMP1 increased in the 200 mg/kg hyperin group(all P<0.05).Conclusion Heart failure can lead to liver fibrosis in rats;hyperin can inhibit liver fibrosis,and its mechanism may be related to regulating the activation of TGF-β1/Smad pathway and the balance of TIMP/MMP axis.