和血柔肝方对肝纤维化大鼠基质金属蛋白酶及其抑制物表达影响的实验研究

The effects of Hexue Rougan formula on the expression of matrix metalloproteinases 2/13 and their inhibitors in rats with liver fibrosis

目的:观察和血柔肝方(HXRGF)对肝纤维化(LF)大鼠基质金属蛋白酶(MMPs)及其特异性抑制物(TIMPs)的影响,评估HXRGF对LF的疗效。方法:将80只SPF级雄性SD大鼠随机分为正常对照组(CL,n=10)和肝纤维化模型组(FL,n=70),在大鼠颈背部皮下以60%CCl 4溶液(CCl 4∶Olive oil=3∶2,3 ml/kg体重,每周2次)连续注射12周进行造模。造模成功后,原FL组大鼠被随机分为模型组、秋水仙碱组、HXRGF低剂量组、HXRGF中剂量组和HXRGF高剂量组,每组10只,分别给予相应的受试药液灌胃4周(1次/d)。比较各组大鼠的体质量、肝功能、病理Metavir评分和肝组织中MMP2、MMP13、TIMP1、TIMP2、TGFβ及Lect2的mRNA和蛋白表达量的差异。结果:①与对照组相比,模型组大鼠的肝脏形态、HE染色、免疫组化和羟脯氨酸测定结果提示肝纤维化模型复制成功,其体质量下降、肝功能恶化、病理Metavir评分升高,MMP2、MMP13、TIMP1、TIMP2、TGFβ和Lect2 mRNA水平和蛋白表达量升高(P<0.05)。②与模型组比较,HXRGF高剂量组大鼠ALT、AST水平降低(P<0.05);HXRGF高剂量组、中剂量组病理Metavir评分降低(P<0.01);HXRGF各剂量组MMP-2、TIMP1/2、TGFβ和Lect2 mRNA水平和蛋白表达量降低(P<0.05),HXRGF各剂量组MMP-13 mRNA水平和蛋白表达量升高(P<0.05)。结论:HXRGF可以显著改善CCl 4诱导的大鼠肝脏的炎症及纤维化程度,其机制可能是通过调控MMPs及其抑制物TIMPs的表达,降低Lect2调节血管生成,减少TGFβ的表达,从而减轻肝纤维化。

Objective:To observe evaluate the effects of Hexue Rougan formula on indexes such as matrix metalloproteinases(MMPs)and their specific inhibitors(TIMPs)in rats with liver fibrosis(LF),and to evaluate the therapeutic effects and the potential mechanism of Hexue Rougan formula on LF.Methods:SPF grade male SD rats were used for modeling experiments,80 rats were randomly divided into a normal control group(CL,n=10)and a liver fibrosis model group(FL,n=70).Rats in the FL group received subcutaneous injections of 60%CCl 4 solution(CCl 4∶olive oil=3∶2,3 ml/kg body weight,twice a week)into the dorsum of the neck for 12 weeks for modeling.After successful modeling,the rats in the original FL group were randomly divided into model group,colchicine group,HXRGF low-dose group,HXRGF medium dose group and HXRGF high-dose group,with 10 rats in each group.They were given the corresponding test solution by gavage for 4 weeks(once a day).The body weight,liver function,pathological METAVIR score and the contents of MMP2,MMP13,TIMP1,TIMP2 and TGF in liver tissue were comparedβAnd the mRNA expression and protein content of LECT2.Results:①The liver morphology,HE staining,immunohistochemistry and hydroxyproline assay results of the model rats compared with the control rats suggested that the liver fibrosis model replicated successfully,as evidenced by decreased body mass,liver function deterioration,higher pathological METAVIR scores,and higher mRNA levels and protein expression of MMP2,MMP13,TIMP1,TIMP2,TGFβand LECT2(P<0.05).②Compared with the model group,the levels of ALT and AST in the liver function of rats in the HXRGF high dose group were decreased(P<0.05),the pathological METAVIR scores were decreased(P<0.01)in the HXRGF high dose group and the mRNA levels and protein expression of MMP-2,TIMP1/2,TGFβand LECT2 were decreased(P<0.05)in each dose group of HXRGF,and the mRNA levels and protein expression of MMP-13 were increased(P<0.05)in each dose group of HXRGF.Conclusion:HXRGF can significantly improve the degree of inflammation and fibrosis in CCl 4 induced rat liver,and the mechanism may be through regulating the expression of MMPs and their inhibitor TIMPs,decreasing LECT2 to regulate angiogenesis and reducing the expression of TGFβ,thereby alleviating liver fibrosis.