【摘要】 目的:探讨螺内酯对实验性肝纤维化的影响及其作用机制.方法:雄性Wistar大鼠24只,随机分为3组,每组8只.模型组:CCl4油2.5mL/kgSC,3次/wk;螺内酯组:CCl4油注射的同时每日予以螺内酯20mg/kg灌胃,1次/d;对照组:橄榄油SC.于4wk取材,VG染色检测大鼠肝组织学改变.Western印迹法检测肝组织TGF-β1的表达.EMSA检测肝组织NF-κB的活性.酶图法测定MMP-2,9的活性.另培养大鼠HSC-T6,分别予醛固酮(Aldo)1μmol/L、螺内酯1μmol/L(预处理60min,再予Aldo刺激1h)和TNFα100μg/L处理1h,设立阴性对照组.EMSA检测NF-κB DNA结合活性;Western印迹检测胞质内IκBα的表达.结果:体内4wk,螺内酯组纤维化分级小于模型组(螺内酯组vs模型组,P<0.01);模型组TGF-β1的表达明显高于螺内酯治疗组(模型组vs螺内酯治疗组,P<0.05).模型组NF-κB结合活性显著增强(模型组vs对照组,P<0.01),螺内酯可抑制肝组织NF-κB结合活性(螺内酯组vs模型组,P<0.05).模型组MMP2,9活性显著增强(模型组vs对照组,P<0.01),螺内酯可抑制MMP2,9活性(螺内酯组vs模型组,P<0.05).体外Aldo和TNFα干预1h后,NF-κB DNA结合活性增强(Aldo处理组vs对照组,P<0.01),螺内酯可抑制NF-κB DNA结合活性(螺内酯+Al-do处理组vsAldo处理组,P<0.05).Aldo可减低胞浆内IκBα表达(Aldo处理组vs对照组,P<0.05).螺内酯则抑制胞质蛋白中IκBα表达的减低(螺内酯+Aldo处理组vs Aldo处理组,P<0.05).结论:螺内酯可抑制实验性肝纤维化形成,其抗肝纤维化作用与抑制肝组织TGF-β1的表达以及NF-κB和MMP2,9的活性有关.更多还原

【Abstract】 AIM:To determine the effect of spironolactone on the progression of rat hepatic fibrosis in vivo and in vitro and its mechanisms.METHODS:Twenty-four Wistar rats weighing about 250 g were divided into 3 groups,with 8 in each group.In model group,the rats received injection of 40% CCl4 2.5 mL/kg subcutaneously,three times a week.In spironolactone treatment group,the rats received injection of 40% CCl4 as well as ig administration of spironolactone 20 mg/(kg·d).In control group,the rats received injection of olive oil only.After 4 weeks,morphological examination was performed.The expression of TGF-β1 protein in liver tissue was examined by Western Blot.Electrophoretic gel mobility shift assay(EMSA)was utilized to detect NF-κB DNA binding activity in liver tissues.The activities of matrix metalloproteinase-2,9(MMP-2,9)were assessed by zymography.In vitro,HSC(hepatic stellate cell)-T6 cells were preincubated for 1 h with or without spironolactone 1 μmol/L(an inhibitor of aldosterone)prior to exposure to aldosterone(Aldo)1 μmol/L for 1 h.DNA binding activity of NF-κB was analyzed by EMSA.The expression of IκBα protein was examined by Western Blot.RESULTS:In vivo,spironolactone treatment significantly reduced mean fibrosis score and protein levels of TGF-β1 in the 4th week(P<0.01,P<0.05).Spironolactone reduced DNA binding activity of NF-κB and MMP-2,9 activities(P<0.01).In vitro,stimulation of HSC by Aldo increased NF-κB DNA binding activity(P<0.01)and decreased IκBα protein expression(P<0.05).On the contrary,spironolactone significantly reduced NF-κB DNA binding activity and increased IκBα protein expression(P<0.05).CONCLUSION:Spironolactone may partly exert inhibiting effects on CCl4-induced hepatic fibrogenesis by reducing TGF-β1 expression,inhibiting DNA binding activity of NF-κB and activities of MMP-2,9.Stimulation of HSC by Aldo promotes NF-κB binding activity which can be inhibited by spironolactone.更多还原