螺内酯体内外抗大鼠肝纤维化的作用

In vivo and in vitro effect of spironolactone on hepatic fibrosis of rat

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.

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 IKBα 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 IKBα protein expression （P 〈 0.05 ）. On the contrary, spironolactone significantly reduced NF-κB DNA binding activity and increased IKBα 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.