白花蛇舌草醇提物对矽肺大鼠肺组织TGF-β1、Smad、MMP-2表达的影响

Effects of alcohol extract from Oldenlandia diffusa on the expression of TGF-β1,Smad and MMP-2 in lung tissue of silicosis rats

目的探究白花蛇舌草醇提物对矽肺大鼠肺组织转化生长因子-β1(TGF-β1)、母亲DPP同源物(Smad)、基质金属蛋白酶2(MMP-2)表达的影响。方法以气管内滴注二氧化硅混悬液的方法构建矽肺大鼠模型60只,随机分为模型组、白花蛇舌草醇提物低(1.5 g/kg)、中(3 g/kg)、高(6 g/kg)剂量组、吡非尼酮(100 mg/kg)组,每组12只,另选取12只大鼠气管内滴注等剂量生理盐水作为对照组。采用药物干预后,检测大鼠肺功能,比较其指标:每分通气量(MV)、呼气峰流值(PEF)、吸气阻力(Ri);检测各组大鼠双肺质量及其脏器系数;免疫组织化学染色检测各组大鼠肺组织淋巴细胞浸润情况,比较各组淋巴细胞功能相关抗原-1(LFA-1)阳性细胞比例;天狼星红染色检测各组大鼠肺组织胶原沉积及纤维化变性情况,比较胶原容积分数(CVF);采用试剂盒检测大鼠血清白细胞介素-18(IL-18)、环氧化酶-2(COX-2)、TGF-β1水平;蛋白免疫印迹法检测大鼠肺组织TGF-β1、Smad、MMP-2蛋白表达情况。结果与对照组相比,模型组大鼠MV、PEF显著降低,Ri、双肺质量及其脏器系数、肺组织LFA-1阳性细胞比例、CVF、血清IL-18、COX-2及TGF-β1水平、肺组织TGF-β1、Smad及MMP-2蛋白表达水平显著升高,差异均有统计学意义(P<0.05)。与模型组相比,白花蛇舌草醇提物低、中、高剂量组和吡非尼酮组大鼠MV、PEF均升高,Ri、双肺质量及其脏器系数、肺组织LFA-1阳性细胞比例、CVF、血清IL-18、COX-2及TGF-β1水平、肺组织TGF-β1、Smad及MMP-2蛋白表达水平均降低,差异均有统计学意义(P<0.05),且白花蛇舌草醇提物随剂量升高而作用增强并呈剂量依赖性(P<0.05)。白花蛇舌草醇提物高剂量组与吡非尼酮组相比,大鼠各指标水平无明显变化,差异无统计学意义(P>0.05)。结论白花蛇舌草醇提物可抑制肺组织TGF-β1、Smad、MMP-2表达,降低矽肺大鼠炎性因子水平,减轻肺组织炎症反应损伤和纤维化变性,修复肺功能。

Objective To explore the effects of alcohol extract from Oldenlandia diffusa on the expression of transforming growth factor-β1(TGF-β1),mothers against decapentaplegic homolog(Smad)and matrix metalloproteinase-2(MMP-2)in lung tissue of silicosis rats.Methods Sixty silicosis rat models were constructed by instilling silica suspension into the trachea,and they were randomly divided into model group,alcohol extract from Oldenlandia diffusa low(1.5 g/kg),medium(3 g/kg),high(6 g/kg)dose groups and pirfenidone(100 mg/kg)group,with 12 rats in each group,in addition,12 rats were selected to instill the same dose of normal saline into the trachea in control group.After drug intervention,the lung function of rats was tested,including minute ventilation(MV),peak expiratory flow(PEF),and resistance inspiratory(Ri).The double lung weights and their organ coefficients of rats in each group were detected.Immunohistochemical staining was used to observe the lymphocyte infiltration in the lung tissues of rats and the proportion of lymphocyte function-related antigen-1(LFA-1)positive cells in each group.Sirius red staining was used to observe the collagen deposition and the fibrosis degeneration in lung tissues of rats in each group,and detect the collagen volume fraction(CVF).Kits were used to detect the serum levels of interleukin-18(IL-18),cyclooxygenase-2(COX-2)and TGF-β1.Western blotting was used to detect the expression of TGF-β1,Smad and MMP-2 proteins in rat lung tissue.Results Compared with control group,MV and PEF were significantly reduced in model group(P<0.05),while Ri,double lung weights and their organ coefficients,the proportion of LFA-1 positive cells in lung tissue,CVF,serum IL-18,COX-2 and TGF-β1 levels,lung tissue TGF-β1,Smad and MMP-2 protein expression levels were significantly increased(P<0.05).Compared with model group,MV and PEF of rats were increased in alcohol extract from Oldenlandia diffusa low,medium,high dose groups and pirfenidone group(P<0.05),while Ri,the double lung weights and their organ coefficients,the proportion of LFA-1 positive cells in lung tissue,CVF,serum IL-18,COX-2 and TGF-β1 levels,lung tissue TGF-β1,Smad and MMP-2 protein expression levels were reduced(P<0.05),and the effect of alcohol extract from Oldenlandia diffusa increased in a dose-dependent manner(P<0.05).The levels of all indicators in rats showed no significant difference between pirfenidone group and high dose group(P>0.05).Conclusion Alcohol extract from Oldenlandia diffusa can inhibit the expression of TGF-β1,Smad and MMP-2 in lung tissue,reduce the levels of inflammatory factors in silicosis rats,reduce the inflammatory reaction damage and fibrosis degeneration of lung tissue,and repair the lung function.