SB431542抑制TGF-β/Smad3信号通路在大鼠矽肺纤维化中的干预作用

Inhibition of the TGF-β/Smad3 signaling pathway by SB431542:A study of the intervention effect of SB431542 on silicotic fibrosis in rats

目的探索SB431542抑制转化生长因子β(TGF-β)/Smad3信号通路在大鼠矽肺纤维化中的干预作用。方法将40只SPF级SD大鼠按随机数字表法分为4组:生理盐水对照组、模型组、SB431542抑制剂组和SB431542抑制剂对照组,每组10只。除生理盐水对照组外,其他3组采用非暴露气管注入法一次性气管内注入游离二氧化硅(SiO2)粉尘悬浊液1 ml(50 mg/ml);SB431542抑制剂组于染尘后第7、30天腹腔注射(5 mg/kg)SB431542;SB431542抑制剂对照组同样于染尘后第7、30天腹腔注射(5 mg/kg)SB431542助溶剂;生理盐水对照组气管内注入等量生理盐水(5 mg/kg)。在染尘后第60天,取大鼠右上叶肺组织石蜡包埋切片行苏木素-伊红(HE)染色;取左上叶肺组织用于实时荧光定量聚合酶链反应(qPCR)法检测纤维黏连蛋白(FN)和胶原蛋白(COL)Ⅰ、COLⅢmRNA水平;取右肺下叶经蛋白免疫印记(Western blot)法检测肺组织中FN、COLⅠ、COLⅢ、磷酸化Smad3(p-Smad3)和Smad3蛋白水平。结果与生理盐水对照组比较,模型组大鼠肺组织有大小不等的结节状结构,部分肺泡间隔断裂,呈肺气肿改变,肺间质纤维化,肺组织FN、COLⅠ、COLⅢmRNA表达水平均升高,FN、COLⅠ、COLⅢ、p-Smad3和Smad3蛋白表达水平均升高,差异均有统计学意义(P<0.05);与SB431542抑制剂对照组比较,SB431542抑制剂组肺组织基本结构完整,未见明显结节,肺泡腔及细支气管腔内可见少量渗出物,肺组织FN、COLⅠ、COLⅢmRNA表达水平均降低,FN、COLⅠ、COLⅢ、p-Smad3和Smad3蛋白表达水平均降低,差异均有统计学意义(P<0.05);模型组和SB431542抑制剂对照组各因子mRNA和蛋白水平差异均无统计学意义(P>0.05)。结论SB431542可能通过阻断TGF-β/Smad3信号通路降低下游纤维化因子FN、COLⅠ、COLⅢ的表达,进而干预大鼠矽肺纤维化过程。

ObjectiveTo investigate the intervention effect of SB431542,which inhibits the TGF-β/Smad3 signaling pathway,on silicotic fibrosis in rats.MethodsA total of 40 specific pathogen-free Sprague-Dawley rats were divided into normal saline control group,model group,SB431542 inhibitor group,and SB431542 inhibitor control group using a random number table,with 10 rats in each group.All rats except those in the normal saline control group were given non-exposed single intratracheal instillation of free silicon dioxide dust suspension 1 mL(50 mg/mL);the rats in the SB431542 inhibitor group were given intraperitoneal injection of SB431542(5 mg/kg)on days 7 and 30 after dust exposure,those in the SB431542 inhibitor control group were given intraperitoneal injection of SB431542 cosolvent(5 mg/kg)on days 7 and 30 after dust exposure,and those in the normal saline control group were given intratracheal instillation of an equal volume of normal saline(5 mg/kg).On day 60 after dust exposure,the paraffin-embedded section of the right upper lobe of lung was collected for HE staining;the left upper lobe of lung was collected to measure the mRNA levels of fibronectin(FN),collagen type I(COL-Ⅰ),and collagen type Ⅲ(COL-Ⅲ)by quantitative real-time PCR;the right inferior lobe of lung was collected to measure the protein levels of FN,COL-I,COL-Ⅲ,phosphorylated Smad3(p-Smad3),and Smad3.ResultsCompared with the normal saline control group,the model group had nodules with various sizes in lung tissue,with rupture of some alveolar septa,emphysema changes,and pulmonary interstitial fibrosis,as well as significant increases in the mRNA expression of FN,COL-Ⅰ,and COL-Ⅲ and the protein expression of FN,COL-I,COL-Ⅲ,p-Smad3,and Smad3 in lung tissue(P<0.05).Compared with the SB431542 inhibitor control group,the SB431542 inhibitor group had a relatively complete structure of lung tissue without marked nodules and with a small amount of exudate in alveolar space and the lumen of bronchioles,as well as significant reductions in the mRNA expression of FN,COL-Ⅰ,and COL-Ⅲ and the protein expression of FN,COL-Ⅰ,COL-Ⅲ,p-Smad3,and Smad3 in lung tissue(P<0.05).There were no significant differences in the mRNA expression of FN,COL-Ⅰ,and COL-Ⅲ and the protein expression of FN,COL-Ⅰ,COL-Ⅲ,p-Smad3,and Smad3 between the model group and the SB431542 inhibitor control group(P>0.05).ConclusionSB431542 exerts an intervention effect on silicotic fibrosis by blocking the TGF-β/Smad3 signaling pathway and reducing the expression of the downstream fibrosis factors FN,COL-Ⅰ,and COL-Ⅲ.