大黄[庶虫]虫丸经p38 MAPK/NF-κB/TGF-β1通路抑制小鼠硅肺纤维化的机制探讨

Mechanism of Dahuang Zhechongwan in Inhibiting Silicosis in Mice via p38 MAPK/NF-κB/TGF-β Pathway

目的:运用中医经方大黄[庶虫]虫丸干预小鼠硅肺纤维化模型,观察其对小鼠硅肺纤维化的影响并探讨其作用机制。方法:选用SPF级雄性昆明种小鼠36只,分为正常组,模型组,大黄?虫丸高、中、低剂量(1.560,0.780,0.390 g·kg^(-1))组,汉防己甲素组(0.039 mg·kg^(-1)),每组6只。除正常组外,其余组均用静式染毒法连续40 d吸入二氧化硅(SiO2)粉尘复制小鼠硅肺纤维化模型,并用相应药物进行干预28 d后处死小鼠,获得血清和肺组织样品,运用酶联免疫吸附测定(ELISA)方法检测血清中肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL^(-1)β),IL-6和羟脯氨酸(HYP)的含量,运用肺组织样本进行病理切片观察,并采用蛋白免疫印迹法(Western blot),实时荧光定量聚合酶链反应(Real-time PCR)方法检测肺组织中p38丝裂原活化蛋白激酶(MAPK),核转录因子-κB(NF-κB),转化生长因子-β1(TGF-β1),α-平滑肌肌动蛋白(α-SMA),Smad2,Smad3,Smad7的蛋白和mRNA的表达水平。结果:与正常组比较,模型组中的TNF-α,IL^(-1)β,IL-6和HYP的含量均明显升高,差异具有统计学意义(P<0.05,P<0.01);与模型组比较,大黄[庶虫]虫丸高剂量组能够明显降低小鼠血清中TNF-α,IL-6和HYP的含量(P<0.05,P<0.01),可见大黄[庶虫]虫丸能够减轻硅肺小鼠肺部炎症。同时,与正常组比较,模型组中的p38 MAPK,NF-κB p65,TGF-β1,α-SMA,Smad2和Smad3蛋白和mRNA表达水平均明显升高(P<0.05,P<0.01),Smad7蛋白和mRNA表达水平均显著降低(P<0.01);与模型组比较,大黄?虫丸高剂量组中的p38 MAPK,NF-κB p65,TGF-β1,α-SMA,Smad2和Smad3蛋白和mRNA表达水平均明显降低(P<0.05,P<0.01),Smad7蛋白和mRNA表达水平均明显升高(P<0.05,P<0.01)。结论:大黄[庶虫]虫丸能改善硅肺小鼠肺泡炎症、细胞外基质沉积的情况,因而起到减轻纤维化的作用,这可能是通过调节p38 MAPK/NF-κB/TGF-β1通路来实现的。

Objective:To explore the effects and mechanism of Chinese classical prescription Dahuang Zhechongwan on silicosis in mice. Method: Thirty-six male Kunming mice of SPF grade were randomized into the normal control group,model control group,tetrandrine(Tet,0.039 mg·kg^(-1))group,as well as high-(1.560 g·kg^(-1)),medium-(0.780 g·kg^(-1)),and low-dose(0.390 g·kg^(-1))Dahuang Zhechongwan groups,with six mice in each group. Mice in all groups except for the normal control group underwent static inhalation of silica(SiO2)dust for 40 consecutive days to induce fibrosis. After 28 days of intervention with corresponding drugs,the mice were sacrificed to collect the serum and lung tissues,with the former used for detecting tumor necrosis factor-α(TNF-α),interleukin-1β(IL^(-1)β),IL-6,and hydroxyproline(HYP)levels by enzyme-linked immunosorbent assay(ELISA)and the latter for observing the pathological changes. Meanwhile,the protein and mRNA expression levels of p38 mitogen-activated protein kinase(p38 MAPK),nuclear transcription factor-κB(NF-κB),transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),Smad2,Smad3,and Smad7 in the lung tissues were determined by Western blot and real-time polymerase chain reaction(Real-time PCR). Result: Compared with the normal group,the contents of TNF-α,IL^(-1)β,IL-6 and HYP in the model group were significantly increased,the difference was statistically significant(P<0.05,P<0.01);compared with the model group,the high-dose group of Dahuang Zhechongwan could significantly reduce the contents of TNF-α,IL-6 and HYP in the serum of mice(P<0.05,P<0.01),indicating that Dahuang Zhechongwan could reduce the lung inflammation of silicosis mice. At the same time,compared with the normal group,the protein and mRNA expression levels of p38 MAPK,NF-κB p65,TGF-β1,α-SMA,Smad2 and Smad3 in the model group were significantly increased(P<0.05,P<0.01),while the protein and mRNA expression levels of Smad7 were significantly decreased(P<0.01);compared with the model group,the protein and mRNA expression levels of p38 MAPK,NF-κB p65,TGF-β1,α-SMA,Smad2 and Smad3 in the high-dose Dahuang Zhechongwan group were significantly increased the protein and mRNA expression levels were significantly decreased(P<0.05,P<0.01),while Smad7 protein and mRNA expression levels were significantly increased(P<0.05,P<0.01). Conclusion:Dahuang Zhechongwan ameliorates the alveolar inflammation,extracellular matrix(ECM)deposition,and fibrosis in mice with silicosis possibly by regulating the p38 MAPK/NF-κB/TGF-β1 pathway.