补肾益肺消癥方对肺纤维化上皮间质转化中瘦素相关因子的影响

Study on the Expression of Leptin-related Factors in Pulmonary Fibrosis Epithelial Mesenchymal Transformation Model and the Effect of Bushen Yifei Xiaozheng Formula on It

目的:明确肺纤维化上皮-间质转化(EMT)对瘦素相关因子的影响,对瘦素加速EMT的分子机制进行初探,并观察补肾益肺消癥方对EMT过程中瘦素相关因子的作用。方法:将A549细胞根据干预手段不同分为正常组、模型组[转化生长因子-β_(1)(TGF-β_(1))]、中药组(TGF-β_(1)+补肾益肺消癥方)、尼达尼布组(TGF-β_(1)+尼达尼布),免疫荧光观察α-平滑肌肌动蛋白(α-SMA)、E-钙黏附蛋白(E-cadherin)的表达,酶联免疫吸附试验(ELISA)检测细胞上清中的瘦素水平,Western Blotting检测瘦素受体(LEPR)、过氧化物酶体增殖物激活受体γ(PPAR-γ)的蛋白含量以及STAT3的活化程度。结果:与正常组比较,模型组细胞上清中瘦素水平升高(P<0.01),中药组、尼达尼布组出现不同程度的下降(P<0.001);与正常组比较,模型组LEPR蛋白表达升高(P<0.001),PPAR-γ表达下降(P<0.05),STAT3活化水平降低,中药组、尼达尼布组LEPR蛋白表达下调(P<0.001),PPAR-γ表达上调(P<0.05),中药组STAT3活化程度与模型组比较差异无统计学意义(P>0.05),尼达尼布组与模型组比较,STAT3活化水平升高(P<0.01)。结论:EMT可促进细胞内源性肺瘦素的分泌,增加细胞表面LEPR的表达,下调与瘦素有拮抗作用的PPAR-γ的含量,减少STAT3的活化,为瘦素加速EMT进程提供物质基础。而补肾益肺消癥方则可能通过降低内源性肺瘦素分泌,减少LEPR,提高PPAR-γ的水平逆转此过程。

Objective:To clarify the effect of epithelial mesenchymal transition(EMT)on leptin-related factors in pulmonary fibrosis,explore the molecular mechanism of leptin accelerating EMT,and observe the effects of Bushen Yifei Xiaozheng Formula on leptin-related factors in the process of EMT.Methods:A549 cells were divided into a control group,a model group(TGF-β_(1)),a Chinese medicinal group(TGF-β_(1)+Bushen Yifei Xiaozheng Formula),and Nintedanib group(TGF-β_(1)+Nintedanib)according to different intervention methods.The expressions ofα-smooth muscle actin(α-SMA)and E-cadherin were observed by immunofluorescence.The leptin level in cell supernatant was detected by ELISA,and the protein content of LEPR,PPAR-γand the activation degree of STAT3 were detected by Western blot.Results:Compared with control group,leptin level in cell supernatant of model group was increased(P<0.01),and decreased in Chinese medicine group and Nintedanib group(P<0.001).Compared with control group,the expression of LEPR protein in model group was increased(P<0.001),the expression of PPAR-γwas decreased(P<0.05),the activation level of STAT3 was decreased,the expression of LEPR protein in Chinese medicine group and Nintedanib group was down-regulated(P<0.001),and the expression of PPAR-γwas up-regulated(P<0.05).The activation level of STAT3 in Chinese medicinal group was not significantly different from that in model group(P>0.05),but the activation level of STAT3 in Nintedanib group was increased compared with that in model group(P<0.01).Conclusion:This study confirmed that EMT can promote the secretion of endogenous lung leptin,increase the expression of LEPR on the cell surface,down-regulate the content of PPAR-γ,which has the antagonistic effect of leptin,and reduce the activation of STAT3,providing material basis for leptin to accelerate the process of EMT.However,Bushen Yifei Xiaozheng Formula may reverse this process by reducing endogenous leptin secretion,reducing LEPR and increasing PPAR-γlevel.