红花黄色素通过TGF-β_(1)通路抑制百草枯诱导的肺纤维化研究

Study on inhibition of paraquat-induced pulmonary fibrosis by safflower yellow through TGF-β_(1) pathway

目的探讨红花黄色素(SY)抑制百草枯(PQ)诱导的肺纤维化的可能机制,为其临床应用提供理论依据。方法24只健康清洁级SD大鼠,随机分为对照组(NS组)、PQ组、SY组、PQ+SY组,每组6只。PQ组和PQ+SY组通过腹腔注射PQ溶液(20 mg/kg)建立肺纤维化模型,NS组和SY组给予等体积生理盐水。24 h建模成功后,SY组和PQ+SY组灌胃25 mg/(kg·d)的SY进行干预治疗,NS组和PQ组每天给予等体积的生理盐水。7 d后处死,采用HE和Masson染色观察大鼠肺组织病理变化;逆转录-聚合酶链式反应(RT-PCR)法检测大鼠肺组织匀浆转化生长因子-β_(1)(TGF-β_(1))基因表达水平;蛋白印迹法(Western Blot)法检测肺组织匀浆中TGF-β_(1)蛋白、α-肌动蛋白(α-SMA)、E-Cadherin的表达水平。结果NS组、PQ组、SY组、PQ+SY组SY组大鼠肺组织中TGF-β_(1)相对mRNA表达水平分别为(1.00±0.20)、(2.63±0.12)、(1.28±0.26)、(1.52±0.08)。NS组与SY组大鼠肺组织中TGF-β_(1)相对mRNA表达水平比较差异无统计学意义(P>0.05);PQ组大鼠肺组织中TGF-β_(1)相对mRNA表达水平高于NS组,差异具有统计学意义(P<0.05);SY+PQ组大鼠肺组织中TGF-β_(1)相对mRNA表达水平低于PQ组,差异具有统计学意义(P<0.05)。NS组、PQ组、SY组、PQ+SY组大鼠肺组织中TGF-β_(1)蛋白表达水平分别为(0.150±0.012)、(0.340±0.013)、(0.120±0.034)、(0.290±0.020)μg/ml,E-Cadherin表达水平分别为(0.700±0.066)、(0.280±0.039)、(0.820±0.116)、(0.560±0.050)μg/ml,α-SMA表达水平分别为(0.930±0.021)、(1.840±0.090)、(0.850±0.093)、(1.490±0.060)μg/ml。NS组与SY组大鼠肺组织中TGF-β_(1)蛋白、E-Cadherin、α-SMA表达水平比较差异无统计学意义(P>0.05);PQ组大鼠肺组织中TGF-β_(1)蛋白、α-SMA表达水平高于NS组,E-Cadherin表达水平低于NS组,差异具有统计学意义(P<0.05);SY+PQ组大鼠肺组织中TGF-β_(1)蛋白、α-SMA表达水平低于PQ组,E-Cadherin表达水平高于PQ组,差异具有统计学意义(P<0.05)。结论SY可能通过抑制TGF-β_(1)蛋白、α-SMA的表达来抑制PQ诱导的肺纤维化。

Objective To discuss the possible mechanism of safflower yellow(SY)inhibiting paraquat(PQ)-induced pulmonary fibrosis,so as to provide theoretical basis for its clinical application.Methods A total of 24 healthy and clean SD rats were randomly divided into control group(NS group),PQ group,SY group and PQ+SY group,with 6 rats in each group.PQ group and PQ+SY group were intraperitoneally injected PQ solution(20 mg/kg)to establish pulmonary fibrosis model,while NS group and SY group were given equal volume normal saline.24 h after successful modeling,the SY and PQ+SY group were treated with 25 mg/(kg·d)of SY by gavage for intervention,and the NS group and PQ group were given equal volume of normal saline daily.The rats were executed 7 d later,and the histopathological changes were observed by HE and Masson staining;the expression levels of transforming growth factor-β_(1)(TGF-β_(1))gene in rat lung homogenates were detected by reverse transcription-polymerase chain reaction(RT-PCR);the expression levels of TGF-β_(1) protein,α-smooth muscle actin(α-SMA)and E-Cadherin in lung homogenates were detected by Western blot assay.Results The relative mRNA expression levels of TGF-β_(1) in the lung tissues of rats were(1.00±0.20),(2.63±0.12),(1.28±0.26)and(1.52±0.08)in NS group,PQ group,SY group and PQ+SY group.There was no statistically significant difference in relative mRNA expression levels of TGF-β_(1) in the lung tissues of rats in between NS and SY groups(P>0.05).The relative mRNA expression level of TGF-β_(1) in the lung tissues of rats in the PQ group were higher than that in the NS group,and the differences were statistically significant(P<0.05).The relative mRNA expression level of TGF-β_(1) in the lung tissue of rats in SY+PQ group was lower than that in PQ group,and the difference was statistically significant(P<0.05).The expression levels of TGF-β_(1) protein in the lung tissue of rats in NS group,PQ group,SY group and PQ+SY group were(0.150±0.012),(0.340±0.013),(0.120±0.034)and(0.290±0.020)μg/ml,the expression levels of E-Cadherin were(0.700±0.066)(0.280±0.039),(0.820±0.116)and(0.560±0.050)μg/ml,and the expression levels ofα-SMA were(0.930±0.021),(1.840±0.090),(0.850±0.093)and(1.490±0.060)μg/ml.There were no statistically significant differences in the expression levels of TGF-β_(1) protein,E-Cadherin andα-SMA in lung tissues between NS group and SY group(P>0.05).The expression level of TGF-β_(1) protein andα-SMA in lung tissue of PQ group was higher than that of NS group,while the expression level of E-Cadherin was lower than that of NS group,and the difference was statistically significant(P<0.05).The expression level of TGF-β_(1) protein andα-SMA in lung tissue of SY+PQ group was lower than that of PQ group,while the expression level of E-Cadherin was higher than that of PQ group,the difference was statistically significant(P<0.05).Conclusion SY may inhibit PQ-induced pulmonary fibrosis by suppressing the expression of TGF-β_(1) protein andα-SMA.