补肺益肾组分方对PM2.5诱发慢性阻塞性肺疾病加重模型大鼠上皮-间质转化的影响

Effect of the Effective-Component Compatibility of Bufei Yishen Formula on Epithelial–Mesenchymal Transition in PM2.5-Aggravated Chronic Obstructive Pulmonary Diseases Rats

目的观察PM2.5及补肺益肾组分方对慢性阻塞性肺疾(COPD)大鼠上皮-间质转化(EMT)的作用,并探讨其作用机制。方法将50只健康SD大鼠随机分为:空白组、COPD组、大气颗粒物(PM2.5)组、补肺益肾组分方组(组分方组)和氨茶碱组,每组10只。除空白组外采用香烟熏吸联合反复克雷伯杆菌感染制备COPD大鼠模型,除空白组和COPD组外以大气实时浓缩PM2.5对其进行全身暴露。自暴露第1天起,组分方组和氨茶碱组分别给予补肺益肾组分方[6.48 mg/(kg·d)]或氨茶碱[54 mg/(kg·d)]灌胃,其余各组给予生理盐水,1次/天,持续8周。检测大鼠最大呼气中期流速(MMEF)和功能残气量(FRC);免疫组化法检测肺组织α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COLⅠ)、Ⅲ型胶原蛋白(COLⅢ)表达;Western Blot检测肺组织E-钙黏附蛋白(E-cad)、N-钙黏附蛋白(N-cad)、α-SMA、COLⅠ、Smad家族成员(Smad)4、p-Smad2/3表达;ELISA法检测血清和肺组织匀浆中TGF-β1水平。结果与空白组比较,COPD组MMEF及E-cad蛋白表达降低,FRC、TGF-β1水平及α-SMA、COLⅠ、COLⅢ、α-SMA、p-Smad3、Smad4蛋白表达增加(P<0.05,P<0.01);PM2.5组MMEF及E-cad蛋白表达降低,FRC、TGF-β1水平及α-SMA、COLⅠ、COLⅢ、N-cad、p-Smad2、p-Smad3、Smad4蛋白表达增加(P<0.05,P<0.01)。与COPD组比较,PM2.5组FRC、TGF-β1水平及α-SMA、COLⅠ、COLⅢ表达增加(P<0.05,P<0.01)。与PM2.5组比较,组分方组MMEF及E-cad表达升高,FRC、TGF-β1水平及α-SMA、COLⅠ、COLⅢ、N-cad、p-Smad2、Smad4蛋白表达降低(P<0.05,P<0.01);氨茶碱组FRC、TGF-β1水平及α-SMA、COLⅠ、COLⅢ、p-Smad2、Smad4蛋白表达降低(P<0.05,P<0.01)。结论PM2.5暴露促进COPD大鼠肺组织EMT,进一步加重肺功能损伤;补肺益肾组分方可有效防治PM2.5诱导的COPD肺组织EMT加重,其机制可能与调控TGF-β1/Smad通路有关。

Objective To observe the effect of PM2.5 and the effective-component compatibility of Bufei Yishen Formula(ECC-BYF)on the epithelial–mesenchymal transition(EMT)of chronic obstructive pulmonary diseases(COPD)rats and its possible involved mechanism.Methods Fifty rats were randomly divided into control group,COPD group,PM2.5 group,ECC-BYF and aminophylline group,10 rats in each group.Except for the control group,the COPD rat model was established by repeated cigarette smoke inhalations and bacterial infections,then rats in all groups except for the control and COPD groups were exposed to real-time atmospheric concentrated PM2.5.Meanwhile,rats in the ECC-BYF group and aminophylline group were given ECC-BYF(6.48 mg·kg^(-1)·d^(-1))and aminophylline(54 mg·kg^(-1)·d^(-1))by gavage,respectively,while rats in other groups were given normal saline once daily,the course was 8 weeks.The maximum expiratory flow(MMEF)and functional residual capacity(FRC)were measured.The expression levels of alpha-smooth muscle actin(α-SMA),collagen(COL)Ⅰand COLⅢin lung tissues were examined by immumohistochemical.Evaluation of the expressions of E-cadherin(E-cad),N-cadherin(N-cad),α-SMA,COLⅠ,Smad4,p-Smad2/3 in lung tissues were conducted via Western Blot,and TGF-β1 in the serum and lung tissue homogenate were detected by ELISA.Results Compared with the control group,MMEF and E-cad protein expression decreased,FRC,TGF-β1,α-SMA,COLⅠ,COLⅢ,p-Smad3,Smad4 protein expression increased in the COPD group(P<0.05,P<0.01).Compared with the control group,MMEF and E-cad protein expression dcreased,FRC,TGF-β1 content,andα-SMA,COLⅠ,COLⅢ,N-cad,p-Smad2,p-Smad3 and Smad4 protein expression increased in the PM2.5 group(P<0.05,P<0.01).Compared with the COPD group,FRC and TGF-β1 content,andα-SMA,COLⅠand COLⅢprotein expression increased in the PM2.5 group(P<0.05,P<0.01).Compared with the PM2.5 group,MMEF and E-cad protein expression increased,FRC,TGF-β1 content,α-SMA,COLⅠ,COLⅢ,N-cad,p-Smad2,p-Smad3,and Smad4 protein expression decreased in ECC-BYF group(P<0.05,P<0.01).FRC,TGF-β1 content,α-SMA,COLⅠ,COLⅢ,p-Smad2 and Smad4 protein expression decreased in aminophylline group(P<0.05,P<0.01).Conclusions PM2.5 exposure promotes EMT in the lung tissue of COPD rats,which markedly aggravated lung injury.ECC-BYF could alleviate the EMT of COPD rats caused by PM2.5 exposure,and the mechanism may be related to the regulation of TGFβ1/Smad pathway.