摘要
研究白杨素(ChR)是否通过影响核转录因子κB(NF-κB)/Twist 1信号通路,进而抑制Ⅱ型肺泡上皮细胞间质转分化(EMT)而产生抗肺纤维化作用。动物实验设对照组、博莱霉素组(BLC)、BLC+ChR(50 mg·kg^(-1))和BLC+ChR(100 mg·kg^(-1))剂量组,每组动物15只。气管注射BLC(7500 U·kg^(-1))诱导肺纤维化大鼠模型,造模后连续灌胃给药28 d。细胞实验设对照组、转化生长因子-β1(TGF-β1)(5 ng·mL^(-1))组、TGF-β1+ChR(1,10,100μmol·L^(-1))组,Ⅱ型肺泡上皮细胞先用TGF-β1处理24 h,再用TGF-β1和(或)不同剂量ChR处理48 h。肺组织病理变化和胶原沉积情况分别采用HE染色、Masson染色和免疫组化法进行观察。肺组织和细胞内Ⅰ型胶原(collagenⅠ)、E钙粘蛋白(E-cadherin)、紧密连接蛋白-1(ZO-1)、波形蛋白(vimentin)、α-平滑肌肌动蛋白(α-SMA)、核转录因子κB抑制因子α(IκBα)、NF-κB p65、磷酸化NF-κB p65(p-p65)和Twist 1 mRNA及蛋白表达分别采用qPCR和(或)Western blot法检测。动物实验结果显示,与BLC组相比,ChR给药28 d后,大鼠肺纤维化程度明显减轻;肺组织collagenⅠ表达明显下降(P<0.05或P<0.01);肺组织肺泡上皮细胞EMT程度明显受到抑制[ZO-1和E-cadherin的表达明显升高,α-SMA和vimentin的表达明显降低(P<0.05或P<0.01)];肺组织细胞浆内IκBα和p65磷酸化水平、胞核内NF-κB p65和Twist 1的表达水平明显降低(P<0.05或P<0.01)。细胞实验结果表明,不同剂量ChR能够明显减轻TGF-β1诱导的collagenⅠ的表达(P<0.05或P<0.01),显著抑制细胞EMT[E-cadherin和ZO-1的表达明显升高,vimentin和α-SMA的表达明显降低(P<0.05或P<0.01)],同时能够明显抑制TGF-β1诱导的细胞浆内IκBα和p65磷酸化水平并降低细胞核内NF-κB p65和Twist 1的表达(P<0.05或P<0.01)。综上结果推测ChR能够逆转肺泡Ⅱ型上皮细胞EMT、缓解肺纤维化,其机制可能与其降低细胞内IκBα磷酸化水平、抑制NF-κB p65的磷酸化和其核转移,进而下调Twist 1的表达有关。
This study aimed to assess whether chrysin(ChR)can inhibit epithelial-mesenchymal transition(EMT)of typeⅡalveo lar epithelial cell and produce anti-pulmonary fibrosis effect by regulating the NF-κB/Twist 1 signaling pathway.Sixty rats were randomly divided into the control group,the bleomycin(BLC)group,BLC+ChR(50 mg·kg^(-1))group and BLC+ChR(100 mg·kg^(-1))group,with 15 rats in each group.The pulmonary fibrosis model was induced by intratracheal injection of BLC(7500 U·kg^(-1)).Rats were orally administered with different doses of ChR after BLC injection for 28 days.The cells were divided into control group,TGF-β1 group(5 ng·mL^(-1)),and TGF-β1+ChR(1,10,100μmol·L^(-1))groups.The typeⅡalveolar epithelial cells were treated with TGF-β1 for 24 h,and then treated with TGF-β1 for 48 h in the presence or absence of different doses of ChR(1,10 and 100μmol·L^(-1)).The morphological changes and collagen deposition in lung tissues were analyzed by HE staining,Masson staining and immunohistochemistry.The mRNA and protein expression levels of collagenⅠ,E-cadherin,zonula occludens-1(ZO-1),vimentin,alpha smooth muscle actin(α-SMA),inhibitor of nuclear factor kappa B alpha(IκBα),nuclear factor-kappa B p65(NF-κB p65),phospho-NF-κB p65(pp65)and Twist 1 in lung tissues and cells were detected by qPCR and Western blot,respectively.The animal experiment results showed that as compared with the BLC group,after administration of ChR for 28 days,bleomycin-induced pulmonary fibrosis in rats was significantly relieved,collagenⅠexpression in lung tissues was significantly reduced(P<0.05 or P<0.01),and EMT of alveolar epithelial cells was obviously inhibited[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin andα-SMA were decreased(P<0.05 or P<0.01)],concomitantly with significantly reduced IκBαand p65 phosphorylation level in cytoplasm and decreased NF-κB p65 and Twist 1 expression in nucleus(P<0.05 or P<0.01).The cell experiment results showed that different doses of ChR(1,10 and 100μmol·L^(-1))significantly reduced TGF-β1-induced collagenⅠexpression(P<0.05 or P<0.01),significantly inhibited EMT of typeⅡalveolar epithelial cells[the expression levels of E-cadherin and ZO-1 were increased and the expression levels of vimentin andα-SMA were decreased(P<0.05 or P<0.01)],and inhibited IκBαand p65 phosphorylation in cytoplasm and down-regulated NF-κB p65 and Twist 1 expression in nucleus induced by TGF-β1(P<0.05 or P<0.01).The results suggest that ChR can reverse EMT of typeⅡalveolar epithelial cell and alleviate pulmonary fibrosis in rats,and its mechanism may be associated with reducing IκBαphosphorylation and inhibiting NF-κB p65 phosphorylation and nuclear transfer,thus downregulating Twist 1 expression.
作者
卢林明
付裕
李秋阳
何晓伟
汤娟
李先伟
LU Lin-ming;FU Yu;LI Qiu-yang;HE Xiao-wei;TANG Juan;LI Xian-wei(Department of Pathology,Wannan Medical College,Wuhu 241002,China;Department of Pharmacology,Wannan Medical College,Wuhu 241002,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2021年第1期146-154,共9页
China Journal of Chinese Materia Medica
基金
安徽省高校自然科学研究重点项目(2018A0254,KJ2019A0419)
皖南医学院重点科研项目培育基金项目(LAB201805)
2019年国家级大学生创新创业训练计划项目(201910368002)。
