升陷汤调节Wnt3a/β-catenin信号途径介导的细胞衰老改善特发性肺纤维化大鼠肺功能

Shengxian Decoction improves lung function in rats with idiopathic pulmonary fibrosis by regulating cell senescence mediated by Wnt3a/β-catenin signaling pathway

目的:观察升陷汤对特发性肺纤维化(IPF)大鼠Wnt3a/β-连环蛋白(β-catenin)途径介导的细胞衰老的作用特点,揭示该方改善IPF大鼠肺功能的可能机制。方法:32只SPF级SD大鼠随机分为假手术组、模型组、吡非尼酮组和升陷汤组。气管内滴注博来霉素(0.005 g·kg^(-1))复制IPF大鼠模型。术后次日灌胃,升陷汤组灌胃升陷汤配方颗粒水溶液(0.78 g·kg^(-1)),吡非尼酮组灌胃吡非尼酮混悬液(0.05 g·kg^(-1)),其余各组灌胃去离子水(10 mL·kg^(-1)),连续28 d。测定大鼠肺功能后取肺组织:苏木素-伊红(HE)和马松(Masson)染色观察大鼠肺组织病理形态学变化,并行Szapiel和Ashcroft评分;实时荧光定量聚合酶链式反应(Real-time PCR)检测端粒长度;蛋白免疫印迹法(Western blot)检测上皮-间质转化(EMT)标记物[α-平滑肌肌动蛋白(α-SMA)、E-钙黏蛋白(E-cadherin)]、端粒酶逆转录酶(TERT)、衰老相关蛋白(p53、p21)、衰老相关分泌表型[白细胞介素-6(IL-6)、基质金属蛋白酶-1(MMP-1)]和Wnt信号途径关键蛋白[Wnt3a、糖原合酶激酶-3β(GSK-3β)、β-catenin、细胞周期蛋白D1(Cyclin D1)、c-Myc]表达。结果:与假手术组比较,模型组大鼠最大呼吸流量(PEF)和每分钟通气量(MV)显著降低(P<0.01),呼吸频率(f)显著升高(P<0.01),Szapiel和Ashcroft评分及α-SMA、p53、p21、IL-6、MMP-1、Wnt3a、GSK-3β、β-catenin、Cyclin D1、c-Myc蛋白表达显著升高(P<0.01),E-cadherin和TERT表达及端粒长度显著降低(P<0.01)。与模型组比较,升陷汤组大鼠PEF和MV显著升高(P<0.01),f显著降低(P<0.01),Szapiel和Ashcroft评分及α-SMA、p53、p21、IL-6、MMP-1、Wnt3a、GSK-3β、β-catenin、Cyclin D1、c-Myc蛋白表达明显降低(P<0.05,P<0.01),E-cadherin和TERT表达及端粒长度显著升高(P<0.01)。结论:升陷汤对IPF大鼠肺功能具有显著地保护作用,其机制可能是作用于Wnt3a/β-catenin信号途径,调节TERT诱导的细胞衰老以抑制EMT实现。

Objective:To observe the effect of Shengxiantang(SXT)on cell senescence mediated by wingless/integrated(W nt)3a/β-catenin pathway in rats with idiopathic pulmonary fibrosis(IPF)and reveal the possible mechanism in improving lung function of IPF rats.M ethod::A total of 32 SPF level SD rats were randomly divided into sham group,model group,pirfenidone group,and SXT group.The IPF rat model was established by intratracheal instillation of bleomycin(0.005 g·kg^(-1)).The following day after surgery,rats in the SXT group were given the aqueous solution of SXT granules(0.78 g·kg^(-1)),and the pirfenidone group was given pirfenidone suspension(0.05 g·kg^(-1)).The other groups were given deionized water(10 mL·kg^(-1))for 28 consecutive days.Lung tissue was collected after the lung function was measured.The pathological changes of the lung tissue were observed by hematoxylin-eosin(HE)and Masson staining,and then the Szapiel score and Ashcroft score were performed.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect telomere length.Western blot was applied to detect the expressions of epithelial-mesenchymal transformation(EMT)markers[α-smooth muscle actin(α-SMA)and E-cadherin],telomere reverse transcriptase(TRET),aging-related proteins(p53 and p21),senescence-associated secretory phenotype[interleukin-6(IL-6)and matrix metalloproteinase-1(MMP-1)],and key proteins of W nt signaling pathway[W nt3a,glycogen synthase kinase-3β(GSK-3β),β-catenin,Cyclin D1,and c-Myc].Result:Compared with those in the Sham group,peak expiratory flow(PEF)and minute ventilation volume(MV)in the model group were significantly decreased(P<0.01),and the frequency of respiratory(f)was significantly increased(P<0.01).The Szapiel score,Ashcroft score,and protein expression ofα-SMA,p53,p21,IL-6,MMP-1,W nt3a,GSK3β,β-catenin,Cyclin D1,and c-Myc were increased(P<0.01).The expressions of E-cadherin and TERT,as well as telomere length were significantly decreased(P<0.01).Compared with those in the model group,PEF and MV in the SXT group were significantly increased(P<0.01),while f was significantly decreased(P<0.01).The Szapiel score,Ashcroft score,and protein expression ofα-SMA,p53,p21,IL-6,MMP-1,W nt3a,GSK3β,β-catenin,Cyclin D1,and c-Myc were significantly decreased(P<0.05,P<0.01).Nevertheless,the expression of E-cadherin and TERT,as well as telomere length were significantly increased(P<0.01).Conclusion:SXT presents a significant protective effect on lung function in IPF rats,and the prescription may act on the W nt3a/β-catenin signaling pathway to regulate cell senescence induced by TERT to inhibit EMT.