穿山龙总皂苷经TGF-β1/α-SMA通路减轻博莱霉素所致大鼠肺纤维化的实验研究

Relieving Effect of Total Saponins from Rhizoma Dioscorea Nipponica on Bleomycin-induced Pulmonary Fibrosis by Regulating TGF-β1/α-SMA Pathway in Rats

目的:探讨穿山龙总皂苷(TSRDN)经转化生长因子-β1/α-平滑肌动蛋白(TGF-β1/α-SMA)通路减轻博莱霉素所致大鼠肺纤维化的作用机制。方法:96只大鼠随机分为6组:假手术组、模型组、泼尼松组(3 mg·kg^-1),以及TSRDN低、中、高剂量组(20,40,80 mg·kg^-1),每组16只。以博莱霉素5 mg·kg^-1滴入气管建立肺纤维化动物模型,连续给药28 d后,检测羟脯氨酸含量、肺系数,观察肺组织病理变化,同时采用反转录·聚合酶链反应(RT-PCR)及蛋白免疫印迹试验(Western Blot)检测肺组织中TGF-β1、α-SMA mRNA和蛋白表达量。结果:假手术组肺组织结构正常,无间质纤维组织增生,偶见炎性细胞;模型组大鼠组织巨噬细胞活化,炎性细胞浸润,肺泡隔增宽,表面有较多出血灶,形成弥散性肺纤维化,多呈3级纤维化和2级肺泡炎改变;泼尼松组和TSRDN给药组胶原组织增生降低,肺泡间隔增宽不明显,多数肺组织结构较为清晰,多呈2级和1级纤维化改变,炎性细胞浸润减少。与假手术组比较,模型组大鼠肺系数、肺组织羟脯氨酸含量、肺泡炎评分、肺纤维化评分、TGFβ1、α-SMA mRNA和蛋白表达水平显著升高(P<0.05);与模型组比较,泼尼松组和TSRDN各剂量组大鼠上述相关指标均降低(P<0.05),且随着TSRDN剂量的升高,TSRDN各剂量组大鼠上述相关指标逐渐降低,呈剂量依赖性(P<0.05);与泼尼松组比较,TSRDN低、中剂量组大鼠肺系数、肺组织羟脯氨酸含量、肺泡炎评分、肺纤维化评分、TGFβ1、α-SMA mRNA和蛋白表达水平均升高(P<0.05),TSRDN高剂量组大鼠上述相关指标差异无统计学意义(P>0.05)。结论:TSRDN具有一定的抗肺纤维化作用,其机制可能通过TGF-β1/α-SMA信号途径干预纤维蛋白形成和沉积。

Objective:To investigate the relieving effect of total saponins from Rhizoma dioscorea nipponica(TSRDN)on pulmonary fibrosis in rats induced by bleomycin through TGF-β1/α-SMA pathway.Methods:Totally 96 rats were randomly divided into 6 groups:sham operation group,model group,prednisone group(3 mg·kg^-1),TSRDN low,middle and high dose groups(20,40 and 80 mg·kg^-1)with 16 rats in each group.The animal model of pulmonary fibrosis was established by instilling 5 mg·kg^-1 of bleomycin into trachea.After 28-day continuous administration,the hydroxyproline content and lung coefficient were measured,and the pathological changes were observed.RT-PCR and Western blot were used to detect the expressions of TGF-β1,α-SMA mRNA and protein in lung tissue.Results:In the sham operation group,the lung tissue structure was normal,there was no proliferation of interstitial fibrous tissue,and inflammatory cells were occasionally seen.In the model group,macrophages in lung tissue were activated,inflammatory cells were infiltrated,alveolar septum was widened,there were more bleeding spots on the surface,and diffuse pulmonary fibrosis was formed,and most of them showed grade 3 fibrosis and grade 2 alveolar inflammation.The collagen tissue proliferation in prednisone group and TSRDN groups were reduced,alveolar septum widening was not obvious,and lung tissue structure was clearer with grade 2 and 1 fibrosis changes,and the inflammatory cell infiltration decreased.Compared with those in the sham operation group,the lung coefficient,lung hydroxyproline content,alveolitis score,pulmonary fibrosis score,TGFβ1,α-SMA mRNA and protein expressions were significantly increased in the model group(P<0.05).Compared with those in the model group,the above related indices of rats in prednisone group and all TSRDN groups were decreased(P<0.05),and with the increase of TSRDN dose,the above related indices were gradually decreased in a dose-dependent manner(P<0.05).Compared with those in prednisone group,the lung coefficient,lung proline content,alveolitis score,pulmonary fibrosis score,TGFβ1,α-SMA mRNA and protein expressions in TSRDN low and medium dose groups were significantly increased(P<0.05).There were no significant differences in the above related indices between high dose TSRDN group and prednisone group(P>0.05).Conclusion:The total saponins from Rhizoma dioscorea nipponica has a certain antipulmonary fibrosis effect,which may be related to its inhibition against fibrin formation and deposition and intervention with TGF-β1/α-SMA signal pathway activation.