益肺散结方对肺纤维化大鼠模型自噬及PI3K-AKT-mTOR通路的影响

Effects of Yifei Sanjie Recipe on autophagy and PI3K-AKT-mTOR pathway in pulmonary fibrosis rat model

目的探讨益肺散结方对肺纤维化(PF)大鼠肺组织中磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)/雷帕霉素靶分子(mTOR)信号通路及自噬的影响。方法选取SD大鼠50只,随机选10只行气管插管术并注入生理盐水0.3 mL作为对照组,其余40只大鼠行气管插管并注入博莱霉素(BLM)5 mg/kg,0.3 mL,制备PF模型,造模成功后,随机分为模型组(PF组)、益肺散结方低(7.5 g/kg)、中(15 g/kg)、高(30 g/kg)剂量组,每组各10只。对照组、PF组经灌胃给予生理盐水,益肺散结方各剂量组灌胃给予相应剂量药物1次/d,共给药21 d。末次给药12 h后,处死大鼠取肺组织;用苏木精-伊红染色(HE)观察肺组织病理形态;马松(Masson)染色法评估胶原增殖程度;羟脯氨酸试剂盒测定胶原蛋白含量;蛋白免疫印迹法(Western blot)检测肺组织通路蛋白p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR及自噬相关蛋白beclin-1、泛素和LC3结合蛋白p62(p62)、ɑ-平滑肌动蛋白(ɑ-SMA)、微管相关蛋白轻链3(LC3-Ⅱ)/LC3-Ⅰ表达水平。结果与对照组相比,PF组大鼠肺组织炎性细胞浸润及炎性程度评分、胶原增殖程度及评分、胶原蛋白含量、通路蛋白p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR相对表达水平、自噬相关蛋白ɑ-SMA和p62均升高(均P<0.05),自噬相关蛋白beclin-1和LC3-Ⅱ/LC3-Ⅰ均降低(均P<0.05)。与PF组相比,益肺散结方高、中、低剂量组大鼠肺组织炎性细胞浸润及炎性程度评分、胶原增殖程度及评分、胶原蛋白含量、通路蛋白p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR相对表达水平、自噬相关蛋白ɑ-SMA和p62均降低(均P<0.05),自噬相关蛋白beclin-1和LC3-Ⅱ/LC3-Ⅰ均升高(均P<0.05)。结论益肺散结方可通过抑制PI3K/Akt/mTOR信号通路表达,提高肺组织细胞自噬活性,缓解肺纤维化进程。

Objective To explore the effect of Yifei Sanjie Recipe on the phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/rapamycin target molecule(mTOR)signaling pathway Influence.Methods 50 SD rats were randomly divided into model group(PF group),low(7.5 g/kg),medium(15 g/kg)and high(30 g/kg)groups with 10 rats in each group.The control group and PF group were given normal saline by gavage,while the Yifei Sanjie Decoction groups were given the corresponding dose of drugs once a day for 21 days.12 hours after the last administration,the rats were killed and the lung tissue was taken.The pathological morphology of lung tissue was observed by hematoxylin eosin staining(he).Masson staining was used to evaluate the degree of collagen proliferation.The content of collagen was determined by hydroxyproline kit.Western blot was used to detect the expression of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR,autophagy related protein Beclin 1,ubiquitin and LC3 binding protein p62(p62),αsmooth actin(α-SMA),microtubule associated protein light chain 3(LC3-Ⅱ)/LC3-Ⅰ.Results Compared with the control group,the scores of inflammatory cell infiltration and inflammatory degree,the degree and score of collagen proliferation,the content of collagen,the relative expression levels of pathway proteins p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR,the autophagy related proteins(α-SMA and p62)were increased in PF group(all P<0.05),and the autophagy related proteins Beclin 1 and LC3-Ⅱwere increased in PF group/LC3-Ⅰdecreased(P<0.05).Compared with PF group,the inflammatory cell infiltration and inflammatory degree score,collagen proliferation degree and score,collagen content,relative expression levels of pathway proteins p-PI-3K/PI-3K,p-Akt/Akt,p-mTOR/mTOR,autophagy related protein(a)SMA and p62 were significantly decreased(P<0.05),and autophagy related proteins Beclin 1 and LC3-Ⅱwere decreased in Yifei Sanjie Decoction group/LC3-Ⅰincreased(P<0.05).Conclusion Yifei Sanjie decoction can inhibit the expression of PI3K/Akt/mTOR signaling pathway,improve the autophagy activity of lung tissue cells,and alleviate the pulmonary fibrosis process.