芍药苷对克雷伯菌肺炎大鼠肺功能及mTOR/HIF-1α/VEGF通路的影响

Effects of paeoniflorin on lung function and mTOR/HIF-1α/VEGF pathway in rats with Klebsiella pneumonia

目的 探讨芍药苷(paeoniflorin, PF)对肺炎克雷伯菌(Klebsiella pneumoniae,KP)大鼠肺功能及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)/低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)/血管内皮生长因子(vascular endothelial growth factor, VEGF)通路的影响。方法 将SD大鼠随机分为空白组模型组,雷帕霉素组(3 mg/kg),芍药苷低、中、高剂量组(50、100、200 mg/kg),每组12只。采用克雷伯杆菌气管滴入的方法建立KP大鼠模型,给予相应的药物干预7 d后,检测大鼠肺功能情况;对外周血白细胞(white blood cell, WBC)和中性粒细胞(polymorphonuclear neutrophils, PMN)进行计数,并计算肺指数;采用苏木精-伊红(hematoxylin-eosin, HE)和Masson染色观察肺部病理学变化;采用CD34免疫组化法检测肺组织血管生成,计算微血管密度(microvessel density, MVD);采用ELISA检测肺匀浆中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、HIF-1α、VEGF含量;采用western blotting检测肺组织mTOR/HIF-1α/VEGF通路相关蛋白表达。结果 芍药苷高、中剂量可增加KP大鼠用力肺活量(forced vital capacity, FVC)、第0.3秒用力呼气量(forced expiratory volume in 0.3 second, FEV0.3)和FEV0.3/FVC比值,差异有统计学意义(q=8.394、5.524、11.047、6.656、7.510、5.546,P<0.05);降低外周血WBC和PMN水平、肺指数、MVD、TNF-α、IL-1β、HIF-1α、VEGF水平和p-mTOR/mTOR、磷酸化真核翻译起始因子4E结合蛋白1(phosphorylated-eIF4E binding protein 1,p-4EBP1)/4EBP1比值,差异有统计学意义(q=10.078、7.558,12.891、6.254,5.716、4.528,13.399、6.895,22.616、10.699、38.973、29.814,10.431、4.397,14.913、9.714,6.755、4.504,8.202、5.126,P<0.05);通过减少炎性细胞浸润和胶原沉积,可减轻肺组织损伤。结论 芍药苷可降低肺部炎症反应、抑制肺纤维化、改善KP大鼠的肺功能,其作用机制可能与抑制mTOR/HIF-1α/VEGF信号通路及肺组织血管新生有关。

Objective To investigate the effects of paeoniflorin on lung function and mammalian target of rapamycin(mTOR)/hypoxia inducible factor 1α(HIF-1α)/vascular endothelial growth factor(VEGF) pathway in Klebsiella pneumonia(KP) rats.Methods SD rats were randomly divided into blank group, model group, rapamycin group(3 mg/kg),low, medium and highdose groups(50 mg/kg, 100 mg/kg, 200 mg/kg) with paeoniflorin, with 12 rats in each group.The rat model of KP pneumonia was established by the Klebsiella tracheal drip method, and lung function in rats was detected after 7 days of corresponding drug intervention.The white blood cells(WBC) and polymorphonuclear neutrophils(PMN) in peripheral blood were counted, and the lung index was calculated.Hematoxylin-Eosin(HE) and Masson staining were used to observe the pathological changes in the lungs.Angiogenesis in lung tissue was detected by the CD34 immunohistochemical method and microvessel density(MVD) was calculated.The contents of tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β),HIF-1α,and VEGF in lung homogenate were detected using ELISA.The expression of mTOR/HIF-1α/VEGF pathway-related proteins in lung tissue was tested by western blotting.Results High and medium doses of paeoniflorin increased forced vital capacity(FVC),forced expiratory volume in 0.3 second(FEV 0.3),and FEV 0.3/FVC ratio in KP pneumonia rats with statistically significant differences(q=8.394,5.524,11.047,6.656,7.510,5.546,P<0.05),and reduced the levels of WBC and PMN in peripheral blood, lung index, MVD,TNF-α,IL-1β,HIF-1α,VEGF levels and p-mTOR/mTOR,phosphorylated-eIF4E binding protein 1(p-4EBP1)/4EBP1 ratio with statistically significant differences(q=10.078,7.558,12.891,6.254,5.716,4.528,13.399,6.895,22.616,10.699,38.973,29.814,10.431,4.397,14.913,9.714,6.755,4.504,8.202,5.126,P<0.05).Lung tissue injury was reduced by reducing inflammatory cell infiltration and collagen deposition.Conclusion Paeoniflorin reduced pulmonary inflammation response, inhibited pulmonary fibrosis, and improved the lung function of rats with Klebsiella pneumonia,and its mechanism may be related to the inhibition of mTOR/HIF-1α/VEGF signaling pathway and angiogenesis in lung tissue.