黄芪丹参提取物改善急性呼吸窘迫综合征大鼠肺损伤的作用和机制研究

Effect and Mechanism of Astragalus and Salvia Miltiorrhiza Extract on Lung Injury in Rats with Acute Respiratory Distress Syndrome

目的通过大鼠在体研究明确黄芪丹参提取物对急性呼吸窘迫综合征(acute respiratory distress syndrme,ARDS)大鼠肺损伤的影响及作用机制。方法通过慢病毒途径在大鼠肺部靶向沉默白细胞介素(interleukin,IL)4Rα,经黄芪丹参提取物给药后通过油酸与脂多糖(lipopolysaccharide,LPS)联合诱导建立大鼠ARDS模型,造模后6 h应用称重法检测肺组织湿干重比,HE染色评估肺组织病理学改变,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中肿瘤坏死因子(tumor necrosing factor,TNF)-α、IL-1β、IL-6含量以及肺组织中髓过氧化物酶(myeloperoxidase,MPO)活性。流式细胞术检测BALF来源中性粒细胞凋亡率。免疫印迹法检测BALF来源中性粒细胞中IL-4Rα及半胱氨酸蛋白酶3水解体(Caspase 3-cleaved)蛋白表达,荧光定量PCR检测肺组织中趋化因子含量。结果与假手术组比较,ARDS模型组大鼠肺组织湿干重比明显增加(P<0.01),肺组织损伤评分明显升高(P<0.01),BALF中炎症因子TNF-α、IL-1β和IL-6含量和肺组织中MPO含量明显升高(P<0.01)。BALF来源中性粒细胞凋亡率稍降低(P>0.05)。IL-4Rα与Caspase 3-cleaved蛋白表达明显减弱(P<0.01)。肺组织中趋化因子CCL2、CCL3和CCL17 m RNA表达量明显增加(P<0.01)。10 mg/(kg·d)和30 mg/(kg·d)黄芪丹参提取物连续3 d灌胃给药能够明显改善上述指标的异常变化(P<0.05,P<0.01)。IL-4Rα沉默能有效逆转黄芪丹参提取物的上述作用(P<0.05,P<0.01)。结论黄芪丹参提取物能够通过上调IL-4Rα表达并间接诱导ARDS大鼠肺部中性粒细胞凋亡诱导因子Caspase 3-cleaved表达而导致中性粒细胞程序性坏死,并最终有效降低肺部炎症因子释放和减轻ARDS导致的大鼠肺损伤。

Objective To clarify the effect and mechanism of Astragalus and Salvia miltiorrhiza extract on lung injury in rats with acute respiratory distress syndrome(ARDS).Methods Interleukin 4R alpha(IL-4Rα) was silenced in the lungs of rats via lentiviral pathway,and then rat ARDS model was established by the combined induction of oleic acid and lipopolysaccharide(LPS) after administration of Astragalus salvia miltiorrhiza extract.The wet-dry weight ratio of lung tissue was detected by weighing 6 hours after modeling.HE staining was used to evaluate the pathological changes of lung tissue.Enzyme-linked immunosorbent assay(ELISA) was used to detect the contents of tumor necrosis factor-α(TNF-α),interleukin 1β(IL-1β),interleukin 6(IL-6) in bronchoalveolar lavage fluid(BALF) and myeloperoxidase(MPO)activity in lung tissue.The apoptosis rate of BALF-derived neutrophils was detected by flow cytometry.The expressions of IL-4Rα and Caspase 3-cleaved in BALF-derived neutrophils were detected by western blotting.The content of chemokine in lung tissue was detected by quantitative real-time PCR.Results Compared with the sham-operated group,the wet-dry weight ratio of lung tissue in ARDS rats was significantly increased( P0.05).The protein expressions of 4Rα and Caspase 3-cleaved were significantly decreased(P<0.01),and the m RNA expressionsof chemokines CCL2,CCL3 and CCL17 were significantly increased in lung tissue(P<0.01).10 mg/(kg·d)and 30 mg/(kg·d) of Astragalus and Salvia miltiorrhiza extract by intragastric administration for 3 days could significantly improve the abnormal changes of the above indexes(P<0.05,P<0.01).IL-4Rα silencing could effectively reverse the above effects of Astragalus and Salvia miltiorrhiza extract( P< 0.05,P< 0.01).Conclusion Astragalus and Salvia miltiorrhiza extract can induce the programmed necrosis of neutrophils by up-regulating the expression of IL-4Rα and indirectly inducing the expression of Caspase 3-cleaved,a neutrophil Apoptosis-inducing factor in the lungs of ARDS rats,and ultimately effectively reduce the release of inflammatory factors in the lung and alleviate ARDS-induced lung injury in rats.