五虎汤对RSV诱导的哮喘小鼠模型树突细胞自噬和炎性因子IL-17A和IL-17F水平的影响

Effect of Wuhu Decoction on autophagy in dendritic cells and levels of inflammatory factors IL-17A and IL-17F in RSV-induced asthmatic mice

目的探讨五虎汤对呼吸道合胞病毒(respiratory syncytial virus,RSV)诱导的哮喘小鼠模型树突细胞自噬和炎性因子白细胞介素-17A(interleukin-17A,IL-17A)、白细胞介素-17F(interleukin-17F,IL-17F)的影响。方法采用RSV联合鸡卵清蛋白(ovalbumin,OVA)建立哮喘小鼠模型,将小鼠随机分为对照组、模型组及五虎汤低、中、高剂量(1.6、3.2、6.4g/kg)组和地塞米松(1.82mg/kg)组、雷帕霉素(1mg/kg)组,连续给药14d后采用苏木-伊红(HE)染色法观察各组小鼠肺组织病理变化,电镜观察肺组织自噬小体变化,Westernblotting法检测肺组织树突细胞中微管相关蛋白轻链3I(microtubule-associated protein lightc hain 3I,LC3I)和LC3II表达,ELISA法检测肺泡灌洗液中IL-17A、IL-17F的水平。SD大鼠随机分为对照组及五虎汤低、中、高(1.1、2.2、4.4 g/kg)组,各给药组ig相应药物,2次/d,连续给药7 d后腹主动脉取血,离心取血清,灭活后滤过。将分离的树突细胞与CD4^(+)T细胞共培养,采用RSV联合OVA-17肽建立RSV诱导的树突细胞自噬模型,流式细胞术检测五虎汤含药血清对CD4^(+)T细胞增殖与T细胞活化标志物CD69表达的影响。结果模型组小鼠气道狭窄,血管周围有大量炎症细胞浸润,肺组织自噬小体增加,自噬蛋白LC3II/LC3I表达水平显著升高(P<0.01),IL-17A和IL-17F水平显著升高(P<0.01);与模型组比较,五虎汤组肺组织炎症表现减轻,肺组织自噬小体增加,LC3II/LC3I蛋白表达水平显著升高(P<0.01),IL-17A和IL-17F水平显著降低(P<0.05、0.01)。RSV联合OVA-17肽诱导的树突细胞自噬模型中,与对照组比较,模型组CD4^(+)T细胞增殖与CD69表达均显著升高(P<0.01);与模型组比较,五虎汤中、高剂量含药血清组CD4^(+)T细胞增殖与CD69表达显著降低(P<0.05、0.01)。结论RSV可诱导小鼠肺组织树突细胞自噬水平升高,上调IL-17A和IL-17F水平,从而介导炎症反应;五虎汤可通过上调RSV诱导的哮喘小鼠肺组织树突细胞自噬水平,下调IL-17A和IL-17F水平,从而改善哮喘气道炎症。

Objective To observe the effect of Wuhu Decoction(五虎汤)on dendritic cells autophagy,inflammatory factor interleukin-17A(IL-17A),and interleukin-17F(IL-17F)in RSV-induced asthmatic mice.Methods Asthmatic mice model was established by RSV and ovalbumin(OVA).Mice were randomly divided into control group,model group,Wuhu Decoction low-,medium-,high-dose(1.6,3.2,6.4 g/kg)groups,dexamethasone(1.82 mg/kg)group and rapamycin(1 mg/kg)group.After given with drugs for 14 d,HE staining was used to observe the pathology of lung,electron microscope was used to observe autophagosome changes of lung,Western blotting was used to detect the expressions of microtubule-associated protein lightc hain 3 I(LC3 I)and LC3 II in dendritic cells,ELISA was used to detect the level of IL-17A and IL-17F in bronchoalveolar lavage fluid.SD rats were randomly divided into control group,Wuhu Decoction low-,medium-and high-dose(1.1,2.2,4.4 g/kg)groups,each administration group were ig corresponding drugs,2 times/d,after given with drugs for 7 d,blood was collected from the artery,the serum was collected by centrifugation and filtered after inactivation.Dendritic cells and CD4^(+)T cells were co-cultured,RSV and OVA-17 peptide were used to establish RSV-induced dendritic cells autophagy model.CD4^(+)T cell proliferation and CD69 expression were detected by flow cytometry.Results In model group,airways of mice were narrowed,a large number of inflammatory cells were infiltrated around the blood vessels,autophagosomes in lung tissue was increased,autophagy protein LC3 II/LC3 I expressions were significantly increased(P<0.01),IL-17A and IL-17F levels were significantly increased(P<0.01);Compared with model group,lung tissue inflammation of mice in Wuhu Decoction group was reduced,lung tissue autophagosomes was increased,LC3 II/LC3 I protein expressions were significantly increased(P<0.01),levels of IL-17A and IL-17F were significantly reduced(P<0.05,0.01).In RSV combined with OVA-17 peptide-induced autophagy model of dendritic cells;Compared with control group,CD4^(+)T cell proliferation and CD69 expression in model group were significantly increased(P<0.01);Compared with model group,CD4^(+)T cell proliferation and CD69 expression in drug-containing serum middle-and high-dose groups were significantly reduced(P<0.05,0.01).Conclusion RSV could induce the autophagy of dendritic cells in lung tissue of mice,and upregulate the expression of IL-17A and IL-17F,thereby mediating the inflammatory response;Wuhu Decoction could upregulate the autophagy of dendritic cells in lung tissue of mice induced by RSV,downregulate levels of IL-17A and IL-17F,thereby improving asthmatic airway inflammation.