基于RAF/MEK/ERK信号通路探讨苦杏仁-苦参对特发性肺纤维化大鼠的保护作用OA

Objective To explore the protective effect of herb pair Kuxingren(Armeniacae Semen Amarum)-Kushen(Sophorae Flavescentis Radix)on bleomycin-induced idiopathic pulmonary fibrosis(IPF)in rats and investigate its mechanism.Methods SD rats were randomly divided into control group,model group,pirfenidone(200 mg/kg)group,Armeniacae Semen Amarum-Sophorae Flavescentis Radix low-,medium-and high-dose(1,2,4 g/kg)groups.The IPF rat model was established by one-time tracheal instillation of bleomycin sulfate(5 mg/kg).After drug intervention,the pathological changes in lung tissue were observed using hematoxylin-eosin(HE)and Masson staining.ELISA was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-iβ(IL-1 β),IL-6 and hydroxyproline(Hyp)in serum and bronchoalveolar lavage fluid.TUNEL staining was used to detect cell apoptosis in lung tissue.The expressions of α-smooth muscle actin(α-SMA),collagen type Ⅰ(collagen-Ⅰ)and fibronectin-1(FN-1)in lung tissue were detected by immunofluorescence.Western blotting was used to detect the expressions of p-p38,phosphorylated extracellular signal regulated kinase(p-ERK),phosphorylated c-Jun N-terminal kinase(p-JNK),B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax)in lung tissue.Transforming growth factor-β1(TGF-β1)was used to induce human lung fibroblasts(MRC-5)in vitro model.After intervention with Armeniacae Semen Amarum-Sophorae Flavescentis Radix containing serum,cell proliferation activity was detected using CCK-8 method.TUNEL staining was used to detect cell apoptosis.The expressions of α-SMA,collagen-Ⅰ and FN-1 were detected by immunofluorescence.Western blotting was used to detect the expressions of p-RAF,phosphorylated mitogen activated protein kinase(p-MEK),p-ERK,Cyclin D1,proliferating cell nuclear antigen(PCNA),Bax and Bcl-2 proteins.Results In the in vivo experiment,compared with control group,the body weight of rats in model group was decreased(P＜0.01),the dry wet mass ratio and lung index of lung tissue were increased(P＜0.01),the lung tissue showed significant inflammatory infiltration,thickening and edema of alveolar space,and the cell apoptosis rate of lung tissue was decreased(P＜0.01),the levels of inflammatory factors in serum and bronchoalveolar lavage fluid were increased(P＜0.01),the protein expressions of α-SMA,collagen-Ⅰ,FN-1,p-p38,p-ERK,p-JNK and Bcl-2 in lung tissue were significantly up-regulated(P＜0.01),while the protein expression of Bax was significantly down-regulated(P＜0.01).Compared with model group,the body weight of rats in each treatment group were increased(P＜0.01),the dry wet mass ratio and lung index of lung tissue were decreased(P＜0.01),the pathological changes of lung tissue were significantly improved,the cell apoptosis rate of lung tissue was increased(P＜0.01),the levels of inflammatory factors in serum and bronchoalveolar lavage fluid were decreased(P＜0.05,0.01),the expressions of α-SMA,collagen-Ⅰ,FN-1,p-p38,p-ERK,p-JNK and Bcl-2 proteins in lung tissue were significantly down-regulated(P＜0.01),and the expression of Bax protein was significantly up-regulated(P＜0.01).In the in vitro experiments,compared with control group,cell proliferation activity in model group was increased(P＜0.01),cell apoptosis rate was decreased(P＜0.01),α-SMA,collagen-Ⅰ,FN-1,p-RAF,p-MEK,p-ERK,Cyclin D1,PCNA,Bcl-2 protein expressions were increased(P＜0.01),and Bax protein expression was decreased(P＜0.01).Compared with model group,Armeniacae Semen Amarum-Sophorae Flavescentis Radix containing serum and RAF inhibitor GW5074 intervention resulted in a decrease in cell proliferation activity(P＜0.05,0.01),an increase in cell apoptosis rate(P＜0.01),a decrease in the expressions of α-SMA,collagen-Ⅰ,FN-1,p-RAF,p-MEK,p-ERK,Cyclin D1,PCNA,Bcl-2 proteins(P＜0.05,0.01),and an increase in Bax protein expression(P＜0.05,0.01).Conclusion Armeniacae Semen Amarum-Sophorae Flavescentis Radix exerts an anti-IPF effect by regulating RAF/MEK/ERK signaling pathway,inhibiting inflammatory response,suppressing cell proliferation and promoting cell apoptosis.