双氢青蒿素通过调节TGF-β/Smad通路改善小鼠接触性皮炎

Dihydroartemisinin ameliorates contact hypersensitivity in mice by regulating TGF-β/Smad signaling pathway

目的:探讨双氢青蒿素(DHA)对小鼠接触性皮炎(CHS)的抑制作用及机制。方法:0.5%2,4-二硝基氟苯(DNFB)涂抹小鼠腹部连续2 d致敏,5 d后用0.25%DNFB涂抹左耳发敏,右耳涂抹丙酮和橄榄油混合液作为对照,于致敏前2 d灌胃给予DHA处理。HE染色观察小鼠皮肤组织病理学变化,免疫组化染色观察小鼠耳部皮肤CD4^(+)T、CD8^(+)T细胞浸润情况,测定脾脏指数。ELISA检测血清IL-6、IFN-γ、IL-10、TGF-β和单核细胞趋化蛋白(MCP)-1变化,Western blot检测皮肤Smad2和Smad3磷酸化水平,流式细胞术检测皮肤和脾脏免疫细胞浸润情况。结果:DHA可显著改善CHS小鼠耳朵肿胀、皮肤红斑及脾脏指数(P<0.05)。组织病理学结果显示,DHA处理可明显抑制CHS小鼠皮肤增厚和炎症细胞浸润。流式细胞术结果显示,DHA处理后皮肤和脾脏中浸润的CD4^(+)T细胞、CD8^(+)T细胞、树突状细胞和巨噬细胞显著减少(P<0.05)。ELISA结果显示,相对于模型组,DHA处理组血清IL-6、IFN-γ、TGF-β和MCP-1水平明显降低(P<0.05)。Western blot结果表明DHA处理显著抑制皮肤Smad2和Smad3磷酸化水平(P<0.05)。结论:DHA通过减少免疫细胞浸润和调节TGF-β/Smad信号传导抑制CHS,为治疗CHS提供了新的药物选择和实验依据。

Objective:To investigate inhibition of dihydroartemisinin(DHA)on contact hypersensitivity(CHS)in mice and its mechanisms.Methods:Mice were sensitized with 0.5%2,4-dinitrofluorobenzene(DNFB)on shaved abdominal flank skin for 2 consecutive days,and CHS was elicited by 0.25%DNFB on left ear 5 days later.Right ear was treated with acetone/olive-oil alone as control.Mice were given DHA orally 2 days before sensitization.Skin histopathological changes were observed by HE staining,infiltrations of CD4^(+)T and CD8^(+)T cells in ear skin were observed by immunohistochemical staining,and spleen index was detected.Serum IL-6,IFN-γ,IL-10,TGF-βand monocyte chemotactic protein(MCP)-1 changes were detected by ELISA,skin Smad2 and Smad3 phosphorylation levels were detected by Western blot,and skin and spleen immune cells infiltration were detected by flow cytometry.Results:DHA significantly improved ear swelling,skin erythema and spleen index of CHS mice(P<0.05).Histopathological analysis revealed that degree of edema and cellular infiltration were markedly decreased in DHA-treated CHS mice.Flow cytometry results show that DHA treatment significantly decreased CD4^(+)T cells,CD8^(+)T cells,dendritic cells and macrophages infiltration in ear skin and spleen(P<0.05).ELISA results showed that DHA treatment also diminished serum levels of IL-6,IFN-γ,TGF-βand MCP-1 compared with model group(P<0.05).Western blot showed that Smad2 and Smad3 phosphorylation were normalized by DHA treatment(P<0.05).Conclusion:DHA suppresses CHS by reducing infiltration of immune cells and regulating TGF-β/Smad signaling pathway,which provides a new drug selection and experimental basis for CHS treatment.