根皮素对免疫性肝炎小鼠的保护作用及机制

Protective effects of phloretin on immune hepatitis and its mechanism in mice

目的观察根皮素(Phloretin,PHL)对自身免疫性肝炎(autoimmune hepatitis,AIH)小鼠的保护作用及调控机制。方法SPF级Balb/C小鼠32只,随机分为对照(Control)组、刀豆蛋白A(concanavalin A,ConA)模型组、PHL低剂量组和PHL高剂量组。造模处理24 h后提取小鼠血清及肝脏组织,ELISA法检测血清转氨酶及炎症因子;HE染色观察肝脏组织病理学变化;TUNEL染色检测肝细胞凋亡;qRT-PCR检测转录表达水平;免疫组化和Western-blot检测肝组织炎症因子、自噬与凋亡蛋白及TRAF6-JNK通路信号蛋白表达水平。结果与正常对照组相比,AIH小鼠血清转氨酶及炎症因子表达显著升高(P<0.001),病理学见肝组织结构被广泛破坏,肝细胞大面积坏死及炎性细胞浸润。与ConA组相比,PHL组血清转氨酶及炎症因子水平下降(P<0.05),肝细胞结构完整,肝细胞坏死面积减少(P<0.05)。此外,与ConA组相比,PHL显著下调肝组织促凋亡蛋白及自噬蛋白的表达(P<0.05),下调TRAF6-JNK信号通路激活(P<0.05)。结论PHL可能通过减轻AIH小鼠肝细胞自噬和肝细胞凋亡,缓解AIH小鼠高炎症负荷,发挥对AIH小鼠的保护作用,可能是通过TRAF6-JNK信号通路发挥作用。

Objective To investigate the protective effects and regulatory mechanism of phloretin(PHL)on autoimmune hepatitis(AIH)in mice.Methods 32 SPF Balb/C mice were randomly divided into normal control group,concanavalin A(ConA)model group,PHL low-dose group and PHL high-dose group.The serum and liver tissues were extracted from the mice after 24 hours of modeling treatment.ELISA method was used to detect serum transaminases and inflammatory factors;HE staining was used to observe pathological changes in liver tissue;TUNEL staining was used to detect liver cell apoptosis;qRT-PCR was used to detect transcription expression level;immunohistochemistry and Western blot were used to detect the expression levels of inflammatory factors,autophagy and apoptosis proteins,and TRAF6-JNK pathway signaling proteins in liver tissue.Results Compared with the normal control group,the expressions of serum aminotransferase and inflammatory factors in AIH mice increased significantly(P<0.001),and the pathological findings showed extensive destruction of liver tissue structure,large-scale necrosis of liver cells and inflammatory cell infiltration.Compared with the ConA group,the levels of serum transaminase and inflammatory factors in the PHL group decreased(P<0.05),the structure of hepatocytes was intact,and the necrotic area of hepatocytes was smaller(P<0.05).In addition,compared with the ConA group,PHL significantly down-regulated the expression of pro-apoptotic protein and autophagy protein in liver tissue(P<0.05),and down-regulated the activation of TRAF6-JNK signaling pathway(P<0.05).Conclusion PHL may exert a protective effect on AIH mice by reducing liver cell autophagy and apoptosis,alleviating high inflammatory load.It may act through the TRAF6-JNK signaling pathway.