基于Wnt/β-catenin信号通路探讨槲皮素对自身免疫性葡萄膜炎大鼠的影响机制

Effect and Mechanism of Quercetin on Experimental Autoimmune Uveitis in Rats Based on Wnt/β-catenin Signaling Pathway

目的:探讨槲皮素调控Wnt/β-连环蛋白(β-catenin)信号通路对自身免疫性葡萄膜炎(EAU)的影响机制。方法:运用网络药理学技术对槲皮素与EAU的调控作用关系进行初步判定;通过酶联免疫吸附测定法(ELISA)与临床分级评分对模型进行鉴定,再通过ELISA对信号通路中重要炎性因子白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α进行检测,通过分子对接技术对通路中重要靶点因子进行结合验证,蛋白免疫印迹法(Western blot)检测信号通路下相关靶点蛋白的表达水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测信号通路下相关靶点基因的表达水平。结果:通过ELISA与临床分级评分模型制作成功,ELISA检测信号通路中重要炎性因子IL-6、TNF-α,显示模型组、二甲基亚砜(DMSO)组二者表达最高,槲皮素低剂量和槲皮素高剂量组干预后均降低,趋势相同;分子对接显示槲皮素与β-catenin;低密度脂蛋白受体相关蛋白6(LRP6)对接结合能十分理想;Western blot检测β-catenin、LRP6,模型组表达最高,随着不同剂量的槲皮素使用表达降低,二者趋势相同,槲皮素低剂量与槲皮素高剂量组比较差异无统计学意义;Real-time PCR结果显示β-catenin、MYC、AXIN2和TCF均为模型组表达最高,槲皮素高剂量组最低,槲皮素低剂量组降低表达,槲皮素高剂量组和槲皮素低剂量组之间比较差异无统计学意义。结论:槲皮素通过调控Wnt/β-catenin信号通路可降低EAU疾病的炎性表达,减轻患者的病痛。

Objective:To investigate the effects and mechanism of quercetin on experimental autoimmune uveitis(EAU)by regulating the Wnt/β-catenin signaling pathway.Method:The regulatory relationship between quercetin and EAU was preliminarily determined by network pharmacology.The model was identified by enzyme-linked immunosorbent assay(ELISA)and clinical grading score.The important inflammatory factors,including interleukin(IL)-6 and tumor necrosis factor(TNF)-αin the signaling pathway were detected by ELISA.The binding of important targets in the pathway was verified by molecular docking.The protein and mRNA expression levels of related targets in the signaling pathway were detected by Western blot and Real-time polymerase chain reaction(Real-time PCR),respectively.Result:As evaluated by ELISA and the clinical grading score,the model was properly induced.The results of ELISA showed that the levels of IL-6 and TNF-αwere the highest in the model group and the dimethyl sulfoxide(DMSO)group,and decreased after medium-and high-dose drug interventions,but the changing tend was the same.Molecular docking showed that the binding energies of quercetin toβ-catenin and LRP6 were ideal.Western blot revealed that the expression ofβ-catenin and LRP6 was the highest in the model group,and decreased with drug intervention at different doses,but the changing trend was the same.Besides,there was no significant difference between the low-dose group and the high-dose group.Real-time PCR results showed that the expression ofβ-catenin,MYC,AXIN2,and TCF was the highest in the model group,the lowest in the high-dose group,and decreased in the low-dose group.There was no significant difference between the high-dose group and the low-dose group.Conclusion:Quercetin can reduce the inflammatory expression of EAU and relieve the pain of patients by regulating the Wnt/β-catenin signaling pathway.