清热养阴除湿丸调控焦亡通路治疗痛风的作用机制

Mechanism of Qingre Yangyin Chushi Pills against Gout by Regulating Pyroptosis Pathway

目的:基于NEK7/GSDMD焦亡通路,探讨清热养阴除湿丸抗炎、治疗痛风的作用机制。方法:将48只雄性SD大鼠随机分为6组,用改良Coderre法构建痛风动物模型。造模6 h、12 h、24 h、48 h后测量大鼠踝关节周长,计算肿胀指数。在灌胃第7天收集血清和组织样本。利用免疫组织化学技术、实时荧光定量PCR(Real-time PCR)及蛋白质免疫印迹法检测NEK7/GSDMD通路各标志蛋白的mRNA及蛋白表达量,酶联免疫吸附试验法(ELISA)检测白细胞介素1β(IL-1β),白细胞介素6(IL-6),白细胞介素18(IL-18)及肿瘤坏死因子-α等细胞因子的表达。结果:随着造模时间的延长,与空白组比较,其他各组大鼠均有明显踝关节肿胀;随着治疗时间的延长,各药物处理组大鼠的踝关节肿胀指数逐渐降低,以中药高剂量组下降幅度最大,差异有统计学意义(P<0.05)。与模型组比较,中药高剂量组各细胞因子的含量明显降低(P<0.05),NEK7/GSDMD通路各标志性蛋白mRNA及蛋白表达明显减少(P<0.05)。结论:热养阴除湿丸能够抑制NEK7-NLRP3炎症小体的形成,降低成孔蛋白GSDMD的产生,调控细胞焦亡的发生,减少炎症介质表达,从而减轻关节炎症,清热养阴除湿丸的抗炎作用可能是通过NEK7/GSDMD通路实现的。

Objective:To explore the mechanism of Qingre Yangyin Chushi Pills(QRYYCS)in resisting inflammation and treating gout based on the NEK7/GSDMD pyroptosis pathway.Methods:Forty-eight male SD rats were randomly divided into six groups.The gout model was induced in animals with the modified Coderre method.After 6 h,12 h,24 h,and 48 h of modeling,the ankle circumference was measured and the swelling index was calculated.Serum and tissue samples were collected on the 7th day of drug treatment.The mRNA and protein expression of marker proteins in the NEK7/GSDMD pathway was detected by immunohistochemistry,real-time fluorescence-based quantitative PCR(Real-time PCR),and Western blot.The expression of interleukin(IL)-1β,IL-6,IL-18,and tumor necrosis factor(TNF)-αwas measured by enzyme-linked immunosorbent assay(ELISA).Results:With the prolongation of modeling time,compared with normal rats,the rats in other groups showed obvious ankle swelling.The ankle swelling index of the rats in groups with drug intervention steadily decreased with the prolongation of treatment time,with the high-dose QRYYCS group showing the largest reduction amplitude(P<0.05).Compared with the model group,the high-dose QRYYCS group showed reduced content of cytokines(P<0.05)and reduced mRNA and protein expression levels of marker proteins in the NEK7/GSDMD pathway(P<0.05).Conclusion:QRYYCS can inhibit the formation of NEK7-NLPR3 inflammasome,reduce the production of GSDMD protein,regulate the occurrence of cell pyroptosis,and control the expression of inflammatory factors,thereby reducing joint inflammation.The anti-inflammatory effect of QRYYCS may be achieved through the NEK7/GSDMD pathway.