黄连异喹啉生物碱盐酸巴马汀对NLRP3炎症小体通路调控机制研究

Study on the underlying mechanism of regulating effect on NLRP3 inflammasome pathway of palmatine chloride,an isoquinoline alkaloid from Coptidis Rhizoma

目的:观察黄连主要异喹啉生物碱单体成分盐酸巴马汀的抗炎活性,验证其机制之一为通过抑制NLRP3炎症小体通路激活。方法:本实验通过内毒素(LPS)联合三磷酸腺苷(ATP)刺激小鼠腹膜巨噬细胞系建立炎症模型,采用酶联免疫法(Elisa)检测细胞培养基上清细胞因子白介素1beta(IL-1β)水平对黄连主要生物碱小檗碱、黄连碱、巴马汀、表小檗碱以及药根碱进行抑制活性初筛,综合抑制活性强弱以及研究深入程度,选取巴马汀进行后续机制研究。采用免疫蛋白印迹法分别检测NLRP3、ASC、GAPDH、pro-IL-1β、cleaved-IL-1β、p-JNK和JNK蛋白表达水平。结果:与模型组比较1~30μM/L巴马汀浓度依赖性显著降低细胞液上清中细胞因子IL-1β水平;显著抑制NLRP3、cleaved-IL-1β和p-JNK蛋白表达。结论:巴马汀具有良好的抗炎功效,其机制与其抑制NLRP3、p-JNK和cleaved-IL-1β蛋白水平,从而减少下游细胞因子IL-1β生成有关。

Objective:In the research,anti-inflammatory activity of Isoquinoline alkaloid palmatine,one of the main constituents of Coptidis Rhizoma,was observed;and inhibitory effect on NLRP3 inflammasome pathway was suggested one of the mechanism.Methods:Mice inflammatory model was established by stimulation on Raw264.7 cells with LPS plus ATP,then IL-1β level in culture supernatant was detected by Elisa.We chose the potential alkaloid in Coptidis Rhizoma for further investigation referring to the suppressing effect on IL-1β production.Result:The protein expressions of NLRP3,ASC,GAPDH,pro-IL-1β,cleaved-IL-1β,p-JNK and JNK were tested by Western Blotting.Compared with the model group,1-30μM palmatine decreased the production of IL-1β in a concentration-depended manner(P<0.01).Palmatine(1-30μM) also inhibited the protein expression of NLRP3(P<0.01),cleaved-IL-1β and p-JNK(P<0.05).Conclusion:Palmatine from Coptidis Rhizoma has good anti-inflammatory effect,and the underlying mechanism is associated with its inhibitory effect on NLRP3,pJNK and pro-IL-1β protein expression and the downstream IL-1β production.