【摘要】 目的观察黄连异喹啉生物碱盐酸巴马汀在人急性单核白血病细胞（THP-1）中的抗炎活性,研究其对NF-κB/p38 MAPK信号通路以及NLRP3炎症小体的调控作用机制。方法通过脂多糖（LPS）刺激THP-1细胞建立初筛模型,ELISA法测定细胞上清液中白细胞介素-6（IL-6）水平,初筛黄连中的小檗碱、黄连碱、药根碱、盐酸巴马汀和表小檗碱5种异喹啉生物碱对炎症细胞因子IL-6释放的抑制活性;采用MTT法测定盐酸巴马汀的作用浓度;Western Blot法测定盐酸巴马汀对LPS联合三磷酸腺苷（ATP）刺激THP-1细胞炎症模型中p-IκBα、IκBα、p-IKK、IKK、p-p38、p-38、p-JNK、JNK蛋白表达的影响,同时测定NLRP3、ASC和Caspase-1 p20蛋白的表达,并用Co-IP检测NLRP3炎症小体中pro-caspase-1和ASC的相互作用;ELISA法测定细胞因子IL-1β和IL-18的水平。结果与空白对照组比较,模型组IL-6含量升高（P<0.01）;与模型组比较,黄连碱组、小檗碱组和盐酸巴马汀组IL-6含量明显降低（P<0.01）,由于黄连碱和小檗碱已被大量研究,故选取盐酸巴马汀进行机制研究;盐酸巴马汀在1～60μmol·L^-1浓度范围对THP-1细胞生长没有明显影响（P>0.05）,选取1、10、30μmol·L^-1作为盐酸巴马汀低,中,高剂量。与空白对照组比较,模型组p-IκBα、p-IKK、p-p38、p-JNK、NLRP3、Caspase-1 p20蛋白表达明显升高（P<0.01）,细胞因子IL-1β和IL-18分泌增加（P<0.01）;与模型组比较,盐酸巴马汀中剂量组的p-IκBα、p-p38蛋白和盐酸巴马汀高剂量组的p-p38蛋白表达减少（P<0.05）,盐酸巴马汀高剂量组p-IκBα、p-JNK、NLRP3和Caspase-1 p20蛋白的表达明显降低（P<0.01）,pro-Caspase-1和ASC蛋白的相互作用减弱（P<0.01）,细胞因子IL-1β和IL-18释放减少（P<0.01）。结论盐酸巴马汀降低THP-1细胞炎症模型中p-IκBα、p-p38、p-JNK、NLRP3和Caspase-1 p20蛋白的表达,干扰pro-caspase-1和ASC的绑定,减少IL-1β和IL-18的释放,说明盐酸巴马汀的抗炎作用机制与NF-κB/p38 MAPK信号通路和NLRP3炎症小体信号通路相关。更多还原

【Abstract】 Objective To evaluate the anti-inflammatory activity of palmatine hydrochloride on acute human monocytic leukemia macrophage THP-1,and observe its regulation mechanism on NF-κB/p38 MAPK signaling pathway and NLRP3 inflammasome. Methods Inflammatory model was established by stimulating human monocyte macrophages with LPS. Determination of IL-6 levels in cell supernatants were carried out by ELISA. Preliminary screening of five isoquinoline alkaloids berberine, coptisine, jatrorrhizine, palmatine hydrochloride and epipodamine on the inhibition of inflammatory cytokine IL-6 release was conducted. According to the inhibitory activity, we chose palmatine hydrochloride for further investigation. Determination of the expression levels of IκBα,p-IκBα,IKK,pIKK,p-p38,p-38,JNK,p-JNK,NLRP3,ASC and Caspase-1 proteins in THP-1 stimulated by LPS and ATP was done by Western Blot. Interaction of pro-Caspase-1 and ASC in NLRP3 inflammasome was detected by Co-IP.The levels of downstream cytokine IL-1β and IL-18 were determined by ELISA. Results Compared with the blank control group,the level of IL-6 was increased in model group（P < 0.01）. Compared with the model group,the levels of IL-6 were decreased in the coptisine group,the berberine group and palmatine hydrochloride group（P <0.01）. Plamatine hydrochloride had no effect on the survival of cells in the concentration range of 1-60 μmol·L^-1;and 1,10,30 μmol·L^-1 were takenas low,medium,and high doses of palmatine hydrochloride. Compared with the blank control group,the expression of p-IκBα,p-p38,p-JNK,NLRP3 and Caspase-1 p20 were significantly increased（P < 0.01）. Compared with the model group,the expression levels of p-p38 was decreased in the medium and high dose groups of palmatine hydrochloride（P < 0.05）.The expression of p-IκBα, p-JNK, NLRP3 and Caspase-1 p20 were declined in the high dose group of palmatine hydrochloride（P < 0.01）,the interaction between pro-Caspase-1 and ASC was decreased（P < 0.01）,and the release of cytokines IL-1β and IL-18 were reduced（P <0.01）. Conclusion Palmatine hydrochloride can decrease the expression of p-IκBα,p-p38,p-JNK,NLRP3 and Caspase-1 p20,interfere the binding of pro-Caspase-1 and ASC,and reduced the levels of IL-1β and IL-18 in THP-1. The underlying mechanism focuses on NF-κB/p38 MAPK pathway,and interferes the signal transduction of NLRP3 inflammasome pathway.更多还原