P62-ASC泛素化介导自噬对NLRP3炎症小体调节的机制研究

Study on P62-ASC ubiquitination mediated regulation of NLRP3 inflammasome by autophagy

目的探讨棕榈酸(PA)刺激下P62介导的自噬和含NLR家族Pyrin域蛋白3(NLRP3)炎症小体相互作用机制。方法分离培养小鼠Kupffer细胞(KCs),分对照组、PA组、渥曼青霉素组和雷帕霉素组。Western blot测定各组KCs中LC3、NLRP3、凋亡相关点样蛋白(ASC)、半胱氨酸天冬氨酸酶-1(caspase-1)及Beclin-1表达水平的变化,激光共聚焦显微镜观察P62与ASC,以及P62与LC3在细胞中的共定位情况;免疫共沉淀技术测定PA刺激前后P62与ASC、LC3之间的相互作用;Western blot测定PA处理12 h前后ASC泛素化改变情况。结果与对照组比较,PA组LC3Ⅱ/LC3Ⅰ、NLRP3、ASC、caspase-1和Beclin-1表达水平明显升高(P<0.05)。而渥曼青霉素组LC3Ⅱ/LC3Ⅰ较PA组下降,NLRP3、ASC及caspase-1表达水平较PA组增加(P<0.05);雷帕霉素组LC3Ⅱ/LC3Ⅰ较PA组增加,而NLRP3、ASC及caspase-1表达水平较PA组减少(P<0.05)。激光共聚焦结果显示KCs中P62与ASC、P62与LC3共定位,且在PA处理24 h后明显增强。免疫共沉淀结果表明,与对照组比较,PA组P62、ASC形成的共聚体明显增多。Western blot检测结果表明,PA组ASC泛素化水平较对照组增强。结论KCs在PA刺激下,自噬与NLRP3炎症小体的表达均增强,且促进自噬能降低NLRP3炎症小体的表达,对KCs具有保护作用。P62对ASC泛素蛋白的识别在自噬对NLRP3炎症小体的调节中非常关键。

Objective To investigate the mechanism and interaction of palmitic acid(PA)-induced autophagy and recombinant NLR family,Pyrin domain containing protein 3(NLRP3).Methods Mouse kupffer cells(KCs)were isolated,cultured and divided into the control group,the PA group,the wortmannin group and the rapamycin group.The protein levels of LC3,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),cysteinyl aspartate specific proteinase-1(caspase-1)and Beclin-1 were detected by Western blot.Confocal microscopy was used to observe the co-localization of P62 and ASC protein and the co-localization of P62 and LC3 protein in KCs;co-immunoprecipitation assay was used to detect the interaction between P62 and ASC protein before and after PA stimulation;ASC ubiquitination level were detected by WB before and after PA stimulation.Results The protein levels of LC3Ⅱ/LC3Ⅰ,NLRP3,ASC,capase-1 and Beclin-1 in the PA group significantly increased compared with the control group(P<0.05).In the rapamycin group,LC3Ⅱ/LC3Ⅰwas increased significantly compared with the PA group,but the expression of NLRP3,ASC and caspase-1 protein was decreased compared with the PA group(P<0.05).In the wortmannin group,compared with the PA group,LC3Ⅱ/LC3Ⅰdecreased while NLRP3,ASC and caspase-1 protein expression increased(P<0.05).The co-localization of P62 and ASC protein in KCs was observed by laser scanning confocal microscope,which enhanced after stimulation with PA.Co-immunoprecipitation showed that the amount of P62 intercalating with ASC protein in the PA group increased compared with the control group.The level of ASC ubiquitination in the PA group was higher than that in the control group.Conclusion The interaction between autophagy and NLRP3 inflammasome is enhanced under the stimulation of PA,and the enhanced autophagy could inhibit the expression of NLRP3.P62-ASC ubiquitination plays an important role in the regulation of NLRP3 inflammasome by autophagy.