目的 探讨牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)的代谢产物通过激活炎症小体(NOD-,LRR-and pyrin domain-containing protein 3,NLRP3)促进神经炎症反应的作用及机制.方法 使用P.gingivalis上清处理小胶质细胞HMC3,采用CCK8、实时荧光定量PCR、Western Blot等方法分别检测P.gingivalis上清对小胶质细胞HMC3 的细胞活力、细胞NLRP3 信号通路激活以及线粒体功能验证.结果 P.gingivalis上清在 5%时,显著激活小胶质细胞HMC3 的NLRP3,增加相关凋亡相关斑点样蛋白(apoptosis-associated spot-like protein containing a CARD,ASC)、胱天蛋白酶 1(Caspase1)、炎症因子(IL-1β)(P＜0.05);且线粒体功能相关蛋白表达和线粒体膜电位降低.加入NLRP3 的抑制剂(MCC950)后,10nM MCC950 显著增强细胞活力,抑制相关胱天蛋白酶1(Caspase1)、凋亡相关斑点样蛋白(ASC)、炎症因子(IL-1β)(P＜0.05)的表达;同时恢复了线粒体功能.结论 P.gingivalis通过激活NLRP3 促进小胶质细胞炎症反应发生,其机制可能与线粒体功能有关.
Abstract
Objective To investigate the role and mechanism of Porphyromonas gingivalis(P.gingivalis)metabolites in promoting neuroinflammatory response by activating the NLRP3 inflammatome.Methods Microglia HMC3 cells was treated with P.gingivalis supernatant.CCK8,real-time fluorescent quantitative PCR,and Western blot were used to detect P.gingivalis supernatant's activation of inflammation in microglia HMC3 cells,its validation of inflammation activation and mitochondrial function verification.Results P.gingivalis supernatant,the cell viability of microglia HMC3 was inhibited(P＜0.05).The NLRP3 inflammatome of microglia HMC3 was significantly activated by 5%P.gingivalis supernant,and the apoptosis-associated spot-like protein(ASC),Caspase1 and IL-1β were increased(P＜0.05).The expression of mitochondrial function-related proteins and mitochondrial membrane potential decreased.After addition of NLRP3 inflammation inhibitor(MCC950),the CCK8 cell viability was significantly enhanced at 10nM MCC950,and apoptosis-associated spot-like protein(ASC),Caspase1 and inflammatory factor(IL-1β)were inhibited at 10nM MCC950(P＜0.05).Mitochondrial function was also restored.Conclusion P.gingivalis promotes microglial inflammatory response by activating NLRP3,which may be related to mitochondrial function.
维普
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