黄地安消胶囊含药血清抑制NLRP3介导的细胞焦亡对糖尿病认知功能障碍细胞模型的保护作用  

Huangdi Anxiao Capsules-containing serum protects cell model from cognitive dysfunction in diabetes via inhibiting NLRP3-mediated pyroptosis

作　　者：王晓娟 王雨露 邵楠 叶婷 叶树[1,2] 高华武 王艳[1,2] WANG Xiao-juan;WANG Yu-lu;SHAO Nan;YE Ting;YE Shu;GAO Hua-wu;WANG Yan(School of Integrated Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Provincial Medical Basic Research and Innovation Center for the Prevention and Treatment of Neurodegenerative Diseases by Integrating Traditional Chinese and Western Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学中西医结合学院,安徽合肥230012 [2]中西医结合防治神经退行性疾病安徽省医药基础研究创新中心,安徽合肥230012

出　　处：《中国中药杂志》2023年第19期5315-5325,共11页China Journal of Chinese Materia Medica

基　　金：安徽省教育厅重点项目(2022AH050485);安徽省教育厅安徽省高校优秀青年人才支持计划一般项目(gxyq2021185)。

摘　　要：探讨黄地安消胶囊(Huangdi Anxiao Capsules,HDAX)含药血清对高糖诱导大鼠嗜铬细胞瘤(PC12)细胞认知功能障碍的保护作用及可能的分子机制。构建高糖诱导PC12细胞糖尿病认知功能障碍模型,使用mcc950抑制NLRP3。将PC12随机分为正常(control)组、模型(model)组、HDAX含药血清组、NLRP3受体阻断剂(mcc950)组、HDAX含药血清+NLRP3受体阻断剂(HDAX含药血清+mcc950)组。PC12细胞按照分组分别处理后通过MTT法检测细胞增殖活性,Hoechst 33258/PI染色检测细胞焦亡,ELISA法测定PC12中白细胞介素(interleukin,IL)-1β、IL-18水平,Western blot检测PC12中突触后致密物(postsynaptic density,PSD)、焦亡相关通路蛋白NOD样受体蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、gasdermin D(GSDMD)、GSDMD-N、裂解的半胱天冬酶-1前体(cleaved cysteinyl aspartate specific proteinase-1,cleaved caspase-1)蛋白水平;RT-PCR检测PC12中NLRP3、ASC、GSDMD、caspase-1的mRNA表达量,免疫荧光检测焦亡标志指标GSDMD-N、NLRP3表达情况。结果显示,与control组相比,model组细胞增殖活性显著降低,PI阳性率增加,PSD-95表达降低,促进焦亡相关通路蛋白NLRP3、ASC、GSDMD-N、GSDMD、cleaved caspase-1表达,增加NLRP3、ASC、GSDMD、caspase-1的mRNA含量,同时提升促炎因子IL-18、IL-1β的表达;与model组相比,HDAX含药血清组、mcc950抑制剂组以及HDAX含药血清+mcc950组可以提高细胞存活率,改善PC12细胞形态,显著降低PI阳性率,降低焦亡相关通路蛋白NLRP3、ASC、GSDMD-N、GSDMD、cleaved caspase-1的蛋白表达,减少NLRP3、ASC、GSDMD、caspase-1的mRNA含量,同时抑制促炎因子IL-18、IL-1β的表达。综上,HDAX含药血清可抑制NLRP3介导的细胞焦亡,对高糖诱导PC12细胞认知功能障碍具有保护作用。This study aims to investigate the effects and the molecular mechanism of Huangdi Anxiao Capsules(HDAX)-containing serum in protecting the rat adrenal pheochromocytoma(PC12) cells from diabetes-associated cognitive dysfunction induced by high glucose and whether the mechanism is related to the regulation of NOD-like receptor thermal protein domain associated protein 3(NLRP3)-mediated pyroptosis.The PC12 cell model of diabetes-associated cognitive dysfunction induced by high glucose was established and mcc950 was used to inhibit NLRP3.PC12 cells were randomized into control,model,HDAX-containing serum,mcc950,and HDAX-containing serum+mcc950 groups.Methyl thiazolyl tetrazolium(MTT) assay was employed to determine the viability,and Hoechst 33258/PI staining to detect pyroptosis of PC12 cells.Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1 beta(IL-1β) and IL-18.Western blot was employed to determine the protein levels of postsynaptic density protein 95(PSD-95),NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),gasdermin D(GSDMD),GSDMD-N,and cleaved cysteinyl aspartate specific proteinase-1(caspase-1),and RT-PCR to determine the mRNA levels of NLRP3,ASC,GSDMD,and caspase-1.The immunofluorescence assay was adopted to measure the levels and distribution of NLRP3 and GSDMD-N in PC12 cells.Compared with the control group,the model group showed decreased cell proliferation,increased PI positive rate,down-regulated protein level of PSD-95,up-regulated protein levels of NLRP3,ASC,GSDMD-N,GSDMD,and cleaved caspase-1,up-regulated mRNA levels of NLRP3,ASC,GSDMD,and caspase-1,and elevated levels of IL-1β and IL-18.Compared with the model group,HDAX-containing serum,mcc950,and the combination of them improved cell survival rate and morphology,decreased the PI positive rate,down-regulated the protein levels of NLRP3,ASC,GSDMD-N,GSDMD,and cleaved caspase-1 and the mRNA levels of NLRP3,ASC,GSDMD,and caspase-1,and promoted the secretion of IL-1β and IL-18.The findings

关 键 词：黄地安消胶囊含药血清 细胞焦亡 NLRP3炎症小体 PC12细胞 

分 类 号：R285[医药卫生—中药学]