复方鱼桔颗粒通过抑制BMDM中NLRP3炎症小体激活发挥抗炎效果的作用机制研究OA北大核心CSTPCD
Mechanistic study of anti-inflammatory effect of Fu-Fang-Yu-Jie granules through inhibition of NLRP3 inflammasome activation in BMDM
目的:研究复方鱼桔颗粒(FFYJ)对脂多糖(LPS)诱导的原代骨髓来源的巨噬细胞(BMDM)炎症模型的影响,及其对巨噬细胞中核苷酸结合寡聚结构域样受体蛋白3(NLRP3)炎症小体信号通路的干预作用.方法:从C57BL/6小鼠腿部骨髓中提取并分离出原代细胞,经50 ng/ml M-CSF诱导7 d后得到BMDM.分为对照组(Control)、模型组(LPS+ATP)、FFYJ低剂量组(FFYJ 50 µg/ml)、FFYJ中剂量组(FFYJ 100 µg/ml)、FFYJ高剂量组(FFYJ 200 µg/ml)、阳性药地塞米松组(DEX).FFYJ治疗组和阳性药组预处理BMDM 1 h后开始造模.乳酸脱氢酶(LDH)释放法检测各组细胞上清中LDH的含量;ELISA检测各组细胞上清中炎症因子肿瘤坏死因子-α(TNF-α)、IL-6和IL-1β的含量;qRT-PCR检测各组细胞中TNF-α、IL-6和IL-1β mRNA水平;Western blot检测各组细胞中NF-κB、p-NF-κB、NLRP3、Caspase-1 p45、Caspase-1 p20和GSDMD-N 蛋白水平;倒置荧光显微镜观察各组细胞经Hoechst-PI染色后的细胞焦亡情况.结果:与对照组相比,模型组细胞上清液中LDH、TNF-α、IL-6和IL-1β含量显著升高(P<0.000 1);模型组细胞TNF-α、IL-6和IL-1β的mRNA水平显著升高(P<0.000 1);p-NF-κB、NLRP3、Caspase-1 p20和GSDMD-N的蛋白水平显著升高(P<0.05);PI阳性细胞数明显增多(P<0.05).与模型组比较,FFYJ治疗和阳性药显著降低了BMDMs上清液中LDH、TNF-α、IL-6和IL-1β的含量(P<0.05);下调了细胞中TNF-α、IL-6和IL-1β的mRNA水平(P<0.05);抑制了p-NF-κB、NLRP3、Caspase-1 p20和GSDMD-N蛋白的表达(P<0.05);显著减少了PI阳性细胞数(P<0.05).结论:FFYJ通过抑制BMDM原代巨噬细胞中NLRP3炎症小体的激活发挥抗炎效果.
Objective:To investigate the effect of Fu-Fang-Yu-Jie(FFYJ)granules on LPS-induced inflammation model in bone marrow-derived macrophages(BMDM)and its intervention of FFYJ on nucleotide-bound oligomeric domain-like receptor protein 3(NLRP3)inflammatory signaling pathway in macrophages.Methods:Primary cells were extracted and isolated from the leg bone mar-row of C57BL/6 mice and BMDM macrophages were obtained after 7 days of induction with 50 ng/ml M-CSF.Groups included control group(Control),model group(LPS+ATP),FFYJ low dose group(FFYJ 50 µg/ml),FFYJ medium dose group(FFYJ 100 µg/ml),FFYJ high dose group(FFYJ 200 µg/ml)and positive drug dexamethasone group(DEX).BMDM in FFYJ treatment group and posi-tive drug group were pretreated for 1 hour before modeling.Lactate dehydrogenase(LDH)release assay was used to detect the level of LDH in the supernatant of each group of cells;ELISA was used to detect the level of inflammatory factors TNF-α,IL-6 and IL-1β in the supernatant of each group of cells;qRT-PCR was used to detect the levels of TNF-α,IL-6 and IL-1β in each group of cells;protein levels of NF-κB,p-NF-κB,NLRP3,Caspase-1 p45,Caspase-1 p20 and GSDMD-N in each group of cells were detected by Western blot;inverted fluorescence microscope was used to observe the cell pyroptosis of each group after Hoechst-PI staining.Results:Compared with control group,the levels of LDH,TNF-α,IL-6 and IL-1β in the supernatant of the model group were signifi-cantly higher(P<0.000 1);the mRNA levels of TNF-α,IL-6 and IL-1β in the model group were significantly higher(P<0.000 1);the protein levels of p-NF-κB,NLRP3,Caspase-1 p20 and GSDMD-N were significantly higher(P<0.05);and the number of PI-posi-tive cells was significantly higher(P<0.05).Compared with the model group,FFYJ and DEX significantly reduced the levels of LDH,TNF-α,IL-6 and IL-1β in the supernatant of BMDMs(P<0.05);down-regulated the mRNA levels of TNF-α,IL-6 and IL-1β in the cells(P<0.05);and inhibited the expressions of p-NF-κB,NLRP3,Caspase-1 p20 and GSDMD-N protein expressions(P<0.05);and significantly reduced the number of PI-positive cells(P<0.05).Conclusion:FFYJ exerts anti-inflammatory effects by inhibiting NLRP3 inflammasome activation in BMDM macrophages.
马宇慧;孟佳磊;袁林;齐璐瑶;欧阳冰清;雷鸣
上海中医药大学附属第七人民医院,上海 200137上海中医药大学附属第七人民医院,上海 200137||急救与创伤研究教育部重点实验室,急诊创伤学院,海南医学院,海口 571199
中医学
复方鱼桔颗粒NLRP3炎症小体BMDM炎症因子
Fu-Fang-Yu-Jie granulesNLRP3 inflammasomeBMDMInflammatory factor
《中国免疫学杂志》 2024 (007)
1467-1473 / 7
上海市浦东新区重点亚专科建设(PWzy2020-07);上海市浦东新区中医高端人才引进工程(PDZY-2022-0701);浦东新区卫生健康委员会学科建设计划项目(PWZxq2022-09).
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