二氢杨梅素对LPS/ATP诱导血管内皮细胞NLRP3炎症小体活化的影响及机制  

Effect and mechanism of dihydromyricetin on LPS/ATP-induced activation of NLRP3 inflammasome in vascular endothelial cells

作　　者：乔秀梅 厉彦翔 薛彩彩 陈心茹 袁浩铭 王金红 QIAO Xiumei;LI Yanxiang;XUE Caicai;CHEN Xinru;YUAN Haoming;WANG Jinhong(School of Pharmacy,Shandong Second Medical University,Weifang 261053,China)

机构地区：[1]山东第二医科大学药学院,山东潍坊261053

出　　处：《山东医药》2024年第7期17-21,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(82100489);山东省自然科学基金资助项目(ZR2021QH095);潍坊医学院公派国内访学项目(20227-07)。

摘　　要：目的探讨二氢杨梅素(DHM)对脂多糖(LPS)/三磷酸腺苷(ATP)诱导的血管内皮细胞核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体活化的影响。方法体外培养人脐静脉内皮细胞,将细胞随机分为空白对照组、LPS/ATP组、LPS/ATP+25μmol/L DHM组、LPS/ATP+50μmol/L DHM组和LPS/ATP+100μmol/L DHM组。除空白对照组外,其他各组经LPS(0.5μg/mL)诱导细胞3.5 h后加入ATP(5 mmol/L)处理细胞30 min;DHM各组分别加入25、50、100μmol/L DHM孵育1 h后加入LPS/ATP诱导。用Western blotting法检测NLRP3、ASC、半胱氨酸天冬氨酸特异性蛋白酶1前体蛋白(pro-caspase-1)、剪切体半胱氨酸天冬氨酸特异性蛋白酶1(cleaved caspase-1)、沉默信息调节因子1(SIRT1)蛋白;用基因本体论(GO)、京都基因与基因组百科全书(KEGG)对SIRT1进行富集分析,用转录调控网络分析SIRT1调控的靶基因并构建网络图;用免疫荧光法进行SIRT1定位检测;分子对接分析DHM与SIRT1的作用模式。结果与空白对照组比较,LPS/ATP组NLRP3、ASC、cleaved caspase-1蛋白表达高(P均<0.05);与LPS/ATP组比较,各DHM组NLRP3、ASC、cleaved caspase-1蛋白表达低(P均<0.05)。SIRT1主要分布于细胞核内。与空白对照组比较,LPS/ATP组相对荧光强度弱(P<0.05);与LPS/ATP组比较,LPS/ATP+100μmol/L DHM组相对荧光强度强(P<0.05)。与空白对照组比较,LPS/ATP组SIRT1蛋白表达低(P<0.05);与LPS/ATP组、LPS/ATP+25μmol/L DHM组比较,LPS/ATP+50μmol/L DHM组、LPS/ATP+100μmol/L DHM组SIRT1蛋白表达高(P均<0.05)。GO富集分析结果:生物过程富集分析结果显示与对营养水平的反应等生物过程联系密切;细胞组分富集分析结果显示与染色质沉默复合体等密切相关;分子功能富集分析结果显示与染色质DNA结合等分子功能有关。KEGG通路富集分析结果:SIRT1涉及长寿调节、脂质与动脉粥样硬化等信号通路。DHM可有效嵌入SIRT1(受体生物大分子)的“活性口袋”。结论DHM可抑�Objective To investigate the effect of dihydromyricetin(DHM)on the activation of NLRP3 inflammasome induced by lipopolysaccharides(LPS)/adenosine triphosphate(ATP)in vascular endothelial cells.Methods Human umbilical vein endothelial cells(HUVECs)were cultured and randomly divided into the control group,LPS/ATP group,LPS/ATP+25μmol/L DHM group,LPS/ATP+50μmol/L DHM group and LPS/ATP+100μmol/L DHM group,respectively.Except the control group,HUVECs in the other groups were treated with LPS(0.5μg/mL)for 3.5 h and ATP(5 mmol/L)for 30 min.HUVECs in the DHM groups were pretreated with DHM(25,50,and 100μmol/L)for 1 h before stimulation with LPS/ATP.The expression levels of NLRP3,ASC,pro-caspase-1,cleaved caspase-1 and silent information regulator-1(SIRT1)were determined by Western blotting.Pathway enrichment analysis of SIRT1 was performed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG),target genes regulated by SIRT1 were analyzed and a network diagram was constructed using transcriptional regulatory network analysis;SIRT1 localization was detected by immunofluorescence;and the interaction mode between DHM and SIRT1 was analyzed by molecular docking.Results Compared with the blank control group,the expression levels of NLRP3,ASC,and cleaved caspase-1 protein were higher in the LPS/ATP group(all P<0.05);compared with the LPS/ATP group,the expression levels of NLRP3,ASC,and cleaved caspase-1 protein were lower in all DHM groups(all P<0.05).SIRT1 was mainly distributed in the nucleus of cells.Compared with the blank control group,the relative fluorescence intensity was weaker in the LPS/ATP group(P<0.05);compared with the LPS/ATP group,the relative fluorescence intensity was stronger in the LPS/ATP+100μmol/L DHM group(P<0.05).The LPS/ATP group had significantly lower expression of SIRT1 protein than the blank control group(P<0.05);compared with the LPS/ATP group and the LPS/ATP+25μmol/L DHM group,SIRT1 protein expression levels were higher in the LPS/ATP+50μmol/L DHM group and LPS/ATP+100μ

关 键 词：二氢杨梅素 动脉粥样硬化 核苷酸结合寡聚化结构域样受体蛋白3炎症小体 血管内皮细胞 沉默信息调节因子1 

分 类 号：R972.6[医药卫生—药品]