摘要
目的 探讨生骨再造丸对激素诱导骨髓间充质干细胞(BMSCs)焦亡的保护作用机制以及对微管调控NLRP3炎性小体组装的影响.方法 将BMSCs分为空白组、模型组、模型组+紫杉醇组、模型组+诺考达唑组、模型组+生骨再造丸含药血清组.制备生骨再造丸含药血清干预BMSCs.使用CCK8检测细胞活力;流式细胞术检测凋亡率;WB检测AC-α-tubulin、NLRP3、Caspase-1、GSDMD蛋白表达;免疫荧光检测ASC炎性大斑点表达,α-tubulin与NLRP3共定位表达;ELISA检测IL-ip和IL-18含量.结果 与空白组相比,模型组细胞活力下降、凋亡水平上升,AC-α-tubulin、NLRP3、Caspase-1、GSDMD蛋白的表达上升,ASC炎性斑点的荧光强度上升,α-tubulin与NLRP3共定位的荧光强度上升(P＜0.01),IL-1β和IL-18含量明显增加(P＜0.01);与模型组相比,紫杉醇组细胞活力下降、凋亡水平上升、AC-α-tubulin、NLRP3、Caspase-1、GSDMD蛋白的表达上升(P＜0.01),ASC炎性斑点的荧光强度上升(P＜0.05),α-tubulin与NLRP3共定位的荧光强度上升(P＜0.01),IL-1β和IL-18含量增加(P＜0.01,P＜0.05);诺考达唑和生骨再造丸含药血清组的细胞活力上升、凋亡水平下降,AC-α-tubulin、NLRP3、Caspase-1、GSDMD蛋白的表达下降(P＜0.01)、ASC炎性斑点的荧光强度下降(P＜0.05),α-tubulin与NLRP3共定位的荧光强度下降(P＜0.01,P＜0.05),IL-1β和IL-18含量减少(P＜0.01,P＜0.05).结论 生骨再造丸能抑制α-tubulin乙酰化,破坏微管结构,抑制NLRP3炎性小体的组装,从而减少BMSCs细胞焦亡.
Abstract
Objective To investigate the protective mechanism of the bone regeneration pills(BRP)against hormone-induced pyroptosis of bone marrow mesenchymal stem cells(BMSCs)and the impact on microtubule-mediated NLRP3 inflammasome assembly.Methods BMSCs were divided into blank group,model group,model group+paclitaxel group,model group+nocardazole group,and model group+BRP serum group.Serum intervention BMSCs containing BRP were prepared.Cell viability was assessed using CCK-8 assay,apoptosis rate was determined by flow cytometry,and WB was used to detect the expressions of AC-α-tubulin,NLRP3,Caspase-1 and GSDMD.The expression of ASC in cells and co-localized expression of α-tubulin and NLRP3 were detected with immunofluorescence.ELISA was used to detect the levels of IL-1 β and IL-18.Results Compared to the blank group,the model group exhibited reduced cell viability,increased apoptosis levels,upregulated expression of AC-α-tubulin,NLRP3,Caspase-1,and GSDMD proteins,increased fluorescence intensity of ASC inflammatory specks,and enhanced co-localization of α-tubulin and NLRP3(P＜0.01).In comparison to the model group,the paclitaxel group displayed decreased cell viability,increased apoptosis levels,upregulated expression of AC-α-tubulin,NLRP3,Caspase-1 and GSDMD(P＜0.01),increased fluorescence intensity of ASC inflammatory specks(P＜0.05),and enhanced co-localization of α-tubulin and NLRP3(P＜0.01).IL-1β and IL-18 contents increased(P＜0.01,P＜0.05).Both the nocardazole and BRP serum groups showed increased cell viability,decreased apoptosis levels,downregulated expressions of AC-α-tubulin,NLRP3,Caspase-1,and GSDMD proteins(P＜0.01),decreased fluorescence intensity of ASC inflammatory specks(P＜0.05),and reduced co-localization of α-tubulin and NLRP3(P＜0.01).IL-1β and IL-18 contents decreased(P＜0.01,P＜0.05).Conclusion BRP inhibits α-tubulin acetylation,disrupts microtubule structure,suppresses NLRP3 inflammasome assembly,and reduces BMSC pyroptosis.
基金项目
国家自然科学基金(8216091581860859)
兰州市科技计划(2022-3-23)
甘肃中医药大学创新基金(2023CX06)
NETL
NSTL
万方数据