微管调控NLRP3转运探讨生骨再造丸对BMSCs焦亡水平的影响
Investigation of the pyroptosis level of bone marrow mesenchymal stem cells treated with the bone regeneration pill based on microtubule regulation of NLRP3 transport
  
DOI:10.3969/j.issn.1006-7108.2024.05.003
中文关键词:  骨髓间充质干细胞  焦亡  NLRP3  微管  生骨再造丸
英文关键词:bone marrow mesenchymal stem cells  pyroptosis  NLRP3  microtubule  the bone regeneration pill
基金项目:国家自然科学基金(82160915;81860859) ;兰州市科技计划项目(2022-3-23);甘肃中医药大学创新基金(2023CX06)
作者单位
胡康一1 曹林忠1,2 * 1.甘肃中医药大学甘肃 兰州 730000 2.甘肃中医药大学附属医院甘肃 兰州 730000 
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中文摘要:
      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-1β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。
英文摘要:
      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-1β 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.
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