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| 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 |
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| 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） |
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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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