摘要
目的 探讨异常流体剪切力诱导髓核细胞(NPC)凋亡、炎症和自噬的作用及其可能的机制.方法 对NPC 加载24 dyn/cm2流体剪切力,时间分别为 0、60、90、120、150 min.使用细胞骨架染色研究流体剪切力对细胞骨架及细胞形态的影响;使用 5-乙炔基-2'-脱氧尿苷染色验证NPC的增殖能力;用线粒体膜电位变化评估流体剪切力对线粒体的损伤效应;用赫斯特染色研究凋亡与流体剪切力的关系;用丹酰戊二胺染色研究自噬与流体剪切力的关系;探究细胞培养基酸碱度的变化,验证流体剪切力与炎症的关联;使用蛋白质印迹法研究 cGAS-STING 相关蛋白(cGAS、STING、TBK1)、炎症相关蛋白(肿瘤坏死因子-α、COX-2)、凋亡相关蛋白(Bcl-2、Bax)以及自噬蛋白(IL3-Ⅰ/Ⅱ、p62)的变化.结果 作为一种细胞间的机械刺激,24 dyn/cm2强度的流体剪切力可造成 NPC 形态由梭形转变为多边形,细胞骨架发生重组,细胞增殖能力下降,培养基酸性增强,线粒体JC-1多聚体减少、单体增加;同时,细胞内肿瘤坏死因子-α、COX-2、Bax蛋白表达量上调,LC3 Ⅱ/LC3Ⅰ增加,p62、Bcl-2降解增加,出现凋亡、炎症、自噬以及线粒体损伤;加载 24 dyn/cm2的流体剪切力可以激活 NPC 内的 cGAS-STING 信号通路,且与时间呈正相关关系,120 min 时强度最高.使用 cGAS 抑制剂 RU.521 可以减缓24 dyn/cm2流体剪切力造成的 NPC 的凋亡、炎症、自噬以及线粒体损伤.结论 流体剪切力诱导的 NPC 凋亡、炎症和自噬与cGAS-STING 信号通路激活相关,抑制 cGAS-STING 信号通路的激活可以缓解异常流体剪切力造成的细胞损害.
Abstract
Objective To investigate the effects of the abnormal fluid shear stress(FSS)on apoptosis,inflam-mation and autophagy in nucleus pulposus cells(NPC)and its possible mechanisms.Methods NPC were load-ed with 24 dyn/cm2 FSS for 0,60,90,120 and 150 min respectively.Cytoskeleton staining was used to study the effect of FSS on cytoskeleton and cell morphology.5-acetylene-2'-deoxyuridine staining was used to veri-fy the proliferation ability of NPC;JC-1 mitochondrial membrane potential kit used to study the mitochondrial damage induced by FSS;Hoechst 33342 staining used to study the relationship between apoptosis and FSS,and monodansylcadaverine staining used to study the relationship between autophagy and FSS.The chang-es of pH in cell culture medium were explored to verify the relationship between FSS and inflammation.Western blotting was used to study the expression levels of cGAS-STING related proteins(cGAS,STING,TBK1),inflammation-related proteins(TNF-α,COX-2),apoptosis-related proteins(Bcl-2,Bax)and autopha-gy proteins(IL3-Ⅰ/Ⅱ,p62).Results As an intercellular mechanical stimulus,the 24 dyn/cm2 FSS could cause the morphology of NPC to change from a spindle shape to polygon shape,and cause the reorganization of cytoskeleton,decrease in cell proliferation ability,increase in the acidity of the medium,decrease of mitochondrial JC-1 multimers and increase of mitochondrial monomers.At the same time,the expressions of TNF-α,COX-2 and Bax proteins in the cells were up-regulated,the LC3Ⅱ/LC3Ⅰratio increased,the expres-sions of p62 and Bcl-2 decreased,and apoptosis,inflammation,autophagy and mitochondrial damage were observed.In addition,24 dyn/cm2 FSS could activate the cGAS-STING signaling pathway in NPC in a time-dependent manner,with the highest intensity at 120 min and treatment with the cGAS inhibitor RU.521 could alleviate the apoptosis,inflammation,autophagy and mitochondrial damage of NPC caused by 24 dyn/cm2 FSS.Conclusion FSS-induced apoptosis,inflammation and autophagy of NPC were mediated by the activa-tion of cGAS-STING signaling pathway.Inhibition of cGAS-STING signaling pathway could alleviate the cellular damage caused by the abnormal FSS.
基金项目
甘肃省自然科学基金(22JR11RA082)
兰州大学第二医院"萃英科技创新"计划(CY2022-ZD-02)
兰州大学第二医院"萃英科技创新"计划(CY2021-QN-A06)
国家科技期刊平台
NSTL
万方数据