| 酸敏感离子通道1(ASIC1)通过ERK5信号通路和线粒体紊乱途径介导髓核细胞凋亡的相关机制研究 |
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投稿时间：2023-07-18 修订日期：2024-03-20
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| 期刊信息：《中国骨伤》年，第卷，第期，第-页 |
| DOI： |
| 基金项目:浙江省医药卫生科技项目(2020KY343) |
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| 中文摘要:目的 探究酸敏感离子通道1(ASIC1)通过ERK5信号通路和线粒体紊乱途径介导髓核细胞凋亡的相关机制。 方法 收集自2020年1月至2022年12月收治我院脊柱外科的34例退行性腰椎间盘突出症患者作为研究对象,取术中获得的LDH患者髓核组织进行免疫组织化学染色,检测ASIC1的表达。通过原代培养法,培养髓核细胞,通过甲苯胺蓝染色和免疫组织化学染色法进行鉴定,免疫荧光染色定位ASIC1蛋白表达。根据是否加入siRNA-ASIC1,ASIC1过表达质粒,以及ERK5的抑制剂对髓核细胞进行分组,即siRNA沉默组,过表达组和抑制剂组。利用RT-PCR检测ASIC1,ERK5,以及凋亡相关因子Bad,Bax和抗凋亡因子Bcl-2的表达。利用钙离子试剂盒检测细胞内钙离子水平,JC-1试剂盒检测细胞线粒体膜电位,AV-PI试剂盒检测细胞的凋亡。 结果 Grade IV级LDH患者术中取出的髓核组织弹性较差,呈白色,延展性差,ASIC1免疫组织化学染色较强。免疫荧光染色结果显示,ASIC 1蛋白表达于髓核细胞的细胞质中。siRNA沉默组中ASIC1表达量明显降低(P<0.05),ERK5的表达量明显降低(P<0.05),但是在抑制剂组中,ASIC1表达量未见明显降低(P>0.05),ERK5表达量明显较低(P<0.05),认为ERK5作为ASIC1的下游信号分子。siRNA沉默组和抑制剂组的凋亡促进因子Bad,Bax的mRNA的相对表达量明显降低(P<0.05),过表达组Bad,Bax的mRNA的相对表达量明显升高(P<0.05)。siRNA沉默组和抑制剂组的抗凋亡因子Bcl-2的mRNA的相对表达量明显升高(P<0.05),过表达组的抗凋亡因子Bcl-2的mRNA的相对表达量明显降低(P<0.05)。过表达组细胞中钙离子的含量明显高于siRNA沉默组和抑制剂组(P<0.05),过表达组细胞中线粒体膜电位正常比例明显低于siRNA沉默组和抑制剂组(P<0.05),过表达组细胞的细胞凋亡率明显高于siRNA沉默组和抑制剂组(P<0.05)。 结论 ASIC1通道蛋白激活后,钙离子进入细胞,作为第二信使分子,通过ERK5信号通路和线粒体紊乱途径,调控髓核细胞的凋亡,促进LDH的进展。 |
| 【关键词】腰椎间盘突出症 髓核细胞 酸敏感离子通道1 凋亡 |
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| The research about the mechanism of Acid Sensitive Ion channel 1 (ASIC1) mediating Apoptosis of Nucleus Pulposus Cells through ERK5 Signal Pathway and Mitochondrial Disorder Pathway Luo Xianfang , JIN Zhengyue , ZHANG chi . Department of The bone surgery Jinhua Dongyang Hengdian Wenrong Hospital |
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| ABSTRACT Objective: To explore the mechanisms of acid-sensing ion channel 1 (ASIC1) mediated lumbar nucleus pulposus cell apoptosis through the ERK5 signaling pathway and mitochondrial dysfunction pathway. Methods: A total of 34 patients with degenerative lumbar disc herniation (LDH) treated in our hospital from January 2020 to December 2022 were enrolled as the research subjects. Lumbar nucleus pulposus tissue obtained intraoperatively was used for immunohistochemical staining to detect the expression of ASIC1. Primary cultured lumbar nucleus pulposus cells were identified by toluidine blue staining and immunohistochemical staining, and the localization of ASIC1 protein expression was determined by immunofluorescence staining. The lumbar nucleus pulposus cells were divided into groups according to whether siRNA-ASIC1, ASIC1 overexpression plasmid, or ERK5 inhibitor was added. The expression of ASIC1, ERK5, and apoptosis-related factors Bad, Bax, and anti-apoptotic factor Bcl-2 were detected by RT-PCR. Intracellular calcium levels were measured using a calcium ion assay kit, mitochondrial membrane potential was assessed using the JC-1 assay kit, and cell apoptosis was measured using the AV-PI assay kit. Results: The lumbar nucleus pulposus tissue taken from Grade IV LDH patients during surgery exhibited poor elasticity, white color, and poor extensibility. Immunohistochemical staining showed strong ASIC1 expression. Immunofluorescence staining revealed that ASIC1 protein was expressed in the cytoplasm of lumbar nucleus pulposus cells. The siRNA silence group showed a significant decrease in ASIC1 expression (P<0.05), as well as a significant decrease in ERK5 expression (P<0.05). However, in the inhibitor group, ASIC1 expression did not show a significant decrease (P>0.05), while ERK5 expression was significantly lower (P<0.05), indicating that ERK5 acts as a downstream signaling molecule of ASIC1. The relative expression levels of the apoptosis-promoting factors Bad and Bax mRNA were significantly decreased in the siRNA silence group and the inhibitor group (P<0.05), while in the overexpression group, the relative expression levels of Bad and Bax mRNA were significantly increased (P<0.05). The relative expression level of the anti-apoptotic factor Bcl-2 mRNA was significantly increased in the siRNA silence group and the inhibitor group (P <0.05), while in the overexpression group, the relative expression level of Bcl-2 mRNA was significantly decreased (P <0.05). The calcium content in the cells of the overexpression group was significantly higher than that in the siRNA silence group and the inhibitor group (P <0.05). The proportion of normal mitochondrial membrane potential in the cells of the overexpression group was significantly lower than that in the siRNA silence group and the inhibitor group (P <0.05). The cell apoptosis rate in the overexpression group was significantly higher than that in the siRNA silence group and the inhibitor group (P <0.05). Conclusion: Activation of ASIC1 channel protein leads to calcium entry into cells as a second messenger, regulating lumbar nucleus pulposus cell apoptosis through the ERK5 signaling pathway and mitochondrial dysfunction pathway, thereby promoting the progression of LDH. |
| KEY WORDS Lumbar disc herniation Nucleus pulposus cells Acid-sensing ion channel 1 Apoptosis. |
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