| 酸敏感离子通道1通过细胞外信号调节激酶5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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Received:March 21, 2024
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| 作者 | Author | 单位 | Unit | E-Mail |
| 罗贤芳 |
LUO Xian-fang |
横店文荣医院骨科, 浙江 金华 321000 |
Department of Orthopaedics, Hengdian Wenrong Hospital, Jinhua 321000, Zhejiang, China |
jhluoxianfang@163.com |
| 金正跃 |
JIN Zheng-yue |
横店文荣医院骨科, 浙江 金华 321000 |
Department of Orthopaedics, Hengdian Wenrong Hospital, Jinhua 321000, Zhejiang, China |
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| 张弛 |
ZHANG Chi |
金华市中心医院骨科, 浙江 金华 321000 |
Department of Orthopaedics, Jinhua Central Municipal Hospital, Jinhua 321000, Zhejiang, China |
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| 期刊信息：《中国骨伤》2025年38卷，第3期，第298-305页 |
| DOI：10.12200/j.issn.1003-0034.20230722 |
| 基金项目:浙江省医药卫生科技项目(编号:2020KY343) |
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目的: 探讨酸敏感离子通道1(acid-sensing ion channel 1,ASIC1)通过细胞外信号调节激酶5(extracellular-signalregulated protein kinase 5,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: 自2020年1月至2022年12月收治34例退行性腰椎间盘突出症患者作为研究对象,男21例,女13例;年龄29~52(37.43±4.75)岁;按照Pfirrmann腰椎间盘退变分级标准,Ⅱ级22例,Ⅳ级12例;L4,5 15例,L5S1 19例。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表达水平。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,每组3例。干预72 h后收集各组细胞,采用反转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测ASIC1,ERK5及B淋巴细胞瘤-2基因相关启动子(BCL-xL/BCL-2-associated death promoter,Bad),B淋巴细胞瘤-2基因相关X蛋白(B-cell lymphoma-2 Associated X,Bax)和抗凋亡因子B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)的表达,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: 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,免疫组织化学结果显示ASIC1表达升高。siRNA沉默组ASIC1的mRNA相对表达量(0.31±0.03)与抑制剂组(0.39±0.05)比较,差异无统计学意义(P>0.05);siRNA沉默组ERK5的mRNA相对表达量(0.32±0.05)与抑制剂组(0.15±0.04)比较,差异有统计学意义(P<0.05),提示ERK5为ASIC1的下游分子。siRNA沉默组和抑制剂组凋亡促进因子Bad,Bax的mRNA相对表达量低于过表达组,差异有统计学意义(P<0.05),抗凋亡因子Bcl-2的mRNA的相对表达量明显升高,差异有统计学意义(P<0.05);过表达组细胞中钙离子含量高于siRNA沉默组和抑制剂组,差异有统计学意义(P<0.05),过表达组细胞中线粒体膜电位正常比例低于siRNA沉默组和抑制剂组,差异有统计学意义(P<0.05),过表达组细胞凋亡率高于siRNA沉默组和抑制剂组,差异有统计学意义(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。 |
| [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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| Study on the mechanism of apoptosis mediated by acid sensitive ion channel 1 through extracellular signal regulation of kinase 5 signaling pathway and mitochondrial disorder pathway |
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Abstract:
Objective To explore mechanisms of acid-sensing ion channel 1 (ASIC1) mediated lumbar nucleus pulposus cell apoptosis through extracellular-signalregulated protein kinase 5 (ERK5) signaling pathway and mitochondrial dysfunction pathway.
Methods Totally 34 patients with degenerative lumbar disc herniation (LDH) admitted from January 2020 to December 2022 were collected as research objects,including 21 males and 13 females;aged from 29 to 52 years old with an average of (37.43±4.75) years old;22 patients with grade Ⅱ and 12 patients with grade Ⅳ,according to Pfirrmann grading criteria;15 patients with L4,5 and 19 patients with L5S1. The expression of ASIC1 in nucleus pulposus of LDH patients was measured by immunohistochemical staining. Nucleus pulposus cells were cultured by primary culture method,identified by toluidine blue staining and immunohistochemical staining,and the expression of ASIC1 protein was located by immunofluorescence staining. According to the addition of siRNA-ASIC1,ASIC1 overexpression plasmid,and ERK5 inhibitors,the nucleus pulpocyte was divided into three groups,named as SIRNA-silenced group,overexpression group,and inhibitor group,with 3 patients in each group. Cells of each group were collected at 72 h after intervention,expression of ASIC1,ERK5,BCL-xL/BCL-2-associated Death promoter (Bad),B-cell lymphoma-2 associated X (Bax) and B-cell lymphoblast-2 gene (Bcl-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR);intracellular calcium ion levels were detected by calcium ion kit,mitochondrial membrane potential was detected by JC-1 kit,and apoptosis was observed by AV-PI kit.
Results In LDH patients with grade Ⅳ,nucleus pulposus tissue removed during operation revealed poor elasticity,white color and poor ductility,and immunohistochemical results showed increased ASIC1 expression. There was no significant difference in mRNA relative expression of ASIC1 between siRNA silencing group (0.31±0.03) and inhibitor group (0.39±0.05) (P>0.05). The mRNA relative expression level of ERK5 in siRNA silencing group(0.32±0.05) was significantly higher than that in inhibitor group (0.15±0.04)(P<0.05),which suggested ERK5 was the downstream molecule of ASIC1. The mRNA relative expression levels of apoptosis promoting factor Bad and Bax in siRNA silencing group and inhibitor group were lower than those in overexpression group(P<0.05),the relative expression level of anti-apoptosis factor Bcl-2 mRNA was significantly increased (P<0.05). The calcium content in overexpression group was higher than that in siRNA silencing and inhibitor groups (P<0.05),the normal proportion of mitochondrial membrane potential in overexpression group was lower than that in siRNA silencing and inhibitor group (P<0.05),and the apoptosis rate in overexpression group was higher than that in siRNA silencing and inhibitor group (P<0.05).
Conclusion After the activation of ASIC1 channel protein,calcium ions could enter the cells and act as a second messenger molecule to regulate apoptosis of nucleus pulposus cells by ERK5 signaling pathway and mitochondrial disorder pathway. |
| KEYWORDS:Lumbar disc herniation Nucleus pulposus cells Acid-sensing ion channel 1 Apoptosis |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 罗贤芳,金正跃,张弛.酸敏感离子通道1通过细胞外信号调节激酶5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[J].中国骨伤,2025,38(3):298~305 |
| 英文格式: | LUO Xian-fang,JIN Zheng-yue,ZHANG Chi.Study on the mechanism of apoptosis mediated by acid sensitive ion channel 1 through extracellular signal regulation of kinase 5 signaling pathway and mitochondrial disorder pathway[J].zhongguo gu shang / China J Orthop Trauma ,2025,38(3):298~305 |
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