酸敏感离子通道1a偶联内质网应激在人髓核细胞退变中的作用机制初探

Acid-sensing ion channel 1a mediated acid-induced endoplasmic reticulum stress in apoptosis of nucleus pulposus cells

[目的]模拟人椎间盘髓核(nucleus pulposus,NP)酸性环境,探讨酸敏感离子通道1a(acid-sensing ion channel 1a,ASIC1a)的活化与内质网应激的关系。[方法]体外单层培养人正常髓核细胞(nucleus pulposus cells,NPCs)系,不同p H值培养不同时间,模拟椎间盘酸性微环境,建立酸诱导的退变髓核细胞模型。CCK-8检测细胞增殖能力。Western blot、q PCR检测内质网应激。Western blot检测ASIC1a的表达。Fura-2/AM荧光探针检测ASIC1a活化介导的Ca2+内流。流式细胞术检测ASIC1a活化后细胞凋亡率。Pc TX1(ASIC1a特异性阻断剂)阻断ASIC1a后,观察细胞凋亡及内质网应激指标变化情况。[结果]酸诱导髓核细胞凋亡,酸激活ASIC1a及内质网应激,Pc TX1能降低促凋亡的内质网应激通路和髓核细胞凋亡率(P<0.05),而未折叠蛋白反应相关指标无明显变化(P>0.05)。[结论]ASIC1a能够调控内质网应激中的促凋亡通路,阻断ASIC1能够保护酸诱导的髓核细胞凋亡。

[Objective] To mimic human acidic intervertebral disk environment, and investigate the relationship between acid-sensing ion channel 1 a（ASIC1 a） and endoplasmic reticulum（ER） stress. [Methods] Normal human nucleus pulposus cells（NPCs） were incubated in a monolayer culture with different pH to imitate acidic environment of nucleus pulposus. The proliferation of NPCs was assessed by the CCK-8 assay, while cell apoptosis rate was examined by annexin V/propidium iodide（PI）staining and quantified by flow cytometry. In addition, the ASIC1 a-mediated intracellular calcium was determined by Ca^2＋-imaging by Fura-2/AM, the expression of ASIC1 a in acid stimulus was examined by western blotting. Acidity-induced changes in ER stress markers were studied using Western blotting and q PCR. Pc TX1 was used to block ASIC1 a. [Results] Acid stimulus increased intracellular calcium levels and cell apoptosis which could be reduced by Pc TX1. Acid-induced ER stress was partly inhibited by Pc TX1, especially the pro-apoptotic markers（P〉0.01）. [Conclusion] By regulating ER stress, ASIC1 a promotes apoptosis of NPCs under acid stimulus. Blocking of ASIC1 a attenuates acid-induced apoptosis of NPCs.