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
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摘要
[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 p H 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 Ca2+-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.
[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 p H 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 Ca2+-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.
引文
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[14]Cai F,Wang F,Hong X,et al.Acid-sensing ion channel 1a regulates the survival of nucleus pulposus cells in the acidic environment of degenerated intervertebral discs[J].Iran J Basic Med Sci,2016,19(8):812-820.
[15]Krebs J,Agellon LB,Michalak M.Ca(2+)homeostasis and endoplasmic reticulum(ER)stress:An integrated view of calcium signaling[J].Biochem Biophys Res Commun,2015,460(1):114-121.
[16]Yuan FL,Chen FH,Lu WG,et al.Acid-sensing ion channel 1a mediates acid-induced increases in intracellular calcium in rat articular chondrocytes[J].Mol Cell Biochem,2010,340(1-2):153-159.
[17]Navone SE,Marfia G,Canzi L,et al.Expression of neural and neurotrophic markers in nucleus pulposus cells isolated from degenerated intervertebral disc[J].J Orthop Res,2012,30(9):1470-1477.
[2]王锋,吴小涛,王运涛,等.干细胞移植修复椎间盘退变的研究现状[J].中国修复重建外科杂志,2013,27(5):575-579.
[3]王锋,吴小涛,王运涛,等.非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究[J].中国修复重建外科杂志,2010,24(4):19-26.
[4]王锋,郑陈静美,吴小涛.细胞老化及老化表型改变在椎间盘退行性变中的研究进展[J].中国修复重建外科杂志,2012,26(12):1448-1452.
[5]康新桂,吴小涛,王锋,等.人老化椎间盘髓核细胞体外老化模型的建立与细胞老化表型的初步研究[J].中国矫形外科杂志,2017,25(1):64-72.
[6]Ohshima H,Urban JP.The effect of lactate and p H on proteoglycan and protein synthesis rates in the intervertebral disc[J].Spine(Phila Pa 1976),1992,17(9):1079-1082.
[7]Liang CZ,Li H,Tao YQ,et al.The relationship between low p H in intervertebral discs and low back pain:a systematic review[J].Arch Med Sci,2012,8(6):952-956.
[8]Berridge MJ.The endoplasmic reticulum:a multifunctional signaling organelle[J].Cell Calcium,2002,32(5-6):235-249.
[9]Marchi S,Patergnani S,Pinton P.The endoplasmic reticulum-mitochondria connection:one touch,multiple functions[J].Biochim Biophys Acta,2014,1837(4):461-469.
[10]Oakes SA,Papa FR.The role of endoplasmic reticulum stress in human pathology[J].Annu Rev Pathol,2015,10:173-194.
[11]Xie ZY,Chen L,Wang F,et al.Endoplasmic reticulum stress is involved in nucleus pulposus degeneration and attenuates low ph-induced apoptosis of rat nucleus pulposus cells[J].DNA Cell Biol,2017,36(8):627-637.
[12]Cuesta A,Del Valle ME,Garcia-Suarez O,et al.Acid-sensing ion channels in healthy and degenerated human intervertebral disc[J].Connect Tissue Res,2014,55(3):197-204.
[13]Sherwood TW,Frey EN,Askwith CC.Structure and activity of the acid-sensing ion channels[J].Am J Physiol Cell Physiol,2012,303(7):699-710.
[14]Cai F,Wang F,Hong X,et al.Acid-sensing ion channel 1a regulates the survival of nucleus pulposus cells in the acidic environment of degenerated intervertebral discs[J].Iran J Basic Med Sci,2016,19(8):812-820.
[15]Krebs J,Agellon LB,Michalak M.Ca(2+)homeostasis and endoplasmic reticulum(ER)stress:An integrated view of calcium signaling[J].Biochem Biophys Res Commun,2015,460(1):114-121.
[16]Yuan FL,Chen FH,Lu WG,et al.Acid-sensing ion channel 1a mediates acid-induced increases in intracellular calcium in rat articular chondrocytes[J].Mol Cell Biochem,2010,340(1-2):153-159.
[17]Navone SE,Marfia G,Canzi L,et al.Expression of neural and neurotrophic markers in nucleus pulposus cells isolated from degenerated intervertebral disc[J].J Orthop Res,2012,30(9):1470-1477.