抑制髓核细胞Ⅱ型胶原表达的炎性因子白细胞介素1β、白细胞介素6及肿瘤坏死因子α
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摘要
背景:炎性因子在椎间盘退变中起着重要的作用,其抑制Ⅱ型胶原表达机制尚未完全明确。目的:探讨炎性因子白细胞介素1β、白细胞介素6、肿瘤坏死因子α对髓核细胞Ⅱ型胶原表达的影响及作用机制。方法:成年雄性SD大鼠由新疆医科大学动物实验中心提供,实验方案经新疆医科大学第一附属医院动物实验伦理委员会批准(批准号为IACUC20151203-09)。分离、培养及鉴定SD大鼠髓核细胞,将其随机分为7组:①对照组;②白细胞介素1β(50μg/L)组;③白细胞介素6(100μg/L)组;④肿瘤坏死因子α(20μg/L)组;⑤白细胞介素1β(50μg/L)+740Y-P组(PI3K激活剂);⑥白细胞介素6(100μg/L)+740Y-P组;⑦肿瘤坏死因子α(20μg/L)+740Y-P组;并进行相应干预。48 h后收集髓核细胞进行RT-PCR,Western Blot检测及免疫荧光染色,分别检测白细胞介素1β、白细胞介素6和肿瘤坏死因子α对髓核细胞中基质金属蛋白/金属蛋白酶组织抑制因子,炎性因子表达对Ⅰ型、Ⅱ型胶原蛋白,聚集蛋白聚糖以及PI3K/AKT信号通路蛋白表达的影响。结果与结论:①白细胞介素1β组和肿瘤坏死因子α组中金属蛋白酶组织抑制因子1m RNA表达量明显低于对照组(P <0.05);白细胞介素1β、白细胞介素6和肿瘤坏死因子α组中基质金属蛋白酶9和基质金属蛋白酶13 mRNA表达量明显高于对照组(P <0.05);所有实验干预组中白细胞介素6 mRNA表达量明显高于对照组(P <0.05);白细胞介素1β、白细胞介素6和肿瘤坏死因子α组中白细胞介素1β和肿瘤坏死因子αm RNA表达量明显高于对照组;白细胞介素1β和肿瘤坏死因子α组中白细胞介素1ra表达量明显低于对照组(P<0.05);②与对照组相比,白细胞介素1β和肿瘤坏死因子α组的Ⅱ型胶原和聚集蛋白聚糖表达明显降低,而白细胞介素6组的则略有降低;与白细胞介素1β、白细胞介素6和肿瘤坏死因子α组相比,对应的给予PI3K激活剂组的表达则明显升高;③结果表明:炎性因子白细胞介素1β、白细胞介素6、肿瘤坏死因子α可能通过抑制PI3K/AKT细胞增殖通路来抑制髓核细胞Ⅱ型胶原的表达。
BACKGROUND: The role of inflammatory factors in the degeneration of intervertebral discs has received increasing attention. The mechanism by which inflammatory factors inhibit the expression of type II collagen has not been fully elucidated.OBJECTIVE: To observe the expression of type II collagen in nucleus pulposus cells after intervention with inflammatory factors interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) and the mechanism of action.METHODS: Adult male Sprague-Dawley rats were provided by the Animal Experimental Center of Xinjiang Medical University, and the study protocol was approved by the Animal Experimental Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University(approval No. IACUC20151203-09). Nucleus pulposus cells from rats were isolated, cultured, identified and randomly divided into seven groups: control group, IL-1β(50 μg/L) group, IL-6(100 μg/L) group, TNF-α(20 μg/L) group, IL-1β(50 μg/L)+740 Y-P(a PI3 K activator) group, IL-6(100 μg/L)+740 Y-P group, TNF-α(20 μg/L)+740 Y-P group. After 48 hours of intervention, nucleus pulposus cells were collected. RT-PCR, western blot and immunofluorescence staining were used to detect the effects of IL-1β, IL-6 and TNF-α on expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase, type I and type II collagens, Aggrecan, and PI3 K/AKT signaling pathway proteins in nucleus pulposus cells.RESULTS AND CONCLUSION:(1) The expression of tissue inhibitor of metalloproteinase-1 mRNA in IL-1β and TNF-α groups was significantly lower than that in the control group(P < 0.05). The mRNA levels of matrix metalloproteinases-9 and-13 in the IL-1β, IL-6 and TNF-α groups were significantly higher than those in the control group(P < 0.05). The expression of IL-6 mRNA in all experimental intervention groups was significantly higher than that in the control group(P < 0.05). The mRNA levels of IL-1β, IL-6 and TNF-α in the IL-1β,IL-6 and TNF-α groups were significantly higher than those in the control group. The expression of interleukin-1 ra in the IL-1β and TNF-αgroups was significantly lower than that in the control group(P < 0.05).(2) Immunofluorescence results showed that compared with the control group, the expression of type II collagen and Aggrecan significantly decreased in the IL-1β and TNF-α groups, and slightly decreased in the IL-6 group. Compared with IL-1β, IL-6 and TNF-α groups, the expression of type II collagen and Aggrecan was significantly increased in the three corresponding PI3 K activator groups. These findings indicate that IL-1β, IL-6 and TNF-α as inflammatory factors may inhibit the expression of type II collagen in nucleus pulposus cells by inhibiting the PI3 K/AKT cell proliferation pathway.
BACKGROUND: The role of inflammatory factors in the degeneration of intervertebral discs has received increasing attention. The mechanism by which inflammatory factors inhibit the expression of type II collagen has not been fully elucidated.OBJECTIVE: To observe the expression of type II collagen in nucleus pulposus cells after intervention with inflammatory factors interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) and the mechanism of action.METHODS: Adult male Sprague-Dawley rats were provided by the Animal Experimental Center of Xinjiang Medical University, and the study protocol was approved by the Animal Experimental Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University(approval No. IACUC20151203-09). Nucleus pulposus cells from rats were isolated, cultured, identified and randomly divided into seven groups: control group, IL-1β(50 μg/L) group, IL-6(100 μg/L) group, TNF-α(20 μg/L) group, IL-1β(50 μg/L)+740 Y-P(a PI3 K activator) group, IL-6(100 μg/L)+740 Y-P group, TNF-α(20 μg/L)+740 Y-P group. After 48 hours of intervention, nucleus pulposus cells were collected. RT-PCR, western blot and immunofluorescence staining were used to detect the effects of IL-1β, IL-6 and TNF-α on expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase, type I and type II collagens, Aggrecan, and PI3 K/AKT signaling pathway proteins in nucleus pulposus cells.RESULTS AND CONCLUSION:(1) The expression of tissue inhibitor of metalloproteinase-1 mRNA in IL-1β and TNF-α groups was significantly lower than that in the control group(P < 0.05). The mRNA levels of matrix metalloproteinases-9 and-13 in the IL-1β, IL-6 and TNF-α groups were significantly higher than those in the control group(P < 0.05). The expression of IL-6 mRNA in all experimental intervention groups was significantly higher than that in the control group(P < 0.05). The mRNA levels of IL-1β, IL-6 and TNF-α in the IL-1β,IL-6 and TNF-α groups were significantly higher than those in the control group. The expression of interleukin-1 ra in the IL-1β and TNF-αgroups was significantly lower than that in the control group(P < 0.05).(2) Immunofluorescence results showed that compared with the control group, the expression of type II collagen and Aggrecan significantly decreased in the IL-1β and TNF-α groups, and slightly decreased in the IL-6 group. Compared with IL-1β, IL-6 and TNF-α groups, the expression of type II collagen and Aggrecan was significantly increased in the three corresponding PI3 K activator groups. These findings indicate that IL-1β, IL-6 and TNF-α as inflammatory factors may inhibit the expression of type II collagen in nucleus pulposus cells by inhibiting the PI3 K/AKT cell proliferation pathway.
引文
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[3]Masuda K. Biological repair of the degenerated intervertebral disc by the injection of growth factors. Eur Spine J. 2008;17(Suppl 4):441-451.
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[7]Shen Jieliang,Xu Shengxi,Zhou Hao et al. IL-1βinduces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells. Sci Rep.2017; 7:41067.
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[10] Gorth DJ, Mauck RL, Chiaro JA, et al. IL-1ra delivered from poly(lactic-co-glycolic acid)microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro.Arthritis Res Ther. 2012;14(4):R179.
[11] Cuéllar JM, Borges PM, Cuéllar VG, et al. Cytokine expression in the epidural space:a model of noncompressive disc herniation-induced inflammation. Spine. 2013; 38(1):17-23.
[12] Studer RK, Aboka AM, et al. p38 MAPK inhibition in nucleus pulposus cells:a potential target for treating intervertebral disc degeneration. Spine. 2007; 32(25):2827-2833.
[13] Freemont AJ.The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology(Oxford). 2009;48(1):5-10.
[14] Vergroesen PP,Kingma I,Emanuel KS,et al. Mechanics and biology in intervertebral disc degeneration:a vicious circle.Osteoarthri-tis Cartilage.2015;23(7):1057-1070.
[15] Global Burden of Disease Study 2013 Collaborators. Global,region al,and national incidence,prevalence,and years lived with disability for 301 acute and chronic diseases and injuries in 188countries,1990-2013:a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386(9995):743-800.
[16] Willems N, Tellegen AR, Bergknut N, et al. Inflammatory profiles in canine intervertebral disc degeneration. BMC Vet.Res.2016;12:10.
[17] Gu SX, Li X, Hamilton JL, et al. MicroRNA-146a reduces IL-1dependent inflammatory responses in the intervertebral disc.Gene. 2015; 555(2):80-87.
[18] Zhou T, Lin H, Cheng Z, et al. Mechanism of microRNA-146amediated IL-6/STAT3 signaling in lumbar intervertebral disc degeneration. Exp Ther Med, 2017, 14(2):1131-1135.
[19] Yang H, Gao F, Li X, et al. TGF-β1 antagonizes TNF-αinduced up-regulation of matrix metalloproteinase 3 in nucleus pulposus cells:role of the ERK1/2 pathway. Connect. Tissue Res. 2015;56(6):461-468.
[20] Freemont AJ. Keep up the heat on IL-l. Blood. 2012; 120(13):2538-2539.
[21] Croker BA, Kiu H,Roberts AW.IL-6 promotes acute and chronic inflammatory disease in the absence of SOCS3.Immunol Cell Biol. 2012;90(1):124-129.
[22] Hiyama A, Yokoyama K, Nukaga T,et al. A complex interaction between Wnt signaling and TNF-αin nucleus pulposus cells.Arthritis Res Ther. 2013;15(6):R189.
[23] Holm S,Mackiewicz Z,Holm AK et al. Pro-inflammatory,pleiotropic, and anti-inflammatory TNF-alpha, IL-6, and IL-10 in experimental porcine intervertebral disk degeneration.Vet.Pathol. 2009; 46(6):1292-300.
[24] Kim HJ, Yeom JS, Koh YG, et al.Anti-inflammatory effect of platelet-rich plasma on nucleus pulposus cells with response of TNF-αand IL-1. Orthop. Res.2014; 32(4):551-556.
[25] Catabolic cytokine expression in degenerate and herniated human intervertebral discs:IL-1beta and TNFalpha expression profile.Arthritis Res Ther. 2007; 9(4):R77.
[26] Le Maitre CL, Hoyland JA, Freemont AJ. Catabolic cytokine expression in degenerate and herniated human intervertebral discs:IL-1beta and TNF alpha expression profile. Arthritis Res Ther. 2007;9(4):R77.
[27] Jiang L,Zhang X,Zheng X,et al. Apoptosis,senescence,and autophagy in rat nucleus pulposus cells:implications for diabetic intervertebral disc degeneration. J Orthop Res. 2013;31(5):692-702.
[28] Chen ZH,Jin SH,Wang MY,et al. Enhanced NLRP3,caspase 1,and IL 1βlevels in degenerate human intervertebral disc and their association with the grades of discdegeneration. Anat Rec(Hoboken).2015;298(4):720-726.
[29] Yang W,Yu XH,Wang C, et al. Interleukin-1βin intervertebral disk degeneration.Clin Chim Acta. 2015; 450:262-272.
[30] Phillips KL,Jordan Mahy N,Nicklin MJ,et al. Interleukin 1receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration. Ann Rheum Dis.2013;72(11):1860-1907.
[31] Gruber HE,Hoelscher GL,Ingram JA,et al. Increased IL 17expression in degenerated human discs and increased production in cultured annulus cells exposed to IL 1βand TNFα. Biotech Histochem.2013;88(6):302-310.
[32] Cai F,Zhu L,Wang F, et al. The Paracrine Effect of Degenerated Disc Cells on Healthy Human Nucleus Pulposus Cells Is Mediated by MAPK and NF-κB Pathways and Can Be Reduced by TGF-β1.DNA Cell Biol. 2017; 36:143-158.
[33] Wang C,Yu XH,Yan Y, et al. Tumor necrosis factor-α:a key contributor to intervertebral disc degeneration.Acta Biochim.Biophys Sin. 2017;49:1-13.
[34] Cornejo MC,Cho SK,Giannarelli C,et al. Soluble factors from the notochordal rich intervertebral disc inhibit endothelial cell inva-sion and vessel formation in the presenceand absence of pro inflammatory cytokines. Osteoarthritis Cartilage.2015;23(3):487-496.
[35] Yang H, Liu H, Li X, et al. TNF-αand TGF-β1 regulate Syndecan-4 expression in nucleus pulposus cells:role of the mitogen-activated protein kinase and NF-κB pathways.Connect Tissue Res.2015; 56:281-287.
[36] Wang WJ, Yu XH, Wang C, et al.MMPs and ADAMTSs in intervertebral disc degeneration.Clin Chim Acta. 2015; 448:238-246.
[37] Walter BA,Likhitpanichkul M,Illien Junger S,et al.TNF-αtransport induced by dynamic loading alters biomechanics of intact intervertebral discs. PLoS One.2015;10(3):e0118358.
[2]Kalichman L,Hunter DJ. The genetics of intervertebral disc degeneration:Familial predisposition and heritability estimation.Joint Bone Spine.2008;75:383-387.
[3]Masuda K. Biological repair of the degenerated intervertebral disc by the injection of growth factors. Eur Spine J. 2008;17(Suppl 4):441-451.
[4]Shamji MF, Setton LA, Jarvis W, et al. Proinflammatory cytokine expression profile in degenerated and herniated human intervertebral disc tissues. Arthritis Rheum. 2010;62(7):1974-1982.
[5]Studer RK,Vo N,Sowa G, et al. Human nucleus pulposus cells react to IL-6:independent actions and amplification of response to IL-1 and TNF-α. Spine. 2011; 36(8):593-599.
[6]Scheller J, Garbers C, Rose-John S. Interleukin-6:From basic biology to selective blockade of pro-inflammatory activities.Semin Immunol. 2013;11:002.
[7]Shen Jieliang,Xu Shengxi,Zhou Hao et al. IL-1βinduces apoptosis and autophagy via mitochondria pathway in human degenerative nucleus pulposus cells. Sci Rep.2017; 7:41067.
[8]Studer RK,Gilbertson LG,Georgescu H, et al. p38 MAPK inhibition modulates rabbit nucleus pulposus cell response to IL-1. Orthop. Res. 2008; 26(7):991-998.
[9]Mwale F,Wang HT,Zukor DJ et al. Effect of a Type II Collagen Fragment on the Expression of Genes of the Extracellular Matrix in Cells of the Intervertebral Disc. Open Orthop J.2008;2:1-9.
[10] Gorth DJ, Mauck RL, Chiaro JA, et al. IL-1ra delivered from poly(lactic-co-glycolic acid)microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro.Arthritis Res Ther. 2012;14(4):R179.
[11] Cuéllar JM, Borges PM, Cuéllar VG, et al. Cytokine expression in the epidural space:a model of noncompressive disc herniation-induced inflammation. Spine. 2013; 38(1):17-23.
[12] Studer RK, Aboka AM, et al. p38 MAPK inhibition in nucleus pulposus cells:a potential target for treating intervertebral disc degeneration. Spine. 2007; 32(25):2827-2833.
[13] Freemont AJ.The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain. Rheumatology(Oxford). 2009;48(1):5-10.
[14] Vergroesen PP,Kingma I,Emanuel KS,et al. Mechanics and biology in intervertebral disc degeneration:a vicious circle.Osteoarthri-tis Cartilage.2015;23(7):1057-1070.
[15] Global Burden of Disease Study 2013 Collaborators. Global,region al,and national incidence,prevalence,and years lived with disability for 301 acute and chronic diseases and injuries in 188countries,1990-2013:a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015;386(9995):743-800.
[16] Willems N, Tellegen AR, Bergknut N, et al. Inflammatory profiles in canine intervertebral disc degeneration. BMC Vet.Res.2016;12:10.
[17] Gu SX, Li X, Hamilton JL, et al. MicroRNA-146a reduces IL-1dependent inflammatory responses in the intervertebral disc.Gene. 2015; 555(2):80-87.
[18] Zhou T, Lin H, Cheng Z, et al. Mechanism of microRNA-146amediated IL-6/STAT3 signaling in lumbar intervertebral disc degeneration. Exp Ther Med, 2017, 14(2):1131-1135.
[19] Yang H, Gao F, Li X, et al. TGF-β1 antagonizes TNF-αinduced up-regulation of matrix metalloproteinase 3 in nucleus pulposus cells:role of the ERK1/2 pathway. Connect. Tissue Res. 2015;56(6):461-468.
[20] Freemont AJ. Keep up the heat on IL-l. Blood. 2012; 120(13):2538-2539.
[21] Croker BA, Kiu H,Roberts AW.IL-6 promotes acute and chronic inflammatory disease in the absence of SOCS3.Immunol Cell Biol. 2012;90(1):124-129.
[22] Hiyama A, Yokoyama K, Nukaga T,et al. A complex interaction between Wnt signaling and TNF-αin nucleus pulposus cells.Arthritis Res Ther. 2013;15(6):R189.
[23] Holm S,Mackiewicz Z,Holm AK et al. Pro-inflammatory,pleiotropic, and anti-inflammatory TNF-alpha, IL-6, and IL-10 in experimental porcine intervertebral disk degeneration.Vet.Pathol. 2009; 46(6):1292-300.
[24] Kim HJ, Yeom JS, Koh YG, et al.Anti-inflammatory effect of platelet-rich plasma on nucleus pulposus cells with response of TNF-αand IL-1. Orthop. Res.2014; 32(4):551-556.
[25] Catabolic cytokine expression in degenerate and herniated human intervertebral discs:IL-1beta and TNFalpha expression profile.Arthritis Res Ther. 2007; 9(4):R77.
[26] Le Maitre CL, Hoyland JA, Freemont AJ. Catabolic cytokine expression in degenerate and herniated human intervertebral discs:IL-1beta and TNF alpha expression profile. Arthritis Res Ther. 2007;9(4):R77.
[27] Jiang L,Zhang X,Zheng X,et al. Apoptosis,senescence,and autophagy in rat nucleus pulposus cells:implications for diabetic intervertebral disc degeneration. J Orthop Res. 2013;31(5):692-702.
[28] Chen ZH,Jin SH,Wang MY,et al. Enhanced NLRP3,caspase 1,and IL 1βlevels in degenerate human intervertebral disc and their association with the grades of discdegeneration. Anat Rec(Hoboken).2015;298(4):720-726.
[29] Yang W,Yu XH,Wang C, et al. Interleukin-1βin intervertebral disk degeneration.Clin Chim Acta. 2015; 450:262-272.
[30] Phillips KL,Jordan Mahy N,Nicklin MJ,et al. Interleukin 1receptor antagonist deficient mice provide insights into pathogenesis of human intervertebral disc degeneration. Ann Rheum Dis.2013;72(11):1860-1907.
[31] Gruber HE,Hoelscher GL,Ingram JA,et al. Increased IL 17expression in degenerated human discs and increased production in cultured annulus cells exposed to IL 1βand TNFα. Biotech Histochem.2013;88(6):302-310.
[32] Cai F,Zhu L,Wang F, et al. The Paracrine Effect of Degenerated Disc Cells on Healthy Human Nucleus Pulposus Cells Is Mediated by MAPK and NF-κB Pathways and Can Be Reduced by TGF-β1.DNA Cell Biol. 2017; 36:143-158.
[33] Wang C,Yu XH,Yan Y, et al. Tumor necrosis factor-α:a key contributor to intervertebral disc degeneration.Acta Biochim.Biophys Sin. 2017;49:1-13.
[34] Cornejo MC,Cho SK,Giannarelli C,et al. Soluble factors from the notochordal rich intervertebral disc inhibit endothelial cell inva-sion and vessel formation in the presenceand absence of pro inflammatory cytokines. Osteoarthritis Cartilage.2015;23(3):487-496.
[35] Yang H, Liu H, Li X, et al. TNF-αand TGF-β1 regulate Syndecan-4 expression in nucleus pulposus cells:role of the mitogen-activated protein kinase and NF-κB pathways.Connect Tissue Res.2015; 56:281-287.
[36] Wang WJ, Yu XH, Wang C, et al.MMPs and ADAMTSs in intervertebral disc degeneration.Clin Chim Acta. 2015; 448:238-246.
[37] Walter BA,Likhitpanichkul M,Illien Junger S,et al.TNF-αtransport induced by dynamic loading alters biomechanics of intact intervertebral discs. PLoS One.2015;10(3):e0118358.
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