基于Wnt/β-catenin信号通路的艾拉莫德对白介素1β诱导的大鼠退变软骨细胞基质代谢的影响研究
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摘要
背景 Wnt信号通路广泛存在于软骨细胞中,表现为异常活化的状态,从而影响软骨细胞的增殖和凋亡失衡。艾拉莫德作为一种新的抗炎和免疫调节性质的小分子药物,不仅具有抗风湿药物的免疫调节、抑制关节破坏功能,还可影响骨代谢,但尚未见艾拉莫德作用于软骨细胞相关机制的研究。目的基于Wnt/β-catenin信号通路,从细胞、分子水平探讨艾拉莫德对白介素(IL)-1β诱导的大鼠骨关节炎软骨基质代谢的影响及作用机制。方法2017年2—12月选取SPF级雄性2月龄健康Wistar大鼠10只,将大鼠软骨细胞培养至第2代软骨细胞,获得IL-1β诱导的退变软骨细胞模型。将分离培养的软骨细胞分为空白对照组、溶剂对照组、IL-1β组、艾拉莫德A组、艾拉莫德B组、艾拉莫德C组、塞来昔布A组、塞来昔布B组、塞来昔布C组、IL-1β+Dickkopf-1(DKK-1)组、艾拉莫德+DKK-1组、塞来昔布+DKK-1组。采用实时荧光定量反转录聚合酶链式反应和Western blotting法检测各组β-catenin、基质金属蛋白酶(MMP)-13、Ⅱ型胶原a1(Col2a1)、糖原合成酶激酶3β(GSK-3β)mRNA及蛋白表达水平。结果IL-1β+DKK-1组12、24、48、72 h的β-catenin mRNA表达水平均低于IL-1β组;IL-1β+DKK-1组0、12、24、48、72 h的MMP-13 mRNA表达水平均低于IL-1β组;IL-1β+DKK-1组12、24、48、72 h的Col2a1 mRNA表达水平均高于IL-1β组。IL-1β+DKK-1组12、24、48、72 h的β-catenin、MMP-13表达水平均低于IL-1β组,Col2a1表达水平均高于IL-1β组。加入试验药物24 h后,艾拉莫德A组、塞来昔布A组的β-catenin mRNA、MMP-13mRNA表达水平均低于IL-1β组、IL-1β+DKK-1组,Col2a1 mRNA、GSK-3βmRNA表达水平均高于IL-1β组、IL-1β+DKK-1组;艾拉莫德+DKK-1组的β-catenin mRNA、MMP-13 mRNA表达水平均低于艾拉莫德A组,Col2a1mRNA、GSK-3βmRNA表达水平高于艾拉莫德A组;塞来昔布+DKK-1组的β-catenin mRNA、MMP-13 mRNA表达水平低于塞来昔布A组,Col2a1 mRNA、GSK-3βmRNA表达水平高于塞来昔布A组。艾拉莫德A组、塞来昔布A组的β-catenin、MMP-13、Col2a1表达水平均低于IL-1β组和IL-1β+DKK-1组,GSK-3β表达水平高于IL-1β组、IL-1β+DKK-1组;艾拉莫德+DKK-1组的β-catenin、Col2a1、GSK-3β表达水平均高于艾拉莫德A组,MMP-13表达水平低于艾拉莫德A组;塞来昔布+DKK-1组的β-catenin、MMP-13、Col2a1、GSK-3β表达水平均高于塞来昔布A组。结论 Wnt/β-catenin信号通路在IL-1β诱导的大鼠退变软骨细胞中过表达,促进软骨细胞基质降解代谢,抑制其合成功能。而艾拉莫德通过调控Wnt/β-catenin信号通路发挥保护IL-1β诱导的大鼠退变软骨细胞的作用。
Background Wnt signaling pathway widely exists in chondrocyte,which shows abnormal activation,leading to the unbalanced proliferation and apoptosis of chondrocyte.As a new anti-inflammatory and immunomodulatory small molecule drug,iguratimod not only regulates immunity and inhibits joint destruction,but also affects bone metabolism.However,there is no study on the mechanism of iguratimod's action on chondrocytes.Objective To investigate the effects of iguratimod on the proliferation,apoptosis and matrix metabolism of rat chondrocytes induced by IL-1β via the Wnt/β-catenin signaling pathway at the cellular and molecular level.Methods Ten 2-month-old healthy male SPF Wistar rats were selected.Rat chondrocytes were cultured to the 2 nd generation,and by the inducing of IL-1β,the model of degenerative cartilage cells was obtained.The isolated and cultured chondrocytes were divided into 12 groups:normal control group,solvent control group,IL-1β group,iguratimod group A,iguratimod group B,iguratimod group C,celecoxib group A,celecoxib group B,celecoxib group C,IL-1β+Dickkopf-1(DKK-1) group,iguratimod+DKK-1 group,celecoxib+DKK-1 group.The expression levels of β-catenin,MMP-13,Col2a1,GSK-3β mRNA and protein levels in different groups were detected by qRT-PCR and Western blotting.Results The average expression level of β-catenin mRNA in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1βgroup at 12,24,48 and 72 h after culture.The average expression level of MMP-13 mRNA in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1β group at baseline,12,24,48 and 72 h after culture.The average expression level of Col2a1 mRNA in chondrocytes in IL-1β+DKK-1 group was higher than that of IL-1β group at12,24,48 and 72 h after culture.The average expression level of β-catenin protein in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1β group at 12,24,48 and 72 h after culture.The average expression level of MMP-13 protein in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1β group at 12,24,48 and 72 h after culture.The average expression level of Col2a1 protein in chondrocytes in IL-1β+DKK-1 group was higher than that of IL-1β group at 12,24,48 and 72 h after culture.At 24 h after drug intervention,iguratimod group A and celecoxib group A showed lower average expression levels of β-catenin mRNA and MMP-13 mRNA and higher average expression levels of Col2a1 mRNA and GSK-3β mRNA in chondrocytes compared with IL-1β group and IL-1β+DKK-1 group.Iguratimod+DKK-1 group demonstrated lower average expression levels of β-catenin mRNA and MMP-13 mRNA and higher average expression levels of Col2a1 mRNA and GSK-3β mRNA in chondrocytes compared with iguratimod group A.In comparison with celecoxib group A,celecoxib+DKK-1 group presented lower average expression levels of β-catenin mRNA and MMP-13 mRNA and higher average expression levels of Col2a1 mRNA and GSK-3β mRNA in chondrocytes.In comparison with IL-1β group and IL-1β+DKK-1 group,iguratimod group A and celecoxib group A showed lower average expression levels of β-catenin protein,MMP-13 protein and Col2a1 protein and higher average level of GSK-3β protein in chondrocytes.By comparison with iguratimod group A,iguratimod+DKK1 group manifested higher average expression levels of β-catenin protein,Col2a1 protein and GSK-3β protein and lower average expression level of MMP-13 protein in chondrocytes.The average expression levels of β-catenin protein,MMP-13 protein,Col2a1 protein and GSK-3β protein in chondrocytes in celecoxib+DKK-1 group were higher than those of celecoxib group A.Conclusion Our study shows that the overexpression of Wnt/β-catenin signaling pathway in IL-1β induced rat degenerative chondrocytes,promotes chondrocyte matrix metabolism,and inhibits the synthesis function.Iguratimod protects the chondrocytes of rats from IL-1β-induced degeneration through the regulation of the Wnt/β-catenin signaling pathway.
Background Wnt signaling pathway widely exists in chondrocyte,which shows abnormal activation,leading to the unbalanced proliferation and apoptosis of chondrocyte.As a new anti-inflammatory and immunomodulatory small molecule drug,iguratimod not only regulates immunity and inhibits joint destruction,but also affects bone metabolism.However,there is no study on the mechanism of iguratimod's action on chondrocytes.Objective To investigate the effects of iguratimod on the proliferation,apoptosis and matrix metabolism of rat chondrocytes induced by IL-1β via the Wnt/β-catenin signaling pathway at the cellular and molecular level.Methods Ten 2-month-old healthy male SPF Wistar rats were selected.Rat chondrocytes were cultured to the 2 nd generation,and by the inducing of IL-1β,the model of degenerative cartilage cells was obtained.The isolated and cultured chondrocytes were divided into 12 groups:normal control group,solvent control group,IL-1β group,iguratimod group A,iguratimod group B,iguratimod group C,celecoxib group A,celecoxib group B,celecoxib group C,IL-1β+Dickkopf-1(DKK-1) group,iguratimod+DKK-1 group,celecoxib+DKK-1 group.The expression levels of β-catenin,MMP-13,Col2a1,GSK-3β mRNA and protein levels in different groups were detected by qRT-PCR and Western blotting.Results The average expression level of β-catenin mRNA in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1βgroup at 12,24,48 and 72 h after culture.The average expression level of MMP-13 mRNA in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1β group at baseline,12,24,48 and 72 h after culture.The average expression level of Col2a1 mRNA in chondrocytes in IL-1β+DKK-1 group was higher than that of IL-1β group at12,24,48 and 72 h after culture.The average expression level of β-catenin protein in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1β group at 12,24,48 and 72 h after culture.The average expression level of MMP-13 protein in chondrocytes in IL-1β+DKK-1 group was lower than that of IL-1β group at 12,24,48 and 72 h after culture.The average expression level of Col2a1 protein in chondrocytes in IL-1β+DKK-1 group was higher than that of IL-1β group at 12,24,48 and 72 h after culture.At 24 h after drug intervention,iguratimod group A and celecoxib group A showed lower average expression levels of β-catenin mRNA and MMP-13 mRNA and higher average expression levels of Col2a1 mRNA and GSK-3β mRNA in chondrocytes compared with IL-1β group and IL-1β+DKK-1 group.Iguratimod+DKK-1 group demonstrated lower average expression levels of β-catenin mRNA and MMP-13 mRNA and higher average expression levels of Col2a1 mRNA and GSK-3β mRNA in chondrocytes compared with iguratimod group A.In comparison with celecoxib group A,celecoxib+DKK-1 group presented lower average expression levels of β-catenin mRNA and MMP-13 mRNA and higher average expression levels of Col2a1 mRNA and GSK-3β mRNA in chondrocytes.In comparison with IL-1β group and IL-1β+DKK-1 group,iguratimod group A and celecoxib group A showed lower average expression levels of β-catenin protein,MMP-13 protein and Col2a1 protein and higher average level of GSK-3β protein in chondrocytes.By comparison with iguratimod group A,iguratimod+DKK1 group manifested higher average expression levels of β-catenin protein,Col2a1 protein and GSK-3β protein and lower average expression level of MMP-13 protein in chondrocytes.The average expression levels of β-catenin protein,MMP-13 protein,Col2a1 protein and GSK-3β protein in chondrocytes in celecoxib+DKK-1 group were higher than those of celecoxib group A.Conclusion Our study shows that the overexpression of Wnt/β-catenin signaling pathway in IL-1β induced rat degenerative chondrocytes,promotes chondrocyte matrix metabolism,and inhibits the synthesis function.Iguratimod protects the chondrocytes of rats from IL-1β-induced degeneration through the regulation of the Wnt/β-catenin signaling pathway.
引文
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[7]LóPEZ-ARMADA M J,CARAMéS B,LIRES-DEáN M,et al.Cytokines,tumor necrosis factor-alpha and interleukin-1beta,differentially regulate apoptosis in osteoarthritis cultured human chondrocytes[J].Osteoarthr Cartil,2006,14(7):660-669.DOI:10.1016/j.joca.2006.01.005.
[8]王露霏,白丁,韩向龙.Wnt信号通路拮抗剂Dkk1调节骨代谢的最新进展[J].中国组织工程研究,2013,17(2):337-341.DOI:10.3969/j.issn.2095-4344.2013.02.027.WANG L F,BAI D,HAN X L.Progress in Dickkopf-1-mediated bone metabolism[J].Chinese Journal of Tissue Engineering Research,2013,17(2):337-341.DOI:10.3969/j.issn.2095-4344.2013.02.027.
[9]MUCKE H A.Iguratimod:a new disease-modifying antirheumatic drug[J].Drugs Today(Barc),2012,48(9):577-586.DOI:10.1358/dot.2012.48.9.1855758.
[10]KIS A,YELLON D M,BAXTER G F.Role of nuclear factorkappa B activation in acute ischaemia-reperfusion injury in myocardium[J].Br J Pharmacol,2003,138(5):894-900.DOI:10.1038/sj.bjp.0705108.
[11]AIKAWA Y,TANUMA N,SHIN T,et al.A new antirheumatic drug,T-614,effectively suppresses the development of autoimmune encephalomyelitis[J].J Neuroimmunol,1998,89(1/2):35-42.
[12]WENG X,LIN P,LIU F,et al.Achyranthes bidentata polysaccharides activate the Wnt/β-catenin signaling pathway to promote chondrocyte proliferation[J].Int J Mol Med,2014,34(4):1045-1050.DOI:10.3892/ijmm.2014.1869.
[13]LI X,PENG J,WU M,et al.BMP2 promotes chondrocyte proliferation via the Wnt/β-catenin signaling pathway[J].M o l M e d R e p,2 0 1 1,4(4):6 2 1-6 2 6.D O I:10.3892/mmr.2011.474.
[14]吴思敏,刘庆梅,马彦云,等.人成骨细胞分化及骨保护素分泌:R-脊椎蛋白1通过Wnt/β-catenin通路的作用[J].中国组织工程研究,2015,19(37):5923-5927.DOI:10.3969/j.issn.2095-4344.2015.37.004.WU S M,LIU Q M,MA Y Y,et al.Differentiation and osteoprotegerin secretion of human osteoblasts:R-spondin 1 effect via Wnt/beta-catenin signal pathway[J].Chinese Journal of Tissue Engineering Research,2015,19(37):5923-5927.DOI:10.3969/j.issn.2095-4344.2015.37.004.
[15]LUYTEN F P,TYLZANOWSKI P,LORIES R J.Wnt signaling and osteoarthritis[J].Bone,2009,44(4):522-527.DOI:10.1016/j.bone.2008.12.006.
[16]JIANG Q,QIU Y T,CHEN M J,et al.Synovial TGF-β1and MMP-3 levels and their correlation with the progression of temporomandibular joint osteoarthritis combined with disc displacement:a preliminary study[J].Biomed Rep,2013,1(2):218-222.DOI:10.3892/br.2012.41.
[17]SCARPELLINI M,LURATI A,VIGNATI G,et al.Biomarkers,typeⅡcollagen,glucosamine and chondroitin sulfate in osteoarthritis follow-up:the“magenta osteoarthritis study”[J].J Orthop Traumatol,2008,9(2):81-87.DOI:10.1007/s10195-008-0007-5.
[18]BLANEY DAVIDSON E N,REMST D F,VITTERS E L,et al.Increase in ALK1/ALK5 ratio as a cause for elevated MMP-13 expression in osteoarthritis in humans and mice[J].J Immunol,2009,182(12):7937-7945.DOI:10.4049/jimmunol.0803991.
[2]CHOI Y H,BURDICK M D,STRIETER R M.Human circulating fibrocytes have the capacity to differentiate osteoblasts and chondrocytes[J].Int J Biochem Cell Biol,2010,42(5):662-671.DOI:10.1016/j.biocel.2009.12.011.
[3]DAO D Y,JONASON J H,ZHANG Y,et al.Cartilage-specificβ-catenin signaling regulates chondrocyte maturation,generation of ossification centers,and perichondrial bone formation during skeletal development[J].J Bone Miner Res,2012,27(8):1680-1694.DOI:10.1002/jbmr.1639.
[4]中华医学会骨科学分会关节外科学组.骨关节炎诊疗指南(2018年版)[J].中华骨科杂志,2018,38(12):705-715.DOI:10.3760/cma.j.issn.0253-2352.2018.12.001.Joint Surgery Section of Osteology Branch of Chinese Medical Association.Guideline for the management of osteoarthritis(2018)[J].Chinese Journal of Orthopaedics,2018,38(12):705-715.DOI:10.3760/cma.j.issn.0253-2352.2018.12.001.
[5]SHAKIBAEI M,JOHN T,SEIFARTH C,et al.Resveratrol inhibits IL-1 beta-induced stimulation of caspase-3 and cleavage of PARP in human articular chondrocytes in vitro[J].Ann N Y Acad Sci,2007,1095:554-563.DOI:10.1196/annals.1397.060.
[6]YUDOH K,SHISHIDO K,MURAYAMA H,et al.Watersoluble C60 fullerene prevents degeneration of articular cartilage in osteoarthritis via down-regulation of chondrocyte catabolic activity and inhibition of cartilage degeneration during disease development[J].Arthritis Rheum,2007,56(10):3307-3318.DOI:10.1002/art.22917.
[7]LóPEZ-ARMADA M J,CARAMéS B,LIRES-DEáN M,et al.Cytokines,tumor necrosis factor-alpha and interleukin-1beta,differentially regulate apoptosis in osteoarthritis cultured human chondrocytes[J].Osteoarthr Cartil,2006,14(7):660-669.DOI:10.1016/j.joca.2006.01.005.
[8]王露霏,白丁,韩向龙.Wnt信号通路拮抗剂Dkk1调节骨代谢的最新进展[J].中国组织工程研究,2013,17(2):337-341.DOI:10.3969/j.issn.2095-4344.2013.02.027.WANG L F,BAI D,HAN X L.Progress in Dickkopf-1-mediated bone metabolism[J].Chinese Journal of Tissue Engineering Research,2013,17(2):337-341.DOI:10.3969/j.issn.2095-4344.2013.02.027.
[9]MUCKE H A.Iguratimod:a new disease-modifying antirheumatic drug[J].Drugs Today(Barc),2012,48(9):577-586.DOI:10.1358/dot.2012.48.9.1855758.
[10]KIS A,YELLON D M,BAXTER G F.Role of nuclear factorkappa B activation in acute ischaemia-reperfusion injury in myocardium[J].Br J Pharmacol,2003,138(5):894-900.DOI:10.1038/sj.bjp.0705108.
[11]AIKAWA Y,TANUMA N,SHIN T,et al.A new antirheumatic drug,T-614,effectively suppresses the development of autoimmune encephalomyelitis[J].J Neuroimmunol,1998,89(1/2):35-42.
[12]WENG X,LIN P,LIU F,et al.Achyranthes bidentata polysaccharides activate the Wnt/β-catenin signaling pathway to promote chondrocyte proliferation[J].Int J Mol Med,2014,34(4):1045-1050.DOI:10.3892/ijmm.2014.1869.
[13]LI X,PENG J,WU M,et al.BMP2 promotes chondrocyte proliferation via the Wnt/β-catenin signaling pathway[J].M o l M e d R e p,2 0 1 1,4(4):6 2 1-6 2 6.D O I:10.3892/mmr.2011.474.
[14]吴思敏,刘庆梅,马彦云,等.人成骨细胞分化及骨保护素分泌:R-脊椎蛋白1通过Wnt/β-catenin通路的作用[J].中国组织工程研究,2015,19(37):5923-5927.DOI:10.3969/j.issn.2095-4344.2015.37.004.WU S M,LIU Q M,MA Y Y,et al.Differentiation and osteoprotegerin secretion of human osteoblasts:R-spondin 1 effect via Wnt/beta-catenin signal pathway[J].Chinese Journal of Tissue Engineering Research,2015,19(37):5923-5927.DOI:10.3969/j.issn.2095-4344.2015.37.004.
[15]LUYTEN F P,TYLZANOWSKI P,LORIES R J.Wnt signaling and osteoarthritis[J].Bone,2009,44(4):522-527.DOI:10.1016/j.bone.2008.12.006.
[16]JIANG Q,QIU Y T,CHEN M J,et al.Synovial TGF-β1and MMP-3 levels and their correlation with the progression of temporomandibular joint osteoarthritis combined with disc displacement:a preliminary study[J].Biomed Rep,2013,1(2):218-222.DOI:10.3892/br.2012.41.
[17]SCARPELLINI M,LURATI A,VIGNATI G,et al.Biomarkers,typeⅡcollagen,glucosamine and chondroitin sulfate in osteoarthritis follow-up:the“magenta osteoarthritis study”[J].J Orthop Traumatol,2008,9(2):81-87.DOI:10.1007/s10195-008-0007-5.
[18]BLANEY DAVIDSON E N,REMST D F,VITTERS E L,et al.Increase in ALK1/ALK5 ratio as a cause for elevated MMP-13 expression in osteoarthritis in humans and mice[J].J Immunol,2009,182(12):7937-7945.DOI:10.4049/jimmunol.0803991.