单味药鹿茸调节骨关节炎模型兔软骨细胞外基质主要成分ADAMTS-4/TIMP-3基因的表达
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摘要
背景:大量研究证实鹿茸对于治疗骨关节炎有效,但是其与软骨细胞外基质主要成分ADAMTS-4/TIMP-3基因的相关性却报道较少。目的:探讨鹿茸对兔骨关节炎模型ADAMTS-4/TIMP-3基因表达的影响,进一步阐明鹿茸防治骨关节炎的作用机制。方法:选择健康雌性新西兰大白兔42只,采用前交叉韧带离断法建立兔骨关节炎模型,随机分为3组:对照组(生理盐水)、低剂量鹿茸组(0.21 g/kg)和高剂量鹿茸组(0.84 g/kg)。干预9周后,检测双膝关节中的羟脯氨酸含量,采用RT-PCR检测各组软骨靶基因mRNA的表达,采用Westernblot测量蛋白聚糖和Ⅱ型胶原蛋白的表达。结果与结论:(1)低剂量鹿茸组和高剂量鹿茸组软骨羟脯氨酸含量显著高于对照组,且高剂量鹿茸组显著高于低剂量鹿茸组(P <0.05);(2)高剂量鹿茸组和低剂量鹿茸组ADAMTS-4 mRNA表达较对照组明显降低,且高剂量显著组低于低剂量鹿茸组(P <0.05),而TIMP-3、蛋白聚糖、Ⅱ型胶原蛋白mRNA表达较对照组明显增加,且高剂量鹿茸组显著高于低剂量鹿茸组(P <0.05);(3)高剂量鹿茸组和低剂量鹿茸组蛋白聚糖、Ⅱ型胶原蛋白表达较对照组明显增高,且高剂量鹿茸组显著高于低剂量鹿茸组(P <0.05);(4)结果提示,鹿茸通过抑制ADAMTS-4的分泌,促进保护因子TIMP-3的分泌,阻止蛋白聚糖、Ⅱ型胶原蛋白蛋白的降解,起到修复软骨的作用。
BACKGROUND: Pilose antler has been shown to be effective for osteoarthritis,but its association with ADAMTS-4/TIMP-3 gene(main composition of extracellular matrix) is rarely reported. OBJECTIVE: To investigate the effect of pilose antler on the expression of ADAMTS-4/TIMP-3 gene in rabbit models of osteoarthritis,and to further elucidate the mechanism of pilose antler in the prevention and treatment of osteoarthritis. METHODS: Forty-two healthy female New Zealand white rabbits were selected and used for establishing the models of osteoarthritis by anterior cruciate ligament rupture. The rabbit models were then randomized into three groups: control group(normal saline),low-dose(0.21 g/kg) and high-dose(0.84 g/kg) pilose antler groups. After intervention for 9 weeks,the content of hydroxyproline in both knees was detected. The expression levels of mRNA of cartilage target genes in each group were detected by RT-PCR. Protein expression levels of aggrecan and collagen type II were determined by western blot assay. RESULTS AND CONCLUSION: Compared with the control group,the content of hydroxyproline in cartilage in the low-dose and high-dose pilose antler groups was significantly increased,and the content in the high-dose pilose antler group was significantly higher than that in the low-dose pilose antler group(P < 0.05). The order of mRNA expression level of ADAMTS-4 was as follows: high-dose pilose antler group < low-dose pilose antler group < control group(P < 0.05). The order of mRNA expression levels of metallopeptidase inhibitor 3,aggrecan and collagen type II was as follows: high-dose pilose antler group > low-dose pilose antler group > control group(P < 0.05). The expression levels of aggrecan and collagen type II were the highest in the high-dose pilose antler group,followed by the low-dose pilose antler group,and the lowest in the control group(P < 0.05). Our findings indicate that pilose antler can repair the injured cartilage by inhibiting the secretion of ADAMTS-4,increasing the secretion of metallopeptidase inhibitor 3 and preventing the degradation of aggrecan and collagen type II.
BACKGROUND: Pilose antler has been shown to be effective for osteoarthritis,but its association with ADAMTS-4/TIMP-3 gene(main composition of extracellular matrix) is rarely reported. OBJECTIVE: To investigate the effect of pilose antler on the expression of ADAMTS-4/TIMP-3 gene in rabbit models of osteoarthritis,and to further elucidate the mechanism of pilose antler in the prevention and treatment of osteoarthritis. METHODS: Forty-two healthy female New Zealand white rabbits were selected and used for establishing the models of osteoarthritis by anterior cruciate ligament rupture. The rabbit models were then randomized into three groups: control group(normal saline),low-dose(0.21 g/kg) and high-dose(0.84 g/kg) pilose antler groups. After intervention for 9 weeks,the content of hydroxyproline in both knees was detected. The expression levels of mRNA of cartilage target genes in each group were detected by RT-PCR. Protein expression levels of aggrecan and collagen type II were determined by western blot assay. RESULTS AND CONCLUSION: Compared with the control group,the content of hydroxyproline in cartilage in the low-dose and high-dose pilose antler groups was significantly increased,and the content in the high-dose pilose antler group was significantly higher than that in the low-dose pilose antler group(P < 0.05). The order of mRNA expression level of ADAMTS-4 was as follows: high-dose pilose antler group < low-dose pilose antler group < control group(P < 0.05). The order of mRNA expression levels of metallopeptidase inhibitor 3,aggrecan and collagen type II was as follows: high-dose pilose antler group > low-dose pilose antler group > control group(P < 0.05). The expression levels of aggrecan and collagen type II were the highest in the high-dose pilose antler group,followed by the low-dose pilose antler group,and the lowest in the control group(P < 0.05). Our findings indicate that pilose antler can repair the injured cartilage by inhibiting the secretion of ADAMTS-4,increasing the secretion of metallopeptidase inhibitor 3 and preventing the degradation of aggrecan and collagen type II.
引文
[1]田雪秋.骨关节炎相关危险因素研究进展[J].中国社区医师,2016,32(15):14-17.
[2]刘康宝.中老年膝骨关节炎发病的相关因素分析[J].世界最新医学信息文摘,2015,15(31):6-8.
[3]尹丽.中老年膝骨关节炎发病的相关因素分析[J].吉林医学,2015,36(18):4139-4141.
[4]Li J,Anemaet W,Diaz MA,et al.Knockout of ADAMTS5 does not eliminate cartilage aggrecanase activity but abrogates joint fibrosis and promotes cartilage aggrecan deposition in murine osteoarthritis models.J Orthop Res.2011;29(4):516-522.
[5]朱剑,黄烽,张一平.类风湿关节炎患者蛋白聚糖aggrecan+G1区特异性T细胞诱导SCID小鼠产生膝关节炎的实验研究[J].解放军医学杂志,2006,31(8):787-790.
[6]周惠琼,钱宏伟,杨文芳.外周血蛋白聚糖代谢片段检测水平与类风湿关节炎影像学进展Sharp评分的相关性[J].中华风湿病学杂志,2017,21(3):172-178.
[7]杜国辉,屈爱存,陈建英.透明质酸对关节软骨蛋白聚糖aggrecan的影响[J].中国生化药物杂志,2011,32(2):125-8.
[8]高宗强,郭雄.透明质酸对体外培养大骨节病软骨细胞Ⅱ型胶原和聚集蛋白聚糖m RNA表达的影响[J].中国组织工程研究与临床康复,2009,13(46):9061-9066.
[9]周惠琼,张奉春.肿瘤坏死因子-α和白细胞介素-1β对人骨关节炎软骨细胞蛋白聚糖代谢的影响[J].中华风湿病学杂志,2008,12(2):83-88.
[10]Stambas J,Stanton H,Rogerson FM,et al.ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.PLo S Biol.2005;434(7033):648-652.
[11]孙志涛.不同方法提取SD大鼠膝关节软骨组织的总RNA[J].中国组织工程研究,2014,18(51):8201-8205.
[12]娄思权.骨关节炎的病理与发病因素[J].中华骨科杂志,1996,16(1):56-60.
[13]谷艳超.骨关节炎发病机制和治疗的研究进展[J].中国骨与关节杂志,2016,5(10):770-774.
[14]安丙辰,王有.不同强度张应力刺激影响关节软骨细胞Ⅱ型胶原和聚集蛋白聚糖m RNA表达的研究[J].中华关节外科杂志,2010,4(3):362-368.
[15]陈孟交.丹紫康膝冲剂对兔膝骨性关节炎软骨含水率及羟脯氨酸影响的研究[J].湖南中医药大学学报,2012,32(1):20-3.
[16]王坤.T140靶向阻断SDF-1%2f CXCR4信号通路对豚鼠关节软骨Ⅱ型胶原蛋白及聚集蛋白聚糖的影响[J].中国运动医学杂志,2016,36(1):23-27.
[17]董琪.黄芪桂枝五物汤对肩周炎兔模型羟脯氨酸与总蛋白含量测定的实验研究[J].陕西中医,2015,36(8):1081-1083.
[18]陈喜德,宋丽君.骨关节炎SD大鼠关节软骨中蛋白多糖与YKL-40的相关性[J].重庆医学,2016,43(10):1214-1218.
[19]van den Boom R,Brama PA,Kiers GH,et al.Assessment of the effects of age and joint disease on hydroxyproline and glycosaminoglycan concentrations in synovial fluid from the metacarpophalangeal joint of horses.Am J Vet Res.2004;65(3):296-302.
[20]Smith SM,Shu C,Melrose J.Comparative immunolocalisation of perlecan with collagen II and aggrecan in human foetal,newborn and adult ovine joint tissues demonstrates perlecan as an early developmental chondrogenic marker.Histochem Cell Biol.2010;134(3):251-263.
[21]刘晓斌,候瑞.软硬食性对生长早期小鼠颞下颌关节髁突软骨中Aggrecan与ADAMTS-5表达的影响[J].医学研究杂志,2016,45(6):161-164.
[22]李彦林.体外阻断基质细胞衍生因子1/CXC趋化因子受体4信号通路后人关节软骨组织Ⅱ型胶原和聚集蛋白聚糖的表达[J].中国组织工程研究,2012,16(46):8607-8611.
[23]Feng J,Gu Z,Lin X,et al.Postnatal development of type IIcollagen and aggrecan m RNA expression in a rabbit craniomandibular joint.Anat Rec(Hoboken).2010;293(9):1574-1580.
[24]Azadi M,Nia HT,Gauci SJ,et al.Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse.JBiomech.2016;49(9):1634-1640.
[25]杜清峰,张海宁.慢病毒介导的aggrecanase-2+sh RNA转染对类风湿关节炎患者软骨细胞aggrecan的影响[J].中华骨科杂志,2014,34(9):936-945.
[26]袁红,陈榕.平衡针灸对肩周炎模型兔转化生长因子β1、前列腺素+E2、羟脯氨酸影响的实验研究[J].环球中医药,2015,8(1):42-46.
[27]牛维.单味药鹿茸调控大鼠骨关节炎软骨组织Smad2_3表达的研究[J].中国中西医结合杂志,2014,34(2):209-214.
[28]牛维.单味中药鹿茸调控骨关节炎软骨组织TGF-β1表达的研究[J].时珍国医国药,2014,25(3):528-532.
[29]牛维.鹿茸归经与早期骨关节炎软骨靶器官TGF_受体的相关性研究[J].中华中医药杂志,2014,29(11):3626-3630.
[2]刘康宝.中老年膝骨关节炎发病的相关因素分析[J].世界最新医学信息文摘,2015,15(31):6-8.
[3]尹丽.中老年膝骨关节炎发病的相关因素分析[J].吉林医学,2015,36(18):4139-4141.
[4]Li J,Anemaet W,Diaz MA,et al.Knockout of ADAMTS5 does not eliminate cartilage aggrecanase activity but abrogates joint fibrosis and promotes cartilage aggrecan deposition in murine osteoarthritis models.J Orthop Res.2011;29(4):516-522.
[5]朱剑,黄烽,张一平.类风湿关节炎患者蛋白聚糖aggrecan+G1区特异性T细胞诱导SCID小鼠产生膝关节炎的实验研究[J].解放军医学杂志,2006,31(8):787-790.
[6]周惠琼,钱宏伟,杨文芳.外周血蛋白聚糖代谢片段检测水平与类风湿关节炎影像学进展Sharp评分的相关性[J].中华风湿病学杂志,2017,21(3):172-178.
[7]杜国辉,屈爱存,陈建英.透明质酸对关节软骨蛋白聚糖aggrecan的影响[J].中国生化药物杂志,2011,32(2):125-8.
[8]高宗强,郭雄.透明质酸对体外培养大骨节病软骨细胞Ⅱ型胶原和聚集蛋白聚糖m RNA表达的影响[J].中国组织工程研究与临床康复,2009,13(46):9061-9066.
[9]周惠琼,张奉春.肿瘤坏死因子-α和白细胞介素-1β对人骨关节炎软骨细胞蛋白聚糖代谢的影响[J].中华风湿病学杂志,2008,12(2):83-88.
[10]Stambas J,Stanton H,Rogerson FM,et al.ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.PLo S Biol.2005;434(7033):648-652.
[11]孙志涛.不同方法提取SD大鼠膝关节软骨组织的总RNA[J].中国组织工程研究,2014,18(51):8201-8205.
[12]娄思权.骨关节炎的病理与发病因素[J].中华骨科杂志,1996,16(1):56-60.
[13]谷艳超.骨关节炎发病机制和治疗的研究进展[J].中国骨与关节杂志,2016,5(10):770-774.
[14]安丙辰,王有.不同强度张应力刺激影响关节软骨细胞Ⅱ型胶原和聚集蛋白聚糖m RNA表达的研究[J].中华关节外科杂志,2010,4(3):362-368.
[15]陈孟交.丹紫康膝冲剂对兔膝骨性关节炎软骨含水率及羟脯氨酸影响的研究[J].湖南中医药大学学报,2012,32(1):20-3.
[16]王坤.T140靶向阻断SDF-1%2f CXCR4信号通路对豚鼠关节软骨Ⅱ型胶原蛋白及聚集蛋白聚糖的影响[J].中国运动医学杂志,2016,36(1):23-27.
[17]董琪.黄芪桂枝五物汤对肩周炎兔模型羟脯氨酸与总蛋白含量测定的实验研究[J].陕西中医,2015,36(8):1081-1083.
[18]陈喜德,宋丽君.骨关节炎SD大鼠关节软骨中蛋白多糖与YKL-40的相关性[J].重庆医学,2016,43(10):1214-1218.
[19]van den Boom R,Brama PA,Kiers GH,et al.Assessment of the effects of age and joint disease on hydroxyproline and glycosaminoglycan concentrations in synovial fluid from the metacarpophalangeal joint of horses.Am J Vet Res.2004;65(3):296-302.
[20]Smith SM,Shu C,Melrose J.Comparative immunolocalisation of perlecan with collagen II and aggrecan in human foetal,newborn and adult ovine joint tissues demonstrates perlecan as an early developmental chondrogenic marker.Histochem Cell Biol.2010;134(3):251-263.
[21]刘晓斌,候瑞.软硬食性对生长早期小鼠颞下颌关节髁突软骨中Aggrecan与ADAMTS-5表达的影响[J].医学研究杂志,2016,45(6):161-164.
[22]李彦林.体外阻断基质细胞衍生因子1/CXC趋化因子受体4信号通路后人关节软骨组织Ⅱ型胶原和聚集蛋白聚糖的表达[J].中国组织工程研究,2012,16(46):8607-8611.
[23]Feng J,Gu Z,Lin X,et al.Postnatal development of type IIcollagen and aggrecan m RNA expression in a rabbit craniomandibular joint.Anat Rec(Hoboken).2010;293(9):1574-1580.
[24]Azadi M,Nia HT,Gauci SJ,et al.Wide bandwidth nanomechanical assessment of murine cartilage reveals protection of aggrecan knock-in mice from joint-overuse.JBiomech.2016;49(9):1634-1640.
[25]杜清峰,张海宁.慢病毒介导的aggrecanase-2+sh RNA转染对类风湿关节炎患者软骨细胞aggrecan的影响[J].中华骨科杂志,2014,34(9):936-945.
[26]袁红,陈榕.平衡针灸对肩周炎模型兔转化生长因子β1、前列腺素+E2、羟脯氨酸影响的实验研究[J].环球中医药,2015,8(1):42-46.
[27]牛维.单味药鹿茸调控大鼠骨关节炎软骨组织Smad2_3表达的研究[J].中国中西医结合杂志,2014,34(2):209-214.
[28]牛维.单味中药鹿茸调控骨关节炎软骨组织TGF-β1表达的研究[J].时珍国医国药,2014,25(3):528-532.
[29]牛维.鹿茸归经与早期骨关节炎软骨靶器官TGF_受体的相关性研究[J].中华中医药杂志,2014,29(11):3626-3630.