川芎嗪对膝骨性关节炎大鼠软骨下骨中miR-20b/VEGF和BMP2/Smad1通路的影响
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摘要
目的:研究川芎嗪对膝骨性关节炎(KOA)模型大鼠软骨下骨中微小核糖核酸miR-20b/血管内皮生长因子(VEGF)和骨形态发生蛋白2(BMP2)/Smad1通路的影响,并探讨川芎嗪防治KOA的作用机制。方法:取健康雄性SD大鼠18只,随机分为正常对照组、模型组、川芎嗪组,每组6只。对后两组大鼠通过膝关节腔注射4%木瓜蛋白酶溶液建立KOA模型。末次注射后第2天起,川芎嗪组大鼠灌胃川芎嗪混悬液(100 mg/kg)2 m L,正常对照组、模型组大鼠灌胃等体积生理盐水,每天1次,连续6周。给药结束后,暴露大鼠双侧膝关节软骨进行大体情况观察。截取大鼠膝关节,进行切片和苏木精-伊红(HE)染色,在显微镜下观察组织病理学变化,并采用改良的Mankin’s评分进行组织学评分。采用逆转录-聚合酶链反应(RT-PCR)法检测大鼠软骨下骨组织中VEGF、BMP2、Smad1的mRNA表达和miR-20b表达水平;采用Western Blot法检测VEGF、BMP2、Smad1的蛋白表达水平。结果:模型组和川芎嗪组大鼠的膝关节均出现不同程度的软骨损伤;与正常对照组比较,模型组大鼠膝关节软骨组织Mankin’s评分显著升高(P<0.01);软骨下骨组织中BMP、Smad1的m RNA和蛋白表达以及miR-20b表达水平均显著降低,VEGF的mRNA和蛋白表达水平均显著升高(P<0.01)。与模型组比较,川芎嗪组大鼠关节软骨组织Mankin’s评分显著降低(P<0.01);软骨下骨组织中BMP、Smad1的mRNA和蛋白表达以及miR-20b表达水平均显著升高,VEGF的mRNA和蛋白表达水平均显著降低(P<0.05或P<0.01)。结论:川芎嗪能修复KOA模型大鼠损伤的关节软骨,其机制可能是通过上调软骨下骨中miR-20b表达水平,促进VEGF mRNA降解继而抑制VEGF蛋白的表达,同时激活BMP-2/Smad1信号通路而实现的。
OBJECTIVE:To study the effects of ligustrazine on miR-20 b/VEGF and BMP2/Smad1 pathways in subchondral bone of knee osteoarthritis(KOA)model rats,and to investigate the mechanism of ligustrazine for KOA prevention and treatment.METHODS:Totally 18 healthy male SD rats were randomly divided into normal control group, model group and ligustrazine group,with 6 rats in each group. The rats in the latter two groups were used to establish KOA model by intraarticular injection of 4% papain solution. From the 2 nd day after the last injection, ligustrazine group was given intragastrical administration of Ligustrazine suspension(100 mg/kg) 2 mL;normal control group and model group were given intragastrical administration of isometrical normal saline,once a day,for consecutive 6 weeks. After the last after medication,the situation of bilateral knee articular cartilage of rats were observed after exposure. The knee joints of rats were sectioned and stained with HE. The pathological change of articular cartilage were observed by microscope and scored by modified Mankin's score. mRNA expression of VEGF,BMP2 and Smad1,and the expression of miR-20 b were detected by RT-PCR;the protein expression of VEGF,BMP2 and Smad1 were detected by Western blot assay. RESULTS:Model group and ligustrazine group suffered from cartilage injury of knee joint at varying degrees. Compared with normal control group,Mankin's scores of knee joint and cartilage tissue were increased significantly in model group(P<0.01);mRNA and protein expression of BMP and Smad1,the expression of miR-20 b in subchondral bone of model group were decreased significantly,while mRNA and protein expression of VEGF were increased significantly(P<0.01). Compared with model group,Mankin's score of cartilage tissue were decreased significantly in ligustrazine group(P<0.01);mRNA and protein expression of BMP and Smad1,the expression of miR-20 b in subchondral bone were increased significantly,while mRNA and protein expression of VEGF were decreased significantly(P<0.05 or P<0.01). CONCLUSIONS:Ligustrazine can repair damaged articular cartilage in KOA model rats,the mechanism of which may be associated with inhibiting the protein expression of VEGF and activating BMP-2/Smad1 signaling pathway via upregulating the expression of miR-20 b,and promoting the degradation of VEGF mRNA in subchondral bone.
OBJECTIVE:To study the effects of ligustrazine on miR-20 b/VEGF and BMP2/Smad1 pathways in subchondral bone of knee osteoarthritis(KOA)model rats,and to investigate the mechanism of ligustrazine for KOA prevention and treatment.METHODS:Totally 18 healthy male SD rats were randomly divided into normal control group, model group and ligustrazine group,with 6 rats in each group. The rats in the latter two groups were used to establish KOA model by intraarticular injection of 4% papain solution. From the 2 nd day after the last injection, ligustrazine group was given intragastrical administration of Ligustrazine suspension(100 mg/kg) 2 mL;normal control group and model group were given intragastrical administration of isometrical normal saline,once a day,for consecutive 6 weeks. After the last after medication,the situation of bilateral knee articular cartilage of rats were observed after exposure. The knee joints of rats were sectioned and stained with HE. The pathological change of articular cartilage were observed by microscope and scored by modified Mankin's score. mRNA expression of VEGF,BMP2 and Smad1,and the expression of miR-20 b were detected by RT-PCR;the protein expression of VEGF,BMP2 and Smad1 were detected by Western blot assay. RESULTS:Model group and ligustrazine group suffered from cartilage injury of knee joint at varying degrees. Compared with normal control group,Mankin's scores of knee joint and cartilage tissue were increased significantly in model group(P<0.01);mRNA and protein expression of BMP and Smad1,the expression of miR-20 b in subchondral bone of model group were decreased significantly,while mRNA and protein expression of VEGF were increased significantly(P<0.01). Compared with model group,Mankin's score of cartilage tissue were decreased significantly in ligustrazine group(P<0.01);mRNA and protein expression of BMP and Smad1,the expression of miR-20 b in subchondral bone were increased significantly,while mRNA and protein expression of VEGF were decreased significantly(P<0.05 or P<0.01). CONCLUSIONS:Ligustrazine can repair damaged articular cartilage in KOA model rats,the mechanism of which may be associated with inhibiting the protein expression of VEGF and activating BMP-2/Smad1 signaling pathway via upregulating the expression of miR-20 b,and promoting the degradation of VEGF mRNA in subchondral bone.
引文
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[14]MOBASHERI A,RAYMAN MP,GUALILLO O,et al.The role of metabolism in the pathogenesis of osteoarthritis[J].Nat Rev Rheumatol,2017,13(5):302-311.
[15]谢平金,史桐雨,梁桂洪,等.川芎嗪对膝骨性关节炎大鼠软骨BMP-2、Smad1及BMP-2 mRNA、Smad1 mRNA表达的影响[J].中国骨质疏松杂志,2018,24(6):727-731、825.
[16]谢平金,余翔,柴生颋,等.川芎嗪干预早期膝骨关节炎大鼠软骨Ⅱ型胶原纤维α1基因与血管内皮生长因子mRNA及miR20b的表达[J].中国组织工程研究,2018,22(12):1846-1851.
[17]李飞龙,谢平金,柴生颋,等.川芎嗪对膝骨性关节炎大鼠软骨VEGF表达的影响[J].中国骨质疏松杂志,2018,24(7):904-909.
[18]HAMILTON JL,NAGAO M,LEVINE BR,et al.Targeting VEGF and its receptors for the treatment of osteoarthritis and associated pain[J].J Bone Miner Res,2016,31(5):911-924.
[19]KUBO S,COOPER GM,MATSUMOTO T,et al.Blocking vascular endothelial growth factor with soluble Flt-1improves the chondrogenic potential of mouse skeletal muscle-derived stem cells[J].Arthritis Rheum,2009,60(1):155-165.
[20]LIU SC,CHUANG SM,HSU CJ,et al.CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210expression[J].Cell Death Dis,2014.DOI:10.1038/cddis.2014.453.
[21]CASCIO S,D’ANDREA A,FERLA R,et al.miR-20b modulates VEGF expression by targeting HIF-1 alpha and STAT3 in MCF-7 breast cancer cells[J].J Cell Physiol,2010,224(1):242-249.
[22]陶象男,汪忆梦,宋传旺.miR-20b直接靶向3′-UTR负性调节VEGF的表达[J].华中科技大学学报(医学版),2016,45(1):22-26.
[23]KUMAR Y,BISWAS T,THACKER G,et al.BMP signaling-driven osteogenesis is critically dependent on Prdx-1expression-mediated maintenance of chondrocyte prehypetrophy[J].Free Radic Biol Med,2018.DOI:10.1016/j.freeradbiomed.2018.02.016.
[24]ABEDE,BOUVARD B,MARTINEAU X,et al.Elevated hepatocyte growth factor levels in osteoarthritis steoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced ineralization capacity[J].Bone,2015.DOI:10.1016/j.bone.2015.02.001.
[25]TASCA A,STEMIG M,BROEGE A,et al.Smad1/5 and smad4 expression are important for osteoclast differentiation[J].J Cell Biochem,2015,116(7):1350-1360.
[2]ILAS DC,CHURCHMAN SM,MCGONAGLE D,et al.Targeting subchondral bone mesenchymal stem cell activities for intrinsic joint repair in osteoarthritis[J].Future Sci OA,2017.DOI:10.4155/fsoa-2017-0055.
[3]曹一汀.FGF2调控软骨下骨活性促进关节软骨损伤修复的研究[D].北京:北京协和医学院,2017.
[4]ZHANG Z,YANG W,CAO Y,et al.The functions of BMP3 in rabbit articular cartilage repair[J].Int J Mol Sci,2015,16(1):25934-25946.
[5]PRASADAM I,BATRA J,PERRY S,et al.Systematic identification,characterization and target gene analysis of microRNAs involved in osteoarthritis subchondral bone pathogenesis[J].Calcif Tissue Int,2016,99(1):43-55.
[6]OGINO S,SASHO T,NAKAGAWA K,et al.Detection of pain-related molecules in the subchondral bone of osteoarthritic knees[J].Clin Rheumatol,2009,28(1):1395-1402.
[7]胡旭光,甘仲霖,张毅.川芎嗪对骨关节炎兔软骨细胞增殖与凋亡的影响及其机制研究[J].现代中西医结合杂志,2016,25(22):2418-2421.
[8]梁桂洪,孙赫,黄宇新,等.川芎嗪对实验性骨关节炎模型大鼠关节液中NO和PGE2的调节作用[J].动物医学进展,2014,35(9):66-69.
[9]刘刚,周海宇.川芎嗪注射液灌洗联合玻璃酸钠注射对退行性膝骨关节炎患者关节液中SDF-1、MMPs水平的影响[J].临床医学研究与实践,2018,3(31):48-49.
[10]段文秀,汪宗保,张浩,等.木瓜蛋白酶诱导早期膝骨关节炎模型大鼠软骨超微结构的动态变化[J].中国组织工程研究,2015,19(18):2789-2793.
[11]刘国玲,芦琨,郭辉,等.川芎嗪对大鼠胶原性关节炎及免疫功能的影响[J].中药药理与临床,2014,30(1):52-55.
[12]MANKIN HJ,DORFMAN H,LIPPIELLO L,et al.Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hipsⅡ:correlation of morphology with biochemical and metabolic data[J].JBone Joint Surg,1971,53(3):523-537.
[13]SCHMITTGEN TD,LIVAK KJ.Analyzing real-time PCRdata by the comparative C(T)method[J].Nat Protoc,2008,3(6):1101-1108.
[14]MOBASHERI A,RAYMAN MP,GUALILLO O,et al.The role of metabolism in the pathogenesis of osteoarthritis[J].Nat Rev Rheumatol,2017,13(5):302-311.
[15]谢平金,史桐雨,梁桂洪,等.川芎嗪对膝骨性关节炎大鼠软骨BMP-2、Smad1及BMP-2 mRNA、Smad1 mRNA表达的影响[J].中国骨质疏松杂志,2018,24(6):727-731、825.
[16]谢平金,余翔,柴生颋,等.川芎嗪干预早期膝骨关节炎大鼠软骨Ⅱ型胶原纤维α1基因与血管内皮生长因子mRNA及miR20b的表达[J].中国组织工程研究,2018,22(12):1846-1851.
[17]李飞龙,谢平金,柴生颋,等.川芎嗪对膝骨性关节炎大鼠软骨VEGF表达的影响[J].中国骨质疏松杂志,2018,24(7):904-909.
[18]HAMILTON JL,NAGAO M,LEVINE BR,et al.Targeting VEGF and its receptors for the treatment of osteoarthritis and associated pain[J].J Bone Miner Res,2016,31(5):911-924.
[19]KUBO S,COOPER GM,MATSUMOTO T,et al.Blocking vascular endothelial growth factor with soluble Flt-1improves the chondrogenic potential of mouse skeletal muscle-derived stem cells[J].Arthritis Rheum,2009,60(1):155-165.
[20]LIU SC,CHUANG SM,HSU CJ,et al.CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210expression[J].Cell Death Dis,2014.DOI:10.1038/cddis.2014.453.
[21]CASCIO S,D’ANDREA A,FERLA R,et al.miR-20b modulates VEGF expression by targeting HIF-1 alpha and STAT3 in MCF-7 breast cancer cells[J].J Cell Physiol,2010,224(1):242-249.
[22]陶象男,汪忆梦,宋传旺.miR-20b直接靶向3′-UTR负性调节VEGF的表达[J].华中科技大学学报(医学版),2016,45(1):22-26.
[23]KUMAR Y,BISWAS T,THACKER G,et al.BMP signaling-driven osteogenesis is critically dependent on Prdx-1expression-mediated maintenance of chondrocyte prehypetrophy[J].Free Radic Biol Med,2018.DOI:10.1016/j.freeradbiomed.2018.02.016.
[24]ABEDE,BOUVARD B,MARTINEAU X,et al.Elevated hepatocyte growth factor levels in osteoarthritis steoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced ineralization capacity[J].Bone,2015.DOI:10.1016/j.bone.2015.02.001.
[25]TASCA A,STEMIG M,BROEGE A,et al.Smad1/5 and smad4 expression are important for osteoclast differentiation[J].J Cell Biochem,2015,116(7):1350-1360.