补肾活血汤干预骨质疏松症并骨关节炎模型兔软骨细胞NF-KBp65蛋白的表达
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摘要
背景:临床研究发现肾虚血淤是骨性关节炎、骨质疏松症共同病因、病机,运用补肾活血法治疗该类病变疗效肯定。目的:进一步验证补肾活血汤对兔软骨细胞NF-KBp65蛋白表达的影响。方法:雌性新西兰大白兔37只,由贵阳医学院动物实验中心提供,实验方案经贵阳医学院动物实验伦理委员会批准(批准号:2015-024)。随机选取30只大白兔编号后随机均分为3组,分别建立骨质疏松模型、骨关节炎模型、骨质疏松+骨关节炎模型。剩余7只用于制备7种含药血清(分别为正常组、低剂量补肾活血汤组、中剂量补肾活血汤组、高剂量补肾活血汤组、盐酸氨基葡萄糖组、阿仑膦酸钠组、盐酸氨基葡萄糖+阿仑磷酸钠组)。采用Western-blot检测7组药物对3组模型兔软骨细胞NF-KBp65蛋白表达的影响。结果与结论:①中、高剂量组有下调细胞中NF-KBp65蛋白的作用,与正常组对比差异有显著性(P <0.05),其中以高剂量组作用最为明显,与低、中剂量组对比差异有显著性意义(P<0.05);②高剂量组下调NF-KBp65蛋白表达的作用较阿仑膦酸钠组和盐酸氨基葡萄糖组明显(P <0.05);③高剂量组与盐酸氨基葡萄糖+阿仑膦酸钠组在骨质疏松症组对比差异有显著性意义(t=7.597,P <0.05),但在骨关节炎组及骨关节炎+骨质疏松症组中则差异无显著性意义(P>0.05),说明高剂量组下调NF-KBp65蛋白表达的作用优于阿仑膦酸钠组及盐酸氨基葡萄糖组,与盐酸氨基葡萄糖+阿仑膦酸钠组大体相当;④结果表明,补肾活血汤能下调NF-KBp65蛋白表达,从而减轻或抑制骨质疏松症和骨关节炎的炎症反应,间接或直接起到保护或修复软骨细胞的作用。
BACKGROUND: Kidney deficiency and blood stasis are the common etiology and pathogenesis of osteoarthritis and osteoporosis. The method of nourishing kidney and activating blood circulation is effective for treating these diseases.OBJECTIVE: To further investigate the effects of Bushen Huoxue Decoction on the expression level of nuclear factor-KBp65 protein in rabbit chondrocytes.METHODS: Thirty-seven female New Zealand white rabbits were provided by the Laboratory Animal Center of Guiyang Medical University,and the study was approved by the Experimental Animal Ethics Committee of Guiyang Medical University, approval number: 2015-024. Thirty rabbits were randomly divided into three groups, and the osteoporosis, osteoarthritis and osteoporosis + osteoarthritis models, were separately established. The remaining seven rabbits were used to prepare the serum containing drug, such as normal, low-, moderate-, and high-dose Bushen Huoxue Decoction, glucosamine hydrochloride, alendronate sodium, and glucosamine hydrochloride + alendronate sodium groups. The effect of seven groups of drugs on the expression level of nuclear factor-KBp65 protein in rabbit chondrocytes of three kinds of models was detected by western blot assay.RESULTS AND CONCLUSION:(1) Moderate-and high-dose Bushen Huoxue Decoction down-regulated nuclear factor-KBp65 protein level in cells, which was significantly different from the normal group(P < 0.05). The effect was most obvious in the high-dose Bushen Huoxue Decoction group, compared with the low-and the moderate-dose groups(P < 0.05).(2) The down-regulation effect of unclear factor-KBp65 protein expression in the high-dose Bushen Huoxue Decoction group was significantly higher than that in the glucosamine hydrochloride and alendronate sodium groups(P < 0.05).(3) There was a significant difference between the high-dose Bushen Huoxue Decoction and glucosamine hydrochloride + alendronate sodium groups in the osteoporosis group(t=7.597, P < 0.05), but there was no significant difference between the osteoarthritis and osteoporosis + osteoarthritis groups(P > 0.05). Therefore, the down-regulation effect of unclear factor-KBp65 protein expression in the high-dose Bushen Huoxue Decoction group was better than that in the glucosamine hydrochloride and alendronate sodium groups, which was similar to that in the glucosamine hydrochloride + alendronate sodium group.(4) These results indicate that Bushen Huoxue Decoction can down-regulate the expression of nuclear factor-KBp65 protein, so as to reduce or inhibit the inflammatory reaction of osteoporosis and osteoarthritis, and indirectly or directly plays a role in protecting or repairing chondrocytes.
BACKGROUND: Kidney deficiency and blood stasis are the common etiology and pathogenesis of osteoarthritis and osteoporosis. The method of nourishing kidney and activating blood circulation is effective for treating these diseases.OBJECTIVE: To further investigate the effects of Bushen Huoxue Decoction on the expression level of nuclear factor-KBp65 protein in rabbit chondrocytes.METHODS: Thirty-seven female New Zealand white rabbits were provided by the Laboratory Animal Center of Guiyang Medical University,and the study was approved by the Experimental Animal Ethics Committee of Guiyang Medical University, approval number: 2015-024. Thirty rabbits were randomly divided into three groups, and the osteoporosis, osteoarthritis and osteoporosis + osteoarthritis models, were separately established. The remaining seven rabbits were used to prepare the serum containing drug, such as normal, low-, moderate-, and high-dose Bushen Huoxue Decoction, glucosamine hydrochloride, alendronate sodium, and glucosamine hydrochloride + alendronate sodium groups. The effect of seven groups of drugs on the expression level of nuclear factor-KBp65 protein in rabbit chondrocytes of three kinds of models was detected by western blot assay.RESULTS AND CONCLUSION:(1) Moderate-and high-dose Bushen Huoxue Decoction down-regulated nuclear factor-KBp65 protein level in cells, which was significantly different from the normal group(P < 0.05). The effect was most obvious in the high-dose Bushen Huoxue Decoction group, compared with the low-and the moderate-dose groups(P < 0.05).(2) The down-regulation effect of unclear factor-KBp65 protein expression in the high-dose Bushen Huoxue Decoction group was significantly higher than that in the glucosamine hydrochloride and alendronate sodium groups(P < 0.05).(3) There was a significant difference between the high-dose Bushen Huoxue Decoction and glucosamine hydrochloride + alendronate sodium groups in the osteoporosis group(t=7.597, P < 0.05), but there was no significant difference between the osteoarthritis and osteoporosis + osteoarthritis groups(P > 0.05). Therefore, the down-regulation effect of unclear factor-KBp65 protein expression in the high-dose Bushen Huoxue Decoction group was better than that in the glucosamine hydrochloride and alendronate sodium groups, which was similar to that in the glucosamine hydrochloride + alendronate sodium group.(4) These results indicate that Bushen Huoxue Decoction can down-regulate the expression of nuclear factor-KBp65 protein, so as to reduce or inhibit the inflammatory reaction of osteoporosis and osteoarthritis, and indirectly or directly plays a role in protecting or repairing chondrocytes.
引文
[1]Kraus VB,Kilfoil TM,Hash TW,et al.Atlas of radiographic features of osteoarthritis of the ankle and hindfo.Osteoarthritis Cartilage.2015;23(12):2059-2085.
[2]钱兴皋,刘宇恒,王琴,等.炎症因子对骨质疏松症发病影响的研究进展[J].中国康复理论与实践,2013,19(7):645-646.
[3]任海亮,马剑雄,马信龙.膝骨关节炎时关节滑液中炎症相关物质的表达[J].中国组织工程研究,2015,19(15):2336-2339.
[4]夏婷,李双庆.炎症相关骨质疏松症的发病机制[J].中国骨质疏松杂志,2015,21(1):17-120.
[5]马玉环,郑文伟,林平冬,等.骨关节炎软骨退变与炎症的关系[J].风湿病与关节炎,2015,4(8):50-53.
[6]Roman-Blas JA,Jimenez SA.NF-KB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis.Osteoarthritis Cartilage.2006;14(9):839-848.
[7]应俊,张元斌,罗程,等.核转录因子-κB在骨关节炎炎症反应中的作用[J].中医正骨,2015,27(7):40-46.
[8]Sirotkin AV,Alexa R,KisovaG,et al.MicroRNAs control transcription factor NF-KB(p65)expression in human ovarian cells.Funct Integr Genomics.2015;15(3):271-275.
[9]林华,陈新,张咏梅,等.绝经后妇女骨质疏松与骨关节炎的相关性研究[J].中国骨质疏松杂志,2006,12(5):488-489.
[10]沈冯君,李文顺,易洪城.膝骨性关节炎的临床治疗与机理研究[D].贵阳:贵阳中医学院,2003.
[11]刘日光,赵瑜,沈冯君,等.两种不同补肾方法治疗去卵巢大鼠骨质疏松症的对比研究[J].中国中医骨伤科杂志,2003,11(1):12-15.
[12]陈巨鹏.“补肾活血方”对绝经后骨质疏松症影响的实验研究[D].南京:南京中医药大学,2016.
[13]刘维,张磊,吴沅皞.补肾活血法治疗老年骨质疏松症100例随机对照研究[J].中医杂志,2015,56(9):769-772.
[14]马文娟,张开伟,沈冯君,等.骨性关节炎与骨质疏松症共病模型兔的建立及鉴定骨性关节炎与骨质疏松症共病模型兔的建立及鉴定[J].中国组织工程研究,2019,23(3):421-426.
[15]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[16]吴连国,刘康,王定,等.强骨饮治疗骨关节炎合并骨质疏松患者的临床研究[J].中国中医骨伤科杂志,2011,19(12):10-13
[17]吴斌,李平元,董忠,等.膝骨性关节炎与骨质疏松症相关性的研究进展[J].福建中医药,2014,45(3):62-63.
[18]沈冯君.痹和痿与相关骨疾病证的关系[J].中医正骨,2004,16(10):21-22.
[19]熊江华,倪寅,李艳.《医方考》论痹痿病[J].中医药临床杂志,2016,28(1):36-37.
[20]明磊国,陈克明,葛宝丰,等.异补骨脂素对体外培养大鼠成骨细胞增殖分化成熟的影响[J].中药材,2011,34(3):404-407.
[21]Liang H,Yu F,Tong Z,et al.Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA,Smad5m RNA,TGF-β1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease.Mol Biol Rep.2013;40(2):757-763.
[22]宋芹,董小萍,郁小兵.补骨脂提取物对体外培养小鼠成骨细胞株MC3T3-E1细胞分化的影响[J].中国中药杂志,2009,34(5):1264-1267.
[23]Scaffidi JP,Gregas MK,Lauly B,et al.Activity of psoralenfunctionalized nanoscintillators against cancer cells upon X-ray excitation.ACSNano.2011;5(6):4679-4687.
[24]钟秋生,韩临晓,陆向然,等.中西医结合对绝经后女性骨质疏松症骨代谢指标的影[C].全国第十二届中西医结合风湿病学术会议论文集,2014.
[25]程孟春,刘艳秋,王莉,等.何首乌和菟丝子对破骨细胞和成骨细胞增殖及分化的影响[J].中国中药杂志,2011,36(19):2737-2740.
[26]郑树.不同加工法影响当归多糖提取及当归多糖对去势大鼠骨质疏松作用研究[D].扬州:扬州大学,2015.
[27]Qin T,Chen J,Wang D,et al.Optimization of selenylation conditions for Chinese angelica polysaccharide based on immune-enhancing activity.Carbohydr Polym.2013;92(1):645-50.
[28]吴方方,汪鋆植,黄年玉,等.没药烷吉酮治疗痛经及机制研究[J].中国药理与临床,2013,29(5):47-49.
[29]Hanus LO,Rezanka T,Dembitskv VM,et al.Myrrh-Com-miphora chemistry.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub.2005;149(1):3-27.
[30]邱建波,徐清,姜笑寒.独活乙醇提取物对环氧化酶的影响[J].中国医药导报,2011,8(16):42-43.
[31]Kim Beum-Seuk,Lim Hyung-Ho et al.Aqueous extract of Lyciifructus suppresses inflammation through the inhibition of nuclear factor kappaB signal pathway in murine raw 264.7macrophages.Oriental Pharmacy and Experimental Medicine.2010;10(3):155-164.
[32]何蕾.红花提取物生物活性成分的研究[D].兰州:兰州理工大学,2016.
[33]金晓东,张鸣.红花注射液对兔膝骨关节炎软骨组织学及关节滑液PGE2/IL-8的影响[J].中国中医骨伤科杂志,2009,17(3):18-19.
[34]齐静姣,张平,路西明,等.山茱萸果核提取物对佐剂性关节炎大鼠免疫调节因子的影响[J].中国临床药理学杂志,2016,32(6):549-552.
[35]Kwak JK,Jung JK,Lee H.Nuclear factor-kappa Binhibitors:a patent review.Expertopin.TherPatents.2011;21(12):1897-1910
[36]Guo D,Hu B,Tang H,et al.Proteomic profiling analysis reveals a link between experimental autoimmune uveitis and complement activation in rats.Scand J Immunol.2017;85(5):331-342.
[37]孔祥,王志学.卵巢癌组织NF-KB家族蛋白的表达和临床意义[J].实用临床医药杂志,2004,8(6):42-44.
[38]Hagemann T,Kulbe H,Li NF,et al.Macrophages induce unvasiveness of epithelial cancer cells via NF-kappa B and JNK.J Immunol.2005;175(2):1197-205.
[39]陈海霞,张开伟,张波,等.补肾活血汤干预骨质疏松并骨关节炎模型兔软骨多种细胞因子m RNA的表达[J].中国组织工程研究,2018,22(33):5123-5131.
[2]钱兴皋,刘宇恒,王琴,等.炎症因子对骨质疏松症发病影响的研究进展[J].中国康复理论与实践,2013,19(7):645-646.
[3]任海亮,马剑雄,马信龙.膝骨关节炎时关节滑液中炎症相关物质的表达[J].中国组织工程研究,2015,19(15):2336-2339.
[4]夏婷,李双庆.炎症相关骨质疏松症的发病机制[J].中国骨质疏松杂志,2015,21(1):17-120.
[5]马玉环,郑文伟,林平冬,等.骨关节炎软骨退变与炎症的关系[J].风湿病与关节炎,2015,4(8):50-53.
[6]Roman-Blas JA,Jimenez SA.NF-KB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis.Osteoarthritis Cartilage.2006;14(9):839-848.
[7]应俊,张元斌,罗程,等.核转录因子-κB在骨关节炎炎症反应中的作用[J].中医正骨,2015,27(7):40-46.
[8]Sirotkin AV,Alexa R,KisovaG,et al.MicroRNAs control transcription factor NF-KB(p65)expression in human ovarian cells.Funct Integr Genomics.2015;15(3):271-275.
[9]林华,陈新,张咏梅,等.绝经后妇女骨质疏松与骨关节炎的相关性研究[J].中国骨质疏松杂志,2006,12(5):488-489.
[10]沈冯君,李文顺,易洪城.膝骨性关节炎的临床治疗与机理研究[D].贵阳:贵阳中医学院,2003.
[11]刘日光,赵瑜,沈冯君,等.两种不同补肾方法治疗去卵巢大鼠骨质疏松症的对比研究[J].中国中医骨伤科杂志,2003,11(1):12-15.
[12]陈巨鹏.“补肾活血方”对绝经后骨质疏松症影响的实验研究[D].南京:南京中医药大学,2016.
[13]刘维,张磊,吴沅皞.补肾活血法治疗老年骨质疏松症100例随机对照研究[J].中医杂志,2015,56(9):769-772.
[14]马文娟,张开伟,沈冯君,等.骨性关节炎与骨质疏松症共病模型兔的建立及鉴定骨性关节炎与骨质疏松症共病模型兔的建立及鉴定[J].中国组织工程研究,2019,23(3):421-426.
[15]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[16]吴连国,刘康,王定,等.强骨饮治疗骨关节炎合并骨质疏松患者的临床研究[J].中国中医骨伤科杂志,2011,19(12):10-13
[17]吴斌,李平元,董忠,等.膝骨性关节炎与骨质疏松症相关性的研究进展[J].福建中医药,2014,45(3):62-63.
[18]沈冯君.痹和痿与相关骨疾病证的关系[J].中医正骨,2004,16(10):21-22.
[19]熊江华,倪寅,李艳.《医方考》论痹痿病[J].中医药临床杂志,2016,28(1):36-37.
[20]明磊国,陈克明,葛宝丰,等.异补骨脂素对体外培养大鼠成骨细胞增殖分化成熟的影响[J].中药材,2011,34(3):404-407.
[21]Liang H,Yu F,Tong Z,et al.Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA,Smad5m RNA,TGF-β1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease.Mol Biol Rep.2013;40(2):757-763.
[22]宋芹,董小萍,郁小兵.补骨脂提取物对体外培养小鼠成骨细胞株MC3T3-E1细胞分化的影响[J].中国中药杂志,2009,34(5):1264-1267.
[23]Scaffidi JP,Gregas MK,Lauly B,et al.Activity of psoralenfunctionalized nanoscintillators against cancer cells upon X-ray excitation.ACSNano.2011;5(6):4679-4687.
[24]钟秋生,韩临晓,陆向然,等.中西医结合对绝经后女性骨质疏松症骨代谢指标的影[C].全国第十二届中西医结合风湿病学术会议论文集,2014.
[25]程孟春,刘艳秋,王莉,等.何首乌和菟丝子对破骨细胞和成骨细胞增殖及分化的影响[J].中国中药杂志,2011,36(19):2737-2740.
[26]郑树.不同加工法影响当归多糖提取及当归多糖对去势大鼠骨质疏松作用研究[D].扬州:扬州大学,2015.
[27]Qin T,Chen J,Wang D,et al.Optimization of selenylation conditions for Chinese angelica polysaccharide based on immune-enhancing activity.Carbohydr Polym.2013;92(1):645-50.
[28]吴方方,汪鋆植,黄年玉,等.没药烷吉酮治疗痛经及机制研究[J].中国药理与临床,2013,29(5):47-49.
[29]Hanus LO,Rezanka T,Dembitskv VM,et al.Myrrh-Com-miphora chemistry.Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub.2005;149(1):3-27.
[30]邱建波,徐清,姜笑寒.独活乙醇提取物对环氧化酶的影响[J].中国医药导报,2011,8(16):42-43.
[31]Kim Beum-Seuk,Lim Hyung-Ho et al.Aqueous extract of Lyciifructus suppresses inflammation through the inhibition of nuclear factor kappaB signal pathway in murine raw 264.7macrophages.Oriental Pharmacy and Experimental Medicine.2010;10(3):155-164.
[32]何蕾.红花提取物生物活性成分的研究[D].兰州:兰州理工大学,2016.
[33]金晓东,张鸣.红花注射液对兔膝骨关节炎软骨组织学及关节滑液PGE2/IL-8的影响[J].中国中医骨伤科杂志,2009,17(3):18-19.
[34]齐静姣,张平,路西明,等.山茱萸果核提取物对佐剂性关节炎大鼠免疫调节因子的影响[J].中国临床药理学杂志,2016,32(6):549-552.
[35]Kwak JK,Jung JK,Lee H.Nuclear factor-kappa Binhibitors:a patent review.Expertopin.TherPatents.2011;21(12):1897-1910
[36]Guo D,Hu B,Tang H,et al.Proteomic profiling analysis reveals a link between experimental autoimmune uveitis and complement activation in rats.Scand J Immunol.2017;85(5):331-342.
[37]孔祥,王志学.卵巢癌组织NF-KB家族蛋白的表达和临床意义[J].实用临床医药杂志,2004,8(6):42-44.
[38]Hagemann T,Kulbe H,Li NF,et al.Macrophages induce unvasiveness of epithelial cancer cells via NF-kappa B and JNK.J Immunol.2005;175(2):1197-205.
[39]陈海霞,张开伟,张波,等.补肾活血汤干预骨质疏松并骨关节炎模型兔软骨多种细胞因子m RNA的表达[J].中国组织工程研究,2018,22(33):5123-5131.
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