补肾壮筋汤联合盐酸氨基葡萄糖对大鼠膝软骨缺损修复机制探讨
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摘要
目的:研究中药补肾壮筋汤联合西药盐酸氨基葡萄糖作用于大鼠,观察大鼠膝软骨缺损修复过程中中西医药物的联合应用是否能够更为有效的促进大鼠膝软骨缺损的修复,为膝软骨损伤能够得到更好的治疗提供新的理论依据及方法。方法:将SPF级大鼠(64只)随机分为5组,sham组,模型组,补肾壮筋汤组(7.5 g·kg~(-1)·d~(-1)),盐酸氨基葡萄糖组(7.5 g·kg~(-1)·d~(-1)),补肾壮筋汤联合盐酸氨基葡萄糖组[(7.5+7.5) g·kg~(-1)·d~(-1)]对大鼠进行药物灌胃,通过大体观察、扫描电镜观察4,8,12周鼠膝软骨缺损修复情况;实时荧光定量PCR (Real-time PCR)检测各组Ⅱ型胶原(CollagenⅡ),聚集蛋白聚糖(Aggrecan) mRNA表达;蛋白免疫印迹法(Western blot)检测CollagenⅡ蛋白表达。结果:4,8,12周与模型组比较,补肾壮筋汤联合盐酸氨基葡萄糖组软骨缺损处透明软骨填充,钻孔处软骨修复平整、光滑,与周边软骨组织融合为一体,明显优于其他各组; Real-time PCR,Western blot结果显示,与模型组比较,4,8,12周各给药组CollagenⅡ,Aggrecan mRNA和蛋白的表达显著增高(P<0.01),补肾壮筋汤联合盐酸氨基葡萄糖组与补肾壮筋汤组、盐酸氨基葡萄糖组比较增高最为显著(P<0.01);通过扫描电镜观察与模型组比较,补肾壮筋汤联合盐酸氨基葡萄糖组可见软骨缺损处虽然可见缺损边界,再生软骨组织表面平坦,基本与周边正常软骨组织融合,结构形态已趋于正常软骨组织,明显优于其他各组。结论:中西医药物联合应用能够明显促进大鼠膝软骨缺损的修复,从mRNA及蛋白表达水平及微观结构分析了中西医结合治疗软骨损伤的可能机制。
Objective:To study the effect of Bushen Zhuangjintang and glucosamine hydrochloride on the repairing of rat knee cartilage defect,the combined application of traditional Chinese and western medicine can promote the repairing of knee cartilage defect more effectively.To provide a new theoretical basis and method for the treatment of knee cartilage injury.Method:SPF rats(64 rats) were randomly divided into 5 groups,sham group,model group,Bushen Zhuangjintang group(7.5 g ·kg~(-1)·d~(-1)),and glucosamine hydrochloride group(7.5 g·kg~(-1)·d~(-1)),Bushen Zhuangjin Decoction combined with glucosamine hydrochloride group[(7.5 + 7.5) g·kg~(-1)·d~(-1)]was administered to rats by drug gavage.The knee cartilage defects were repaired by gross observation and scanning electron microscopy at 4,8 and 12 weeks.Real-time PCR was used to detect the expression of Collagen Ⅱ and Aggrecan mRNA in each group.Western blot was used to detect the expression of Collagen Ⅱ.Result:4,8,12 weeks compared with group,Bushen Zhuangjintang combined with hyaluronic acid in the glucosamine hydrochloride group was filled with hyaline cartilage.The cartilage in the drilled area was smooth and smooth,and integrated with the surrounding cartilage tissue,which was superior to other groups.Realtime PCR and Western blot analysis showed that the expression of Aggrecan protein and mRNA were significantly increased at 4,8 and 12 weeks compared with the blank group(P<0.01).Bushen Zhuangjintang combined with glucosamine hydrochloride group and Bushen Zhuangjintang group,the increase of glucosamine hydrochloride group was the most significant(P<0.01).Compared with model group,Bushen Zhuangjintang combined with glucosamine hydrochloride showed that the defect was found in the cartilage defect,and the surface of the regenerated cartilage was flat.Normal cartilage tissue fusion,structural morphology has tended to normal cartilage tissue,significantly better than the other groups.Conclusion:The combined application of traditional Chinese and western medicine can obviously promote the repair of cartilage defects in the knee of rats.The possible mechanism of the treatment of cartilage injury by the combination of traditional Chinese and western medicine is analyzed from the level of gene protein expression and microstructure.
Objective:To study the effect of Bushen Zhuangjintang and glucosamine hydrochloride on the repairing of rat knee cartilage defect,the combined application of traditional Chinese and western medicine can promote the repairing of knee cartilage defect more effectively.To provide a new theoretical basis and method for the treatment of knee cartilage injury.Method:SPF rats(64 rats) were randomly divided into 5 groups,sham group,model group,Bushen Zhuangjintang group(7.5 g ·kg~(-1)·d~(-1)),and glucosamine hydrochloride group(7.5 g·kg~(-1)·d~(-1)),Bushen Zhuangjin Decoction combined with glucosamine hydrochloride group[(7.5 + 7.5) g·kg~(-1)·d~(-1)]was administered to rats by drug gavage.The knee cartilage defects were repaired by gross observation and scanning electron microscopy at 4,8 and 12 weeks.Real-time PCR was used to detect the expression of Collagen Ⅱ and Aggrecan mRNA in each group.Western blot was used to detect the expression of Collagen Ⅱ.Result:4,8,12 weeks compared with group,Bushen Zhuangjintang combined with hyaluronic acid in the glucosamine hydrochloride group was filled with hyaline cartilage.The cartilage in the drilled area was smooth and smooth,and integrated with the surrounding cartilage tissue,which was superior to other groups.Realtime PCR and Western blot analysis showed that the expression of Aggrecan protein and mRNA were significantly increased at 4,8 and 12 weeks compared with the blank group(P<0.01).Bushen Zhuangjintang combined with glucosamine hydrochloride group and Bushen Zhuangjintang group,the increase of glucosamine hydrochloride group was the most significant(P<0.01).Compared with model group,Bushen Zhuangjintang combined with glucosamine hydrochloride showed that the defect was found in the cartilage defect,and the surface of the regenerated cartilage was flat.Normal cartilage tissue fusion,structural morphology has tended to normal cartilage tissue,significantly better than the other groups.Conclusion:The combined application of traditional Chinese and western medicine can obviously promote the repair of cartilage defects in the knee of rats.The possible mechanism of the treatment of cartilage injury by the combination of traditional Chinese and western medicine is analyzed from the level of gene protein expression and microstructure.
引文
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[15]刘洪波,靖春颖,韩平.驳骨活血方联合盐酸氨基葡萄糖片治疗膝骨性关节炎临床48例临床分析[J].中国实验方剂学杂志,2015,21(15):164-167.
[16]Hochberg M C,Martel-Pelletier J,Monfort J,et al.Combined chondroitin sulfate and glucosamine for painful knee osteoarthritis:a multicentre,randomised,double-blind,non-inferiority trial versus celecoxib[J].Ann Rheum Dis,2016,75(1):37-44.
[17]郝秀华,韩丛成,杨锦竹,等.异硫氰酸苯酯柱前衍生化HPLC测定盐酸氨基葡萄糖的含量[J].中国实验方剂学杂志,2012,18(4):116-119.
[18]韩国栋,蒋再轶,谭洁,等.仙灵骨葆胶囊联合盐酸氨基葡萄糖片治疗膝骨性关节炎[J].中国实验方剂学杂志,2011,17(17):263-265.
[19]Jeong D H,Ullah H M A,Goo M J,et al.Effects of oral glucosamine hydrochloride and mucopolysaccharide protein in a rabbit model of osteoarthritis[J].Int JRheum Dis,2018,21(3):620-628.
[20]Brody L T.Knee osteoarthritis:clinical connections to articular cartilage structure and function[J].Phys Ther Sport,2015 16(4):301-316.
[21]梁仟,朱飞燕,黄江鸿,等.Ⅱ型胶原水凝胶在软骨损伤修复中的研究进展[J].组织工程与重建外科杂志,2017,13(4):228-231.
[22]赵赫,俞兴,唐向胜,等.现代骨科临床应用补肾壮筋汤的Meta分析[J].世界中医药,2017,12(10):2504-2508.
[2]韩广敬,衣玉胜,周凯,等.金乌骨通胶囊对肾虚血瘀型退行性膝骨性关节炎的治疗及对骨代谢指标的影响[J].中国实验方剂学杂志,2016,22(20):168-172.
[3]刘浩.补肾壮筋汤治疗膝关节骨性关节炎疗效观察[J].实用中医药杂志,2013,29(7):543.
[4]张炳冉.补肾壮筋汤对膝骨关节炎患者血清肿瘤坏死因子-α与白细胞介素-6水平的影响[J].中草药,2006,37(9):1389-1390.
[5]Sterzi S,Giordani L,Morrone M,et al.The efficacy and safety of a combination of glucosamine hydrochloride,chondroitin sulfate and bio-curcumin with exercise in the treatment of knee osteoarthritis:a randomized,doubleblind,placebo-controlled study[J].Eur J Phys Rehabil Med,2016,52(3):321-330.
[6]周岚,李燕.氨基葡萄糖对骨关节影响的临床研究进展[J].现代预防医学,2014,41(21):4018-4019,4022.
[7]冯文哲,陆伟,许鉴,等.组织工程化软骨细胞移植修复膝关节大面积全层软骨缺损[J].中国组织工程研究,2018,22(14):2152-2156.
[8]Dausse Y,Grossin L,Miralles G,et al.Cartilage repair using new polysaccharidic biomaterials:macroscopic,histological and biochemical approaches in a rat model of cartilage defect[J].Osteoarthritis Cartilage,2003,11(1):16-28.
[9]应俊,张元斌,劳杨骏,等.补肾活血方联合骨髓间充质干细胞-藻酸钙复合体修复关节软骨缺损研究[J].中华中医药杂志,2017,32(8):3778-3782.
[10]袁琴,阚卫兵,宋朋飞,等.中医药治疗骨关节炎基础研究进展[J].辽宁中医药大学学报,2012,14(1):101-104.
[11]林木南,张亮,武文,等.补肾壮筋汤治疗肝肾亏虚型膝骨关节炎的临床研究[J].风湿病与关节炎,2017,6(2):15-17,52.
[12]刘文成,黄相杰.中医药治疗膝骨性关节炎研究进展[J].中国中医骨伤科杂志,2016,24(12):79-82.
[13]彭时雨,谢雁鸣,黎元元,等.基于文献计量学方法对中医药治疗膝关节骨性关节炎主要流派的研究[J].中国中医骨伤科杂志,2016,24(9):36-41.
[14]周国顺,李雄峰,管国华,等.补肾壮筋汤含药血清对力学刺激诱导软骨细胞凋亡的影响[J].中国中西医结合杂志,2012,32(6):789-792.
[15]刘洪波,靖春颖,韩平.驳骨活血方联合盐酸氨基葡萄糖片治疗膝骨性关节炎临床48例临床分析[J].中国实验方剂学杂志,2015,21(15):164-167.
[16]Hochberg M C,Martel-Pelletier J,Monfort J,et al.Combined chondroitin sulfate and glucosamine for painful knee osteoarthritis:a multicentre,randomised,double-blind,non-inferiority trial versus celecoxib[J].Ann Rheum Dis,2016,75(1):37-44.
[17]郝秀华,韩丛成,杨锦竹,等.异硫氰酸苯酯柱前衍生化HPLC测定盐酸氨基葡萄糖的含量[J].中国实验方剂学杂志,2012,18(4):116-119.
[18]韩国栋,蒋再轶,谭洁,等.仙灵骨葆胶囊联合盐酸氨基葡萄糖片治疗膝骨性关节炎[J].中国实验方剂学杂志,2011,17(17):263-265.
[19]Jeong D H,Ullah H M A,Goo M J,et al.Effects of oral glucosamine hydrochloride and mucopolysaccharide protein in a rabbit model of osteoarthritis[J].Int JRheum Dis,2018,21(3):620-628.
[20]Brody L T.Knee osteoarthritis:clinical connections to articular cartilage structure and function[J].Phys Ther Sport,2015 16(4):301-316.
[21]梁仟,朱飞燕,黄江鸿,等.Ⅱ型胶原水凝胶在软骨损伤修复中的研究进展[J].组织工程与重建外科杂志,2017,13(4):228-231.
[22]赵赫,俞兴,唐向胜,等.现代骨科临床应用补肾壮筋汤的Meta分析[J].世界中医药,2017,12(10):2504-2508.