[1]王乐,徐无忌.六味地黄丸对兔椎间盘退变模型椎间盘组织中Ⅰ、Ⅱ型胶原表达的影响[J].中医正骨,2016,28(08):1-7.
　WANG Le,XU Wuji.Effect of Liuwei Dihuang Wan(六味地黄丸)on typeⅠandⅡcollagen expression in intervertebral disc of rabbit model of intervertebral disc degeneration[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2016,28(08):1-7.
点击复制

六味地黄丸对兔椎间盘退变模型椎间盘组织中Ⅰ、Ⅱ型胶原表达的影响()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第28卷
期数:
2016年08期
页码:
1-7
栏目:
基础研究
出版日期:
2016-08-20

文章信息/Info

Title:
Effect of Liuwei Dihuang Wan(六味地黄丸)on typeⅠandⅡcollagen expression in intervertebral disc of rabbit model of intervertebral disc degeneration
作者:
王乐; 徐无忌
湖南中医药大学第二附属医院,湖南 长沙 410005
Author(s):
WANG Le; XU Wuji
The Second Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Changsha 410005,Hunan,China
关键词:
椎间盘退行性变;  六味地黄丸;  胶原Ⅰ型;  胶原Ⅱ型;  兔;  动物实验
Keywords:
intervertebral disc degeneration;  Liuwei Dihuang Wan;  collagen typeⅠ;  collagen typeⅡ;  rabbits;  animal experimentation
摘要:
目的:观察六味地黄丸对兔椎间盘退变模型椎间盘组织中Ⅰ、Ⅱ型胶原表达的影响。方法:将80只新西兰兔随机分为空白组、假手术组、模型组和六味地黄组,每组20只。模型组、六味地黄组手术暴露L4～5和L5～6椎间隙,随机选择1个椎间盘注射肿瘤坏死因子α进行椎间盘退变造模,并以咬骨钳咬除该椎间盘上位椎体横突进行标记; 假手术组经相同手术入路暴露L4～5和L5～6椎间隙,不做任何处理后缝合; 空白组不进行任何手术干预。造模术后3 d开始,六味地黄组按26 mg·kg-1以六味地黄胶囊混悬液灌胃,其余3组以等量生理盐水灌胃,每天1次,共8周。分别于药物干预开始后2、4、6、8周,从各组随机选取5只兔子处死,模型组和六味地黄组手术取出造模椎间盘,空白组和假手术组随机选择L4～5或L5～6椎间盘取出。分别采用聚合酶链式反应法和Western Blot法测定椎间盘组织中Ⅰ、Ⅱ型胶原的mRNA和蛋白表达水平。结果:药物干预2、4、6、8周后,4组兔子椎间盘组织Ⅰ型胶原mRNA表达水平比较,组间差异均有统计学意义[(0.064 8±0.009 8),(0.068 6±0.012 7),(0.192 0±0.040 6),(0.124 6±0.012 0),F=49.752,P=0.000;(0.066 0±0.010 0),(0.077 1±0.011 8),(0.252 4±0.039 9),(0.164 1±0.018 1),F=79.537,P=0.000;(0.071 2±0.011 2),(0.089 1±0.008 1),(0.326 3±0.028 5),(0.176 3±0.015 0),F=236.726,P=0.000;(0.094 1±0.012 0),(0.155 0±0.003 8),(0.451 4±0.039 6),(0.205 8±0.018 1),F=201.055,P=0.000]; 模型组Ⅰ型胶原mRNA表达水平高于空白组、假手术组和六味地黄组(P=0.000,P=0.015,P=0.002,P=0.000; P=0.013,P=0.002,P=0.000,P=0.015; P=0.002,P=0.012,P=0.000,P=0.000)。空白组和假手术组的Ⅰ型胶原mRNA表达水平各时间点间比较,差异均无统计学意义(F=50.563,P=0.132; F=80.352,P=0.634); 模型组和六味地黄组的Ⅰ型胶原mRNA表达水平各时间点间比较,差异均有统计学意义(F=193.635,P=0.000; F=284.736,P=0.000),Ⅰ型胶原mRNA表达水平均逐渐增高(P=0.004,P=0.002,P=0.000; P=0.000,P=0.003,P=0.001)。药物干预2、4、6、8周后,4组兔子椎间盘组织Ⅱ型胶原mRNA表达水平比较,组间差异均有统计学意义[(0.042 7±0.008 0),(0.041 2±0.005 8),(0.011 6±0.002 1),(0.031 7±0.005 6),F=42.696,P=0.000;(0.038 8±0.004 3),(0.037 3±0.004 3),(0.011 5±0.001 8),(0.031 1±0.003 5),F=108.110,P=0.000;(0.030 3±0.005 7),(0.025 9±0.008 3),(0.007 9±0.002 7),(0.017 2±0.002 1),F=52.436,P=0.000;(0.029 3±0.006 9),(0.023 7±0.004 6),(0.005 3±0.001 0),(0.014 8±0.001 8),F=51.375,P=0.000]; 模型组Ⅱ型胶原mRNA表达水平低于空白组、假手术组和六味地黄组(P=0.002,P=0.001,P=0.000,P=0.001; P=0.001,P=0.000,P=0.000,P=0.001; P=0.001,P=0.002,P=0.013,P=0.000)。空白组和假手术组的Ⅱ型胶原mRNA表达水平各时间点间比较,差异均无统计学意义(F=70.463,P=0.122; F=90.362,P=0.089); 模型组和六味地黄组的Ⅱ型胶原mRNA表达水平各时间点间比较,差异均有统计学意义(F=110.364,P=0.001; F=86.362,P=0.004),模型组和六味地黄组的Ⅱ型胶原mRNA表达水平均逐渐降低(P=0.002,P=0.005,P=0.003; P=0.000,P=0.001,P=0.000)。药物干预2、4、6、8周后,4组兔子椎间盘组织Ⅰ型胶原蛋白表达水平比较,组间差异均有统计学意义[(0.575 9±0.135 6),(0.599 5±0.037 4),(0.620 0±0.072 0),(0.609 6±0.032 1),F=9.288,P=0.001;(0.593 9±0.108 0),(0.647 1±0.044 8),(0.807 0±0.061 6),(0.659 9±0.105 5),F=28.883,P=0.000;(0.614 0±0.224 6),(0.685 6±0.066 5),(1.129 1±0.097 4),(0.700 1±0.086 5),F=34.957,P=0.000;(0.614 8±0.139 3),(0.742 3±0.165 3),(1.322 4±0.351 7),(0.806 0±0.094 3),F=3.296,P=0.048]; 模型组Ⅰ型胶原蛋白表达水平高于空白组、假手术组和六味地黄组(P=0.000,P=0.015,P=0.002,P=0.000; P=0.013,P=0.002,P=0.000,P=0.015; P=0.002,P=0.012,P=0.000,P=0.000)。空白组和假手术组的Ⅰ型胶原蛋白表达水平各时间点间比较,差异均无统计学意义(F=37.485,P=0.365; F=10.376,P=0.583); 模型组和六味地黄组的Ⅰ型胶原蛋白表达水平各时间点间比较,差异均有统计学意义(F=70.362,P=0.000; F=6.375,P=0.001),模型组和六味地黄组的Ⅰ型胶原蛋白表达水平均逐渐增高(P=0.005,P=0.002,P=0.000; P=0.004,P=0.001,P=0.000)。药物干预2、4、6、8周后,4组兔子椎间盘组织Ⅱ型胶原蛋白表达水平比较,组间差异均有统计学意义[(0.605 8±0.066 5),(0.550 0±0.117 9),(0.278 0±0.053 2),(0.498 6±0.149 1),F=10.224,P=0.001;(0.553 7±0.126 5),(0.523 4±0.078 4),(0.258 2±0.037 8),(0.479 7±0.090 8),F=11.627,P=0.000;(0.546 8±0.120 8),(0.494 8±0.139 8),(0.219 0±0.099 2),(0.440 5±0.052 7),F=13.543,P=0.003;(0.494 8±0.042 5),(0.433 7±0.061 9),(0.131 9±0.012 8),(0.392 9±0.107 0),F=13.979,P=0.000]; 模型组Ⅱ型胶原蛋白表达水平低于空白组、假手术组和六味地黄组(P=0.002,P=0.001,P=0.000,P=0.001; P=0.001,P=0.000,P=0.000,P=0.001; P=0.001,P=0.002,P=0.013,P=0.000)。空白组和假手术组的Ⅱ型胶原蛋白表达水平各时间点间比较,差异均无统计学意义(F=17.364,P=0.092; F=15.573,P=0.175); 模型组和六味地黄组的Ⅱ型胶原蛋白表达水平各时间点间比较,差异均有统计学意义(F=17.753,P=0.001; F=13.674,P=0.000),模型组和六味地黄组的Ⅱ型胶原蛋白表达水平均逐渐降低(P=0.002,P=0.004,P=0.001; P=0.000,P=0.001,P=0.000)。结论:六味地黄丸能适度下调兔椎间盘退变模型椎间盘组织中Ⅰ型胶原的表达、上调Ⅱ型胶原的表达,可在一定程度上延缓椎间盘退变进程。
Abstract:
Objective:To observe the effect of Liuwei Dihuang Wan(六味地黄丸,LWDHW)on typeⅠandⅡcollagen expression in intervertebral disc of rabbit model of intervertebral disc degeneration.Methods:Eighty New Zealand rabbits were randomly divided into blank group,sham-operated group,model group and LWDHW group,20 cases in each group.The surgeries were performed on rabbits in model group and LWDHW group to expose L4/L5 and L5/L6 intervertebral space,and tumor necrosis factor α were injected into L4/L5 or L5/L6 intervertebral disc randomly to build intervertebral disc degeneration model.Then the intervertebral disc was labeled by removing the transverse process of upper vertebrae with rongeur.The rabbits in sham-operated group were treated with sham-operation,while the rabbits in blank group were not given any surgical intervention.Since the 3rd day after the modeling operation,the rabbits in LWDHW group were intragastric administrated with LWDH suspension in dosages of 26 mg/kg while the rabbits in other 3 groups were intragastric administrated with normal saline,once a day for 8 weeks.At 2,4,6 and 8 weeks after the beginning of drug intervention,5 rabbits were randomly selected from each group and were executed,and the intervertebral discs were fetched out.The mRNA and protein expression levels of typeⅠandⅡcollagen in intervertebral disc were measured by using polymerase chain reaction(PCR)and Western Blot assays.Results:There was statistical difference in the mRNA expression levels of typeⅠcollagen in intervertebral disc between the 4 groups at 2,4,6 and 8 weeks after the beginning of drug intervention(0.064 8+/-0.009 8,0.068 6+/-0.012 7,0.192 0+/-0.040 6,0.124 6+/-0.012 0,F=49.752,P=0.000; 0.066 0+/-0.010 0,0.077 1+/-0.011 8,0.252 4+/-0.039 9,0.164 1+/-0.018 1,F=79.537,P=0.000; 0.071 2+/-0.011 2,0.089 1+/-0.008 1,0.326 3+/-0.028 5,0.176 3+/-0.015 0,F=236.726,P=0.000; 0.094 1+/-0.012 0,0.155 0+/-0.003 8,0.451 4+/-0.039 6,0.205 8+/-0.018 1,F=201.055,P=0.000).The mRNA expression levels of typeⅠcollagen were higher in model group compared to blank group,sham-operated group and LWDHW group(P=0.000,P=0.015,P=0.002,P=0.000; P=0.013,P=0.002,P=0.000,P=0.015; P=0.002,P=0.012,P=0.000,P=0.000).There was no statistical difference in mRNA expression levels of typeⅠcollagen between different time points in blank group and sham-operated group(F=50.563,P=0.132; F=80.352,P=0.634).There was statistical difference in mRNA expression levels of typeⅠcollagen between different time points in model group and LWDHW group(F=193.635,P=0.000; F=284.736,P=0.000),and the mRNA expression levels of typeⅠcollagen increased gradually(P=0.004,P=0.002,P=0.000; P=0.000,P=0.003,P=0.001).There was statistical difference in the mRNA expression levels of typeⅡcollagen in intervertebral disc between the 4 groups at 2,4,6 and 8 weeks after the beginning of the drug intervention(0.042 7+/-0.008 0,0.041 2+/-0.005 8,0.011 6+/-0.002 1,0.031 7+/-0.005 6,F=42.696,P=0.000; 0.038 8+/-0.004 3,0.037 3+/-0.004 3,0.011 5+/-0.001 8,0.031 1+/-0.003 5,F=108.110,P=0.000; 0.030 3+/-0.005 7,0.025 9+/-0.008 3,0.007 9+/-0.002 7,0.017 2+/-0.002 1,F=52.436,P=0.000; 0.029 3+/-0.006 9,0.023 7+/-0.004 6,0.005 3+/-0.001 0,0.014 8+/-0.001 8,F=51.375,P=0.000).The mRNA expression levels of typeⅡcollagen were lower in model group compared to blank group,sham-operated group and LWDHW group(P=0.002,P=0.001,P=0.000,P=0.001; P=0.001,P=0.000,P=0.000,P=0.001; P=0.001,P=0.002,P=0.013,P=0.000).There was no statistical difference in mRNA expression levels of typeⅡcollagen between different time points in blank group and sham-operated group(F=70.463,P=0.122; F=90.362,P=0.089).There was statistical difference in mRNA expression levels of typeⅡcollagen between different time points in model group and LWDHW group(F=110.364,P=0.001; F=86.362,P=0.004),and the mRNA expression levels of typeⅡcollagen decreased gradually in model group and LWDHW group(P=0.002,P=0.005,P=0.003; P=0.000,P=0.001,P=0.000).There was statistical difference in the protein expression levels of typeⅠcollagen in intervertebral disc between the 4 groups at 2,4,6 and 8 weeks after the beginning of the drug intervention(0.575 9+/-0.135 6,0.599 5+/-0.037 4,0.620 0+/-0.072 0,0.609 6+/-0.032 1,F=9.288,P=0.001; 0.593 9+/-0.108 0,0.647 1+/-0.044 8,0.807 0+/-0.061 6,0.659 9+/-0.105 5,F=28.883,P=0.000; 0.614 0+/-0.224 6,0.685 6+/-0.066 5,1.129 1+/-0.097 4,0.700 1+/-0.086 5,F=34.957,P=0.000; 0.614 8+/-0.139 3,0.742 3+/-0.165 3,1.322 4+/-0.351 7,0.806 0+/-0.094 3,F=3.296,P=0.048).The protein expression levels of typeⅠcollagen were higher in model group compared to blank group,sham-operated group and LWDHW group(P=0.000,P=0.015,P=0.002,P=0.000; P=0.013,P=0.002,P=0.000,P=0.015; P=0.002,P=0.012,P=0.000,P=0.000).There was no statistical difference in protein expression levels of typeⅠcollagen between different time points in blank group and sham-operated group(F=37.485,P=0.365; F=10.376,P=0.583).There was statistical difference in protein expression levels of typeⅠcollagen between different time points in model group and LWDHW group(F=70.362,P=0.000; F=6.375,P=0.001),and the protein expression levels of typeⅠcollagen increased gradually in model group and LWDHW group(P=0.005,P=0.002,P=0.000; P=0.004,P=0.001,P=0.000).There was statistical difference in the protein expression levels of typeⅡcollagen in intervertebral disc between the 4 groups at 2,4,6 and 8 weeks after the beginning of the drug intervention(0.605 8+/-0.066 5,0.550 0+/-0.117 9,0.278 0+/-0.053 2,0.498 6+/-0.149 1,F=10.224,P=0.001; 0.553 7+/-0.126 5,0.523 4+/-0.078 4,0.258 2+/-0.037 8,0.479 7+/-0.090 8,F=11.627,P=0.000; 0.546 8+/-0.120 8,0.494 8+/-0.139 8,0.219 0+/-0.099 2,0.440 5+/-0.052 7,F=13.543,P=0.003; 0.494 8+/-0.042 5,0.433 7+/-0.061 9,0.131 9+/-0.012 8,0.392 9+/-0.107 0,F=13.979,P=0.000).The protein expression levels of typeⅡcollagen were lower in model group compared to blank group,sham-operated group and LWDHW group(P=0.002,P=0.001,P=0.000,P=0.001; P=0.001,P=0.000,P=0.000,P=0.001; P=0.001,P=0.002,P=0.013,P=0.000).There was no statistical difference in protein expression levels of type Ⅱcollagen between different time points in blank group and sham-operated group(F=17.364,P=0.092; F=15.573,P=0.175).There was statistical difference in protein expression levels of type Ⅱcollagen between different time points in model group and LWDHW group(F=17.753,P=0.001; F=13.674,P=0.000),and the protein expression levels of typeⅡcollagen decreased gradually in model group and LWDHW group(P=0.002,P=0.004,P=0.001; P=0.000,P=0.001,P=0.000).Conclusion:Application of LWDHW can moderately down-regulate the expression of typeⅠcollagen and up-regulate the expression of typeⅡcollagen in intervertebral disc of rabbit model of intervertebral disc degeneration,so it can postpone the degenerative process of intervertebral disc to some extent.

参考文献/References:

[1] Cheung KM,Karppinen J,Chan D,et al.Prevalence and pattern of lumbar magnetic resonance imaging changes in a population study of one thousand forty-three individuals[J].Spine(Phila Pa 1976),2009,34(9):934-940. [2] Freemont AJ.The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain[J].Rheumatology,2009,48(1):5-10. [3] 孟祥宇,夏建龙,杨挺,等.椎间盘退变的机制及修复[J].中国组织工程研究,2015,19(11):1768-1773. [4] 刘强,张军,张慧,等.不同腰椎扳法对椎间盘内压的影响[J].中医正骨,2014,26(1):11-16. [5] Eyre DR,Muir H.Quantitative analysis of typesⅠandⅡcollagens in human intervertebral discs at various ages[J].Biochim Biophys Acta,1977,492(1):29-42. [6] Oegema TR Jr.Biochemistry of the intervertebral disc[J].Clin Sports Med,1993,12(3):419-439. [7] Walker MH,Anderson DG.Molecular basis of intervertebral disc degeneration[J].Spine J,2004,4(6 Suppl):158S-166S. [8] 吕振华,胡有谷,冯传汉.腰椎间盘纤维性主胶原基因表达的观察[J].中华外科杂志,1998,36(2):68-71. [9] 胡绪江,邵增务.外源性肿瘤坏死因子-α对腰椎间盘退变影响的实验研究[J].中国脊柱脊髓杂志,2006,16(7):541-544. [10] Haschtmann D,Stoyanov JV,Ettinger L,et al.Establishment of a novel intervertebral disc/endplate culture model:analysis of an ex vivo in vitro whole-organ rabbit culture system[J].Spine(Phila Pa 1976),2006,31(25):2918-2925. [11] Molinos M,Almeida CR,Caldeira J,et al.Inflammation in intervertebral disc degeneration and regeneration[J].J R Soc Interface,2015,12(104):20141191. [12] Manfreda E,Mitteroecker P,Bookstein FL,et al.Functional morphology of the first cervical vertebra in humans and nonhuman primates[J].Anat Rec B New Anat,2006,289(5):184-194. [13] 徐无忌,李悦,原超.六味地黄丸含药血清对椎间盘Ⅰ型和Ⅱ型胶原表达的影响[J].中国组织工程研究,2013,15(26):4857-4864.

相似文献/References:

[1]张莉,秦丹霞,张细姣.腹针治疗椎间盘源性腰痛[J].中医正骨,2015,27(10):38.
[2]吴青坡,孙国绍,王林杰.后路椎管减压联合腰椎椎弓根钉动态稳定装置内固定 治疗单节段腰椎退行性疾病[J].中医正骨,2015,27(10):42.
[3]夏炳江,童培建.适宜中医药研究的椎间盘退变动物模型的构建[J].中医正骨,2016,28(11):71.
[4]赵赫,高誉珊,胡振国,等.补肾壮筋汤对腰椎间盘退变大鼠椎间盘及行为学特征影响的实验研究[J].中医正骨,2017,29(07):12.
　ZHAO He,GAO Yushan,HU Zhenguo,et al.Effect of Bushen Zhuangjin Tang(补肾壮筋汤)on intervertebral disc and behavioral characteristics of rats with lumbar intervertebral disc degeneration[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(08):12.
[5]许勇,薛彬,孟祥超,等.伸筋活血汤对低氧诱导因子-1α缺失小鼠椎间盘退变的影响…[J].中医正骨,2018,30(01):12.
　XU Yong,XUE Bin,MENG Xiangchao,et al.Effect of Shenjin Huoxue Tang(伸筋活血汤)on intervertebral disc degeneration in mice with hypoxia-inducible factor-1α deficiency[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(08):12.
[6]夏炳江,侯明生,张磊,等.五福饮对大鼠尾部退变椎间盘的影响及其作用机制…[J].中医正骨,2018,30(01):18.
　XIA Bingjiang,HOU Mingsheng,ZHANG Lei,et al.Effects of Wufu Yin(五福饮)on caudal degenerative intervertebral disc in rats and its mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2018,30(08):18.
[7]刁志君,姜宏,刘锦涛,等.炎症因子在椎间盘退变中的作用[J].中医正骨,2018,30(07):32.
[8]梁博程,史晓林,许超,等.基于中药系统药理学方法研究六味地黄丸治疗骨质疏松症的药效成分、作用靶点及作用特点[J].中医正骨,2019,31(04):1.
　LIANG Bocheng,SHI Xiaolin,XU Chao,et al.A study of pharmacodynamic components,action targets and characteristics of Liuwei Dihuang Wan(六味地黄丸)in treatment of osteoporosis using traditional chinese medicine systems pharmacology approach[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(08):1.
[9]刘汝专,张磊,潘汉升,等.中药复方干预椎间盘退变的实验研究进展[J].中医正骨,2019,31(04):36.
[10]劳杨骏,裘一丹,徐彬,等.轴向负载诱导小鼠椎间盘退变模型的建立[J].中医正骨,2019,31(05):1.
　LAO Yangjun,QIU Yidan,XU Bin,et al.A mouse model of intervertebral disc degeneration induced by axial loads[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(08):1.

备注/Memo

备注/Memo:
基金项目:国家自然科学基金青年科学基金项目(81302993); 湖南中医药科技计划重点项目(201317) 通讯作者:王乐 E-mail:546762399@qq.com
更新日期/Last Update: 1900-01-01