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[1]王樂,徐無忌.六味地黃丸對兔椎間盤退變模型椎間盤組織中Ⅰ、Ⅱ型膠原表達的影響[J].中醫(yī)正骨,2016,28(08):1-7.
　WANG Le,XU Wuji.Effect of Liuwei Dihuang Wan(六味地黃丸)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.
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六味地黃丸對兔椎間盤退變模型椎間盤組織中Ⅰ、Ⅱ型膠原表達的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第28卷
期數(shù):
2016年08期
頁碼:
1-7
欄目:
基礎(chǔ)研究
出版日期:
2016-08-20

文章信息/Info

Title:
Effect of Liuwei Dihuang Wan(六味地黃丸)on typeⅠandⅡcollagen expression in intervertebral disc of rabbit model of intervertebral disc degeneration
作者:
王樂; 徐無忌
湖南中醫(yī)藥大學(xué)第二附屬醫(yī)院,湖南 長沙 410005
Author(s):
WANG Le; XU Wuji
The Second Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Changsha 410005,Hunan,China
關(guān)鍵詞:
椎間盤退行性變;  六味地黃丸;  膠原Ⅰ型;  膠原Ⅱ型;  兔;  動物實驗
Keywords:
intervertebral disc degeneration;  Liuwei Dihuang Wan;  collagen typeⅠ;  collagen typeⅡ;  rabbits;  animal experimentation
摘要:
目的:觀察六味地黃丸對兔椎間盤退變模型椎間盤組織中Ⅰ、Ⅱ型膠原表達的影響。方法:將80只新西蘭兔隨機分為空白組、假手術(shù)組、模型組和六味地黃組,每組20只。模型組、六味地黃組手術(shù)暴露L4～5和L5～6椎間隙,隨機選擇1個椎間盤注射腫瘤壞死因子α進行椎間盤退變造模,并以咬骨鉗咬除該椎間盤上位椎體橫突進行標記; 假手術(shù)組經(jīng)相同手術(shù)入路暴露L4～5和L5～6椎間隙,不做任何處理后縫合; 空白組不進行任何手術(shù)干預(yù)。造模術(shù)后3 d開始,六味地黃組按26 mg·kg-1以六味地黃膠囊混懸液灌胃,其余3組以等量生理鹽水灌胃,每天1次,共8周。分別于藥物干預(yù)開始后2、4、6、8周,從各組隨機選取5只兔子處死,模型組和六味地黃組手術(shù)取出造模椎間盤,空白組和假手術(shù)組隨機選擇L4～5或L5～6椎間盤取出。分別采用聚合酶鏈式反應(yīng)法和Western Blot法測定椎間盤組織中Ⅰ、Ⅱ型膠原的mRNA和蛋白表達水平。結(jié)果:藥物干預(yù)2、4、6、8周后,4組兔子椎間盤組織Ⅰ型膠原mRNA表達水平比較,組間差異均有統(tǒng)計學(xué)意義[(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]; 模型組Ⅰ型膠原mRNA表達水平高于空白組、假手術(shù)組和六味地黃組(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)。空白組和假手術(shù)組的Ⅰ型膠原mRNA表達水平各時間點間比較,差異均無統(tǒng)計學(xué)意義(F=50.563,P=0.132; F=80.352,P=0.634); 模型組和六味地黃組的Ⅰ型膠原mRNA表達水平各時間點間比較,差異均有統(tǒng)計學(xué)意義(F=193.635,P=0.000; F=284.736,P=0.000),Ⅰ型膠原mRNA表達水平均逐漸增高(P=0.004,P=0.002,P=0.000; P=0.000,P=0.003,P=0.001)。藥物干預(yù)2、4、6、8周后,4組兔子椎間盤組織Ⅱ型膠原mRNA表達水平比較,組間差異均有統(tǒng)計學(xué)意義[(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]; 模型組Ⅱ型膠原mRNA表達水平低于空白組、假手術(shù)組和六味地黃組(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)。空白組和假手術(shù)組的Ⅱ型膠原mRNA表達水平各時間點間比較,差異均無統(tǒng)計學(xué)意義(F=70.463,P=0.122; F=90.362,P=0.089); 模型組和六味地黃組的Ⅱ型膠原mRNA表達水平各時間點間比較,差異均有統(tǒng)計學(xué)意義(F=110.364,P=0.001; F=86.362,P=0.004),模型組和六味地黃組的Ⅱ型膠原mRNA表達水平均逐漸降低(P=0.002,P=0.005,P=0.003; P=0.000,P=0.001,P=0.000)。藥物干預(yù)2、4、6、8周后,4組兔子椎間盤組織Ⅰ型膠原蛋白表達水平比較,組間差異均有統(tǒng)計學(xué)意義[(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]; 模型組Ⅰ型膠原蛋白表達水平高于空白組、假手術(shù)組和六味地黃組(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)。空白組和假手術(shù)組的Ⅰ型膠原蛋白表達水平各時間點間比較,差異均無統(tǒng)計學(xué)意義(F=37.485,P=0.365; F=10.376,P=0.583); 模型組和六味地黃組的Ⅰ型膠原蛋白表達水平各時間點間比較,差異均有統(tǒng)計學(xué)意義(F=70.362,P=0.000; F=6.375,P=0.001),模型組和六味地黃組的Ⅰ型膠原蛋白表達水平均逐漸增高(P=0.005,P=0.002,P=0.000; P=0.004,P=0.001,P=0.000)。藥物干預(yù)2、4、6、8周后,4組兔子椎間盤組織Ⅱ型膠原蛋白表達水平比較,組間差異均有統(tǒng)計學(xué)意義[(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]; 模型組Ⅱ型膠原蛋白表達水平低于空白組、假手術(shù)組和六味地黃組(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)。空白組和假手術(shù)組的Ⅱ型膠原蛋白表達水平各時間點間比較,差異均無統(tǒng)計學(xué)意義(F=17.364,P=0.092; F=15.573,P=0.175); 模型組和六味地黃組的Ⅱ型膠原蛋白表達水平各時間點間比較,差異均有統(tǒng)計學(xué)意義(F=17.753,P=0.001; F=13.674,P=0.000),模型組和六味地黃組的Ⅱ型膠原蛋白表達水平均逐漸降低(P=0.002,P=0.004,P=0.001; P=0.000,P=0.001,P=0.000)。結(jié)論:六味地黃丸能適度下調(diào)兔椎間盤退變模型椎間盤組織中Ⅰ型膠原的表達、上調(diào)Ⅱ型膠原的表達,可在一定程度上延緩椎間盤退變進程。
Abstract:
Objective:To observe the effect of Liuwei Dihuang Wan(六味地黃丸,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.

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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金青年科學(xué)基金項目(81302993); 湖南中醫(yī)藥科技計劃重點項目(201317) 通訊作者:王樂 E-mail:546762399@qq.com
更新日期/Last Update: 1900-01-01