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[1]鄭文偉,翁霞萍,黃綏心,等.獨活寄生湯對骨關節(jié)炎軟骨退變的影響及其作用機制[J].中醫(yī)正骨,2017,29(07):5-11.
　ZHENG Wenwei,WENG Xiaping,HUANG Suixin,et al.Effects of Duhuo Jisheng Tang on cartilage degeneration in the process of osteoarthritis and the mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(07):5-11.
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獨活寄生湯對骨關節(jié)炎軟骨退變的影響及其作用機制 ()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期數(shù):
2017年07期
頁碼:
5-11
欄目:
基礎研究
出版日期:
2017-07-20

文章信息/Info

Title:
Effects of Duhuo Jisheng Tang on cartilage degeneration in the process of osteoarthritis and the mechanism of action
作者:
鄭文偉1; 翁霞萍1; 黃綏心2; 鄭春松1; 葉錦霞1; 吳追樂2; 許惠鳳2; 吳明霞2; 李西海1; 劉獻祥1
1.福建中醫(yī)藥大學,福建 福州 350122; 2.福建省中西醫(yī)結合老年性疾病重點實驗室,福建 福州 350122
Author(s):
ZHENG Wenwei1; WENG Xiaping1; HUANG Suixin2; ZHENG Chunsong1; YE Jinxia1; WU Zhuile2; XU Huifeng2; WU Mingxia2; LI Xihai1; LIU Xianxiang1
1.Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China 2.Fujian Key Laboratory of Integrated Medicine on Geriatrics,Fuzhou 350122,Fujian,China
關鍵詞:
骨關節(jié)炎;  獨活寄生湯;  軟骨退變;  G蛋白偶聯(lián)信號通路;  動物實驗
Keywords:
Key words osteoarthritis;  Duhuo Jisheng Tang;  cartilage degeneration;  G protein coupled signaling pathway;  animal experimentation
摘要:
目的:探討獨活寄生湯對骨關節(jié)炎軟骨退變的影響及其作用機制。方法:將45只8周齡雄性SD大鼠隨機分為空白組、模型組、獨活寄生湯組,每組15只。模型組、獨活寄生湯組采用改良Hulth法建立膝骨關節(jié)炎大鼠模型,空白組不做任何處理。獨活寄生湯組給予9.3 g·kg-1的獨活寄生湯水煎濃縮液進行灌胃,模型組和空白組給予等劑量生理鹽水灌胃; 每天灌胃1次,連續(xù)灌胃12周。末次灌胃后分別取各組大鼠雙側股骨髁,采用HE染色觀察關節(jié)軟骨組織形態(tài),并采用Mankin's關節(jié)軟骨組織學評分法評價軟骨退變程度; 采用Western blot法檢測軟骨中G蛋白偶聯(lián)信號傳導系統(tǒng)的關鍵調控因子的蛋白表達情況。分別從模型組和獨活寄生湯組各取50 mg關節(jié)軟骨組織,采用基因芯片檢測軟骨基質降解基因表達情況。結果:①軟骨組織形態(tài)。空白組關節(jié)軟骨結構層次清晰,潮線完整,軟骨表面較為平滑; 模型組關節(jié)軟骨結構層次較為紊亂,未見潮線,表層軟骨膜破損,膜樣結構消失并呈絲絨樣; 獨活寄生湯組關節(jié)軟骨結構層次較為清晰,軟骨破壞程度比模型組輕。②軟骨退變程度。3組大鼠關節(jié)軟骨的Mankin's組織學評分比較,差異有統(tǒng)計學意義[(3.46±2.04)分,(12.58±2.55)分,(7.75±1.91)分,F=13.114,P=0.006]; 空白組和獨活寄生湯組低于模型組(t=-5.114,P=0.002; t=2.709,P=0.035); 空白組與獨活寄生湯組比較,差異無統(tǒng)計學意義(t=-2.406,P=0.053)。③藥物干預后G蛋白偶聯(lián)信號傳導系統(tǒng)關鍵調控因子的蛋白表達。3組大鼠G蛋白偶聯(lián)信號傳導系統(tǒng)關鍵調控因子Gαs、Gαq、Gαo、Gαi蛋白表達量比較,組間差異均有統(tǒng)計學意義[(1.59±0.09)kDa,(1.01±0.04)kDa,(1.45±0.14)kDa,F=29.760,P=0.001;(1.03±0.06)kDa,(0.53±0.04)kDa,(0.75±0.09)kDa,F=14.969,P=0.027;(0.36±0.02)kDa,(0.11±0.01)kDa,(0.17±0.02)kDa,F=204.105,P=0.000;(0.20±0.02)kDa,(0.56±0.05)kDa,(0.33±0.07)kDa,F=28.357,P=0.001]; 空白組Gαs、Gαq、Gαo表達量均高于模型組(t=0.407,P=0.000; t=0.546,P=0.012; t=1.933,P=0.000),Gαi表達量低于模型組(t=-0.750,P=0.000); 模型組Gαs、Gαq、Gαo表達量均低于獨活寄生湯組(t=-5.584,P=0.001; t=-2.431,P=0.093; t=-4.584,P=0.004),Gαi表達量高于獨活寄生湯組(t=4.377,P=0.005); 獨活寄生湯組Gαs、Gαq表達量與空白組比較,組間差異均無統(tǒng)計學意義(t=-1.816,P=0.119; t=-3.029,P=0.056); 獨活寄生湯組Gαo表達量低于空白組(t=-14.749,P=0.000),Gαi表達量高于空白組(t=3.119,P=0.021)。④藥物干預后關節(jié)軟骨基質降解基因的表達。藥物干預后,表達上調2倍以上的軟骨基質降解基因為基質金屬蛋白酶(matrix metalloproteinase,MMP)8、硒蛋白P(selenoprotein P,SELP),表達下調2倍以上的軟骨基質降解基因為蛋白聚糖酶(a disintegrin and metalloproteinase with thrombospondin motifs,ADAMTS)1、ADAMTS2、ADAMTS8、鈣黏蛋白3(cadherin 3,CDH3)、膠原(collagen,COL)1A1、COL5A1、結締組織生長因子(connective tissue growth factor,CTGF)、整合素(integrin,ITG)b1、ITGb4、MMP12、MMP14。模型組MMP8、SELP基因表達量均低于獨活寄生湯組(1.00±0.00,2.81±0.60,t=-5.225,P=0.035; 1.00±0.00,2.31±0.81,t=-2.808,P=0.107),ADAMTS1、ADAMTS2、ADAMTS8、CDH3、COL1A1、COL5A1、CTGF、ITGb1、ITGb4、MMP12、MMP14基因表達量均高于獨活寄生湯組(1.00±0.00,0.21±0.05,t=27.366,P=0.001; 1.00±0.00,0.31±0.16,t=7.458,P=0.018; 1.00±0.00,0.11±0.04,t=38.538,P=0.001; 1.00±0.00,0.30±0.23,t=5.271,P=0.034; 1.00±0.00,0.12±0.09,t=17.380,P=0.003; 1.00±0.00,0.09±0.16,t=10.168,P=0.010; 1.00±0.00, 0.21±0.30,t=4.605,P=0.044; 1.00±0.00,0.37±0.20,t=5.456,P=0.032; 1.00±0.00,0.13±0.22,t=6.960,P=0.020; 1.00±0.00,0.05±0.08,t=19.388,P=0.003; 1.00±0.00,0.05±0.10,t=15.671,P=0.004)。結論:獨活寄生湯可以明顯延緩骨關節(jié)炎軟骨的退變,其作用機制可能是通過激活G蛋白偶聯(lián)信號傳導通路而抑制軟骨基質降解,從而使受損的軟骨組織修復,但其具體作用靶點有待進一步深入研究。
Abstract:
ABSTRACT Objective:To explore the effects of Duhuo Jisheng Tang(獨活寄生湯,DHJST)on cartilage degeneration in the process of osteoarthritis(OA)and the mechanism of action.Methods:Forty-five 8-week-old male SD rats were randomly divided into blank group,model group and DHJST group,15 cases in each group.The knee OA models were built by modified Hulth method in rats of model group and DHJST group,while the rats in blank group were not given any surgical intervention.The rats in DHJST group were intragastric administrated with DHJST concentrated solution in dosages of 9.3 g/kg,while the rats in model group and blank group were intragastric administrated with isodose normal saline,once a day for consecutive 12 weeks.The bilateral femoral condyles of rats in each group were fetched out respectively after the last intragastric administration.The articular cartilage tissue forms were observed after HE staining and the degrees of cartilage degeneration were evaluated by using Mankin's articular cartilage histological scores.The protein expression of key regulating factor of G protein coupled signal transduction systems in the cartilage were detected by using Western blot method.The articularological scores.The protein expression of key regulating factor of G protein coupled signal transduction systems in the cartilage were detected by using Western blot method.The articular cartilage(50 mg)were taken from the rats in model group and DHJST group respectively,and the cartilage matrix degradation gene expression were detected by using gene chips.Results:Clear articular cartilage structure,complete tidal line and smooth cartilage surface were found in blank group.Relatively disorderly articular cartilage structure and damaged surface perichondrium were found and tidal line and membrane structure disappeared in model group.Relatively clear articular cartilage structure and slighter cartilage destruction were found in DHJST group.There was statistical difference in Mankin's histological scores of articular cartilage between the 3 groups(3.46+/-2.04,12.58+/-2.55,7.75+/-1.91 points,F=13.114,P=0.006).The Mankin's histological scores were lower in blank group and DHJST group compared to model group(t=-5.114,P=0.002; t=2.709,P=0.035).There was no statistical difference in Mankin's histological scores between blank group and DHJST group(t=-2.406,P=0.053).There was statistical difference in the protein expression of key regulating factor Gαs,Gαq,Gαo and Gαi of G protein coupled signal transduction systems between the 3 groups(1.59+/-0.09,1.01+/-0.04,1.45+/-0.14 kDa,F=29.760,P=0.001; 1.03+/-0.06,0.53+/-0.04,0.75+/-0.09 kDa,F=14.969,P=0.027; 0.36+/-0.02,0.11+/-0.01,0.17+/-0.02 kDa,F=204.105,P=0.000; 0.20+/-0.02,0.56+/-0.05,0.33+/-0.07 kDa,F=28.357,P=0.001).The expressions of Gαs,Gαq and Gαo were higher and the expressions of Gαi were lower in blank group compared to model group(t=0.407,P=0.000; t=0.546,P=0.012; t=1.933,P=0.000; t=-0.750,P=0.000).The expressions of Gαs,Gαq and Gαo were lower and the expressions of Gαi were higher in model group compared to DHJST group(t=-5.584,P=0.001; t=-2.431,P=0.093; t=-4.584,P=0.004; t=4.377,P=0.005).There was no statistical difference in the expressions of Gαs and Gαq between blank group and DHJST group(t=-1.816,P=0.119; t=-3.029,P=0.056).The expressions of Gαo were lower and the expressions of Gαi were higher in DHJST group compared to blank group(t=-14.749,P=0.000; t=3.119,P=0.021).After drug intervention,the expression of some cartilage matrix degradation genes,including matrix metalloproteinase(MMP)8 and selenoprotein P(SELP),were up-regulated more than 2 times; while the expression of some cartilage matrix degradation genes,including a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS)1,ADAMTS2,ADAMTS8,cadherin 3(CDH3),collagen(COL)1A1,COL5A1,connective tissue growth factor(CTGF),integrin(ITG)b1,ITGb4,MMP12 and MMP14 were down-regulated more than 2 times.The gene expression levels of MMP8 and SELP were lower in model group compared to DHJST group(1.00+/-0.00 vs 2.81+/-0.60,t=-5.225,P=0.035; 1.00+/-0.00 vs 2.31+/-0.81,t=-2.808,P=0.107),while the gene expression levels of ADAMTS1,ADAMTS2,ADAMTS8,CDH3,COL1A1,COL5A1,CTGF,ITGb1,ITGb4,MMP12 and MMP14 were higher in model group compared to DHJST group(1.00+/-0.00 vs 0.21+/-0.05,t=27.366,P=0.001; 1.00+/-0.00 vs 0.31+/-0.16,t=7.458,P=0.018; 1.00+/-0.00 vs 0.11+/-0.04,t=38.538,P=0.001; 1.00+/-0.00 vs 0.30+/-0.23,t=5.271,P=0.034; 1.00+/-0.00 vs 0.12+/-0.09,t=17.380,P=0.003; 1.00+/-0.00 vs 0.09+/-0.16,t=10.168,P=0.010; 1.00+/-0.00 vs 0.21+/-0.30,t=4.605,P=0.044; 1.00+/-0.00 vs 0.37+/-0.20,t=5.456,P=0.032; 1.00+/-0.00 vs 0.13+/-0.22,t=6.960,P=0.020; 1.00+/-0.00 vs 0.05+/-0.08,t=19.388,P=0.003; 1.00+/-0.00 vs 0.05+/-0.10,t=15.671,P=0.004).Conclusion:DHJST can obviously delay cartilage degeneration in the process of OA.The mechanisms of action may be that it can inhibit cartilage matrix degradation through activating G protein coupled signaling pathway,so it can repair the damaged cartilage tissues.However,its specific action targets need to be further studied.

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備注/Memo

備注/Memo:
基金項目:國家自然科學基金項目(81373818) 通訊作者:李西海 E-mail:[email protected] mechanism of action
更新日期/Last Update: 2017-12-29