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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:

第24卷

期數(shù):

2012年12期

頁碼:

15-19

欄目:

基礎(chǔ)研究

出版日期:

2012-12-20

文章信息/Info

Title:

Effect of achyranthes bidentata medicated serum on signal transduction pathway of p38 mitogen-activated protein kinases within osteoarthritis chondrocytes

作者:

周江濤¹; 王慶來¹; 趙依娜¹; 吳惠明¹; 王維佳²; 徐海孺²

1.浙江省溫州市中醫(yī)院,浙江 溫州 325000;
2.浙江中醫(yī)藥大學(xué),浙江 杭州 310053

Author(s):

ZHOU Jiang-tao^*; WANG Qing-lai; ZHAO Yi-na; WU Hui-ming; WANG Wei-jia; XU Hai-ru.

^*Traditional Chinese Medical Hospital of Wenzhou City,Wenzhou 325000,Zhejiang,China

關(guān)鍵詞:

骨關(guān)節(jié)炎 軟骨細(xì)胞 p38絲裂原活化蛋白激酶類 膠原Ⅱ型 信號傳導(dǎo) 牛膝

Keywords:

Osteoarthritis;  Chondrocytes;  p38 mitogen-activated protein kinases;  Collagen typeⅡ;  Signal transduction;  Achyranthes bidentata

摘要:

目的:探討牛膝含藥血清對骨關(guān)節(jié)炎軟骨細(xì)胞P38絲裂原活化蛋白激酶信號轉(zhuǎn)導(dǎo)通路的影響。方法:將20只雄性新西蘭大白兔隨機(jī)分為空白對照組、模型組、P38阻斷劑組及含藥血清組,每組5只。分組后含藥血清組大白兔以牛膝水煎劑灌胃(2.5 g·kg^-1),其余各組以等量生理鹽水灌胃,每天2次,共3 d。3 d后靜脈采血,分別配成10%含藥血清和10%空白血清的DMEM培養(yǎng)液保存?zhèn)溆谩３�空白對照組外,其余各組大白兔均采用Hulth法行兔膝骨關(guān)節(jié)炎造模,空白對照組行假手術(shù)。造模成功后處死實(shí)驗(yàn)兔分離兔膝關(guān)節(jié)軟骨進(jìn)行軟骨細(xì)胞培養(yǎng),對空白對照組、模型組的第3代軟骨細(xì)胞以空白血清進(jìn)行干預(yù),P38阻斷劑組以SB203580進(jìn)行干預(yù),含藥血清組以牛膝含藥血清進(jìn)行干預(yù)。血清干預(yù)結(jié)束后,分別采用免疫熒光技術(shù)和Western Blot技術(shù)檢測各組軟骨細(xì)胞磷酸化P38絲裂原活化蛋白激酶及Ⅱ型膠原蛋白含量。結(jié)果:①免疫熒光檢測結(jié)果。各組細(xì)胞均有磷酸化P38絲裂原活化蛋白激酶陽性表達(dá),其中模型組呈強(qiáng)陽性表達(dá),P38阻斷劑組及含藥血清組略弱,空白對照組最弱; 各組細(xì)胞均有Ⅱ型膠原蛋白陽性表達(dá),其中空白對照組呈強(qiáng)陽性表達(dá),P38阻斷劑組及含藥血清組略弱,模型組最弱。②Western Blot檢測結(jié)果。各組磷酸化P38絲裂原活化蛋白激酶含量比較,差異有統(tǒng)計(jì)學(xué)意義(F=3.872,P=0.038)。進(jìn)一步兩兩比較,空白對照組(0.463±0.007)小于模型組(0.856±0.007)、P38阻斷劑組(0.576±0.005)及含藥血清組(0.508±0.004),差異有統(tǒng)計(jì)學(xué)意義(P=0.008; P=0.036; P=0.042); 模型組大于P38阻斷劑組和含藥血清組(P=0.025; P=0.019); P38阻斷劑組與含藥血清組比較,差異無統(tǒng)計(jì)學(xué)意義(P=0.058)。各組細(xì)胞Ⅱ型膠原蛋白含量比較,差異有統(tǒng)計(jì)學(xué)意義(F=3.963,P=0.034)。進(jìn)一步兩兩比較,空白對照組(0.884±0.007)大于模型組(0.434±0.007)、P38阻斷劑組(0.616±0.003)及含藥血清組(0.565±0.008),差異有統(tǒng)計(jì)學(xué)意義(P=0.007; P=0.031; P=0.038); 模型組小于P38阻斷劑組和含藥血清組(P=0.035; P=0.028); P38阻斷劑組與含藥血清組比較,差異無統(tǒng)計(jì)學(xué)意義(P=0.066)。結(jié)論:牛膝含藥血清能阻斷骨關(guān)節(jié)炎軟骨細(xì)胞P38絲裂原活化蛋白激酶信號轉(zhuǎn)導(dǎo)通路,進(jìn)而保護(hù)軟骨細(xì)胞,其效果與SB203580相當(dāng)。

Abstract:

Objective:To explore the effect of achyranthes bidentata medicated serum on signal transduction pathway of p38 mitogen-activated protein kinases(MAPK)within osteoarthritis chondrocytes.Methods:Twenty male New Zealand rabbits were randomly divided into blank control group,model group,p38 blocker group and medicated serum group,5 cases in each group.After grouping,rabbits in the medicated serum group were intragastric administrated with achyranthes bidentata decoction(2.5 g·kg^-1),while the others in the rest 3 groups were all intragastric administrated with equivalent normal saline,twice a day for 3 days. Then blood were drawn from vein and were prepared into DMEM culture solution with 10% medicated serum and 10% blank serum respectively for standby application.The rabbits in each group were all built models of knee osteoarthritis through Hulth method except for cases in the blank control group which were carried out sham operation.After successful modeling,all of the rabbits were executed and their knee articular cartilages were separated for chondrocytes culture.The 3-generation chondrocytes in blank control group and model group were intervened with blank serum,those in p38 blocker group were intervened with SB203580 and those in medicated serum group were intervened with achyranthes bidentata medicated serum.After serum intervention,the contents of phosphorylated p38 MAPK and collagen protein typeⅡin chondrocytes for all the groups were detected through immunofluorescence technique and Western Blot technique respectively.Results:①Results of immunofluorescence detection:positive expressions of phosphorylated p38 MAPK were found in all the groups,strong positive expression was found in model group,slightly weak positive expression was found in p38 blocker group,and medicated serum group,and most weak positive expression was found in the blank control group.Positive expressions of collagen typeⅡwere found in cells of all of the groups,strong positive expression was found in blank control group,slightly weak positive expression in p38 blocker group and medicated serum group,and most weak positive expression in the model group.②Western Blot test results:there was statistical difference in phosphorylated p38 MAPK contents among the 4 groups(F=3.872,P=0.038).As the results of further pairwise comparison showed,the phosphorylated p38 MAPK content of blank control group(0.463±0.007)was less than that of model group(0.856±0.007),p38 blocker group(0.576±0.005)and medicated serum group(0.508±0.004)respectively,and there was statistical difference between them(P=0.008; P=0.036; P=0.042); and phosphorylated p38 MAPK content of model group was more than that of p38 blocker group and medicated serum group respectively(P=0.025; P=0.019); while there was no statistical difference between p38 blocker group and medicated serum group(P=0.058).There was statistical difference in collagen typeⅡcontents among the 4 groups(F=3.963,P=0.034).Further pairwise comparison showed that the content of collagen typeⅡof blank control group(0.884±0.007)was more than that of model group(0.434±0.007),p38 blocker group(0.616±0.003)and medicated serum group(0.565±0.008)respectively,and there was statistical difference between them(P=0.007; P=0.031; P=0.038); and the content of collagen typeⅡof model group was less than that of p38 blocker group and medicated serum group respectively(P=0.035; P=0.028); while there was no statistical difference between p38 blocker group and medicated serum group(P=0.066).Conclusion:The achyranthes bidentata medicated serum can block the signal transduction pathway of p38 MAPK within osteoarthritis chondrocytes,and then protect chondrocytes,and it has the comparable effect of SB203580.

備注/Memo

備注/Memo:

基金項(xiàng)目:浙江省中醫(yī)藥科技計(jì)劃(2007YA024)

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更新日期/Last Update: 2012-12-20