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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:

第24卷

期数:

2012年12期

页码:

15-19

栏目:

基础研究

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:

周江涛¹; 王庆来¹; 赵依娜¹; 吴惠明¹; 王维佳²; 徐海孺²

1.浙江省温州市中医院,浙江 温州 325000;
2.浙江中医药大学,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

关键词:

骨关节炎 软骨细胞 p38丝裂原活化蛋白激酶类 胶原Ⅱ型 信号传导 牛膝

Keywords:

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

摘要:

目的:探讨牛膝含药血清对骨关节炎软骨细胞P38丝裂原活化蛋白激酶信号转导通路的影响。方法:将20只雄性新西兰大白兔随机分为空白对照组、模型组、P38阻断剂组及含药血清组,每组5只。分组后含药血清组大白兔以牛膝水煎剂灌胃(2.5 g·kg^-1),其余各组以等量生理盐水灌胃,每天2次,共3 d。3 d后静脉采血,分别配成10%含药血清和10%空白血清的DMEM培养液保存备用。除空白对照组外,其余各组大白兔均采用Hulth法行兔膝骨关节炎造模,空白对照组行假手术。造模成功后处死实验兔分离兔膝关节软骨进行软骨细胞培养,对空白对照组、模型组的第3代软骨细胞以空白血清进行干预,P38阻断剂组以SB203580进行干预,含药血清组以牛膝含药血清进行干预。血清干预结束后,分别采用免疫荧光技术和Western Blot技术检测各组软骨细胞磷酸化P38丝裂原活化蛋白激酶及Ⅱ型胶原蛋白含量。结果:①免疫荧光检测结果。各组细胞均有磷酸化P38丝裂原活化蛋白激酶阳性表达,其中模型组呈强阳性表达,P38阻断剂组及含药血清组略弱,空白对照组最弱; 各组细胞均有Ⅱ型胶原蛋白阳性表达,其中空白对照组呈强阳性表达,P38阻断剂组及含药血清组略弱,模型组最弱。②Western Blot检测结果。各组磷酸化P38丝裂原活化蛋白激酶含量比较,差异有统计学意义(F=3.872,P=0.038)。进一步两两比较,空白对照组(0.463±0.007)小于模型组(0.856±0.007)、P38阻断剂组(0.576±0.005)及含药血清组(0.508±0.004),差异有统计学意义(P=0.008; P=0.036; P=0.042); 模型组大于P38阻断剂组和含药血清组(P=0.025; P=0.019); P38阻断剂组与含药血清组比较,差异无统计学意义(P=0.058)。各组细胞Ⅱ型胶原蛋白含量比较,差异有统计学意义(F=3.963,P=0.034)。进一步两两比较,空白对照组(0.884±0.007)大于模型组(0.434±0.007)、P38阻断剂组(0.616±0.003)及含药血清组(0.565±0.008),差异有统计学意义(P=0.007; P=0.031; P=0.038); 模型组小于P38阻断剂组和含药血清组(P=0.035; P=0.028); P38阻断剂组与含药血清组比较,差异无统计学意义(P=0.066)。结论:牛膝含药血清能阻断骨关节炎软骨细胞P38丝裂原活化蛋白激酶信号转导通路,进而保护软骨细胞,其效果与SB203580相当。

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.

备注/Memo

备注/Memo:

基金项目:浙江省中医药科技计划(2007YA024)

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更新日期/Last Update: 2012-12-20