[1]王明喜,张丽霞,王长平.马钱子总生物碱修复膝骨关节炎大鼠软骨损伤的效果观察及作用机制研究[J].中医正骨,2021,33(05):11-18.
　WANG Mingxi,ZHANG Lixia,WANG Changping.An experimental study of the effects and mechanism of action of total alkaloids extracted from nux-vomica for repairing cartilage injury in rats with knee osteoarthritis[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2021,33(05):11-18.
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马钱子总生物碱修复膝骨关节炎大鼠软骨损伤的效果观察及作用机制研究()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期数:
2021年05期
页码:
11-18
栏目:
基础研究
出版日期:
2021-05-20

文章信息/Info

Title:
An experimental study of the effects and mechanism of action of total alkaloids extracted from nux-vomica for repairing cartilage injury in rats with knee osteoarthritis
作者:
王明喜; 张丽霞; 王长平
(安阳市中医院,河南 安阳 455000)
Author(s):
WANG Mingxi; ZHANG Lixia; WANG Changping
Anyang Hospital of Traditional Chinese Medicine,Anyang 455000,Henan,China
关键词:
骨关节炎; 膝;  马钱子;  生物碱;  软骨损伤;  Wnt/β-catenin信号通路;  大鼠;  动物实验
Keywords:
osteoarthritis; knee;  semen strychni;  alkaloid;  cartilage injury;  Wnt/β-catenin signaling pathway;  rats;  animal experimentation
摘要:
目的:探讨马钱子总生物碱修复膝骨关节炎(knee osteoarthritis,KOA)大鼠软骨损伤的效果及作用机制。方法:将60只SD大鼠随机分为正常对照组、模型组、塞来昔布组和马钱子总生物碱低、中、高剂量组,每组10只。模型组、塞来昔布组和马钱子总生物碱低、中、高剂量组大鼠采用双侧后肢膝关节腔注射木瓜蛋白酶溶液建立KOA模型,造模完成1周后,从各组中随机选取1只大鼠,处死后取膝关节软骨,观察膝关节软骨组织形态,判断造模是否成功。造模成功后,马钱子总生物碱低、中、高剂量组大鼠分别以马钱子总生物碱混悬液进行灌胃,每日剂量分别为50 mg·kg-1、100 mg·kg-1和150 mg·kg-1; 塞来昔布组大鼠以塞来昔布溶液(塞来昔布胶囊粉剂溶于去离子水中)进行灌胃,每日剂量为24 mg·kg-1; 正常对照组和模型组大鼠以生理盐水进行灌胃,每日1 mL。药物干预6周后,处死所有大鼠,取大鼠一侧膝关节,观察膝关节软骨组织形态,并采用Mankin’s评分法评价膝关节软骨损伤程度。根据膝关节软骨损伤程度评价结果,选取马钱子总生物碱低、中、高剂量组中软骨修复效果最佳的一组以及正常对照组、模型组和塞来昔布组进行膝关节软骨损伤相关基因mRNA和蛋白的表达分析。分别采用实时定量PCR和蛋白印迹法检测基质金属蛋白酶(matrix metalloproteinase,MMP)-9、MMP-13、白细胞介素(interleukin,IL)-1β、糖原合成酶激酶(glycogen synthase kinase,GSK)-3β和β-联蛋白(β-catenin)的mRNA和蛋白表达水平。结果:①模型鉴定结果。造模完成1周后,直视下观察,正常大鼠膝关节软骨呈胶冻样,表面光滑; KOA模型大鼠膝关节软骨色泽暗淡,表面粗糙。膝关节软骨组织切片显示,正常大鼠膝关节软骨组织结构完整、潮线清晰,软骨细胞均匀分布; KOA模型大鼠膝关节软骨组织结构模糊,潮线不完整或丢失,软骨细胞聚集,排列紊乱,提示造模成功。②膝关节软骨组织病理学检查结果。药物干预6周后,正常对照组大鼠膝关节软骨组织结构完整、潮线清晰,软骨细胞均匀分布; 模型组大鼠膝关节软骨结构破坏严重,潮线模糊,软骨细胞聚集; 马钱子总生物碱低、中、高剂量组大鼠膝关节软骨结构可辨识,软骨细胞聚集现象较模型组减少,且马钱子总生物碱中剂量组的大鼠软骨改善效果最明显; 塞来昔布组大鼠膝关节软骨结构较完整、清晰,软骨细胞聚集现象不明显。③膝关节软骨损伤Mankin’s评分结果。6组大鼠膝关节软骨损伤Mankin’s评分的组间差异有统计学意义[(0.28±0.20)分,(8.97±0.46)分,(7.12±0.23)分,(3.23±0.18)分,(6.21±0.25)分,(0.77±0.17)分,F=8.025,P=0.000]。正常对照组和马钱子总生物碱低、中、高剂量组及塞来昔布组的膝关节软骨损伤Mankin’s评分均低于模型组(LSD-t=54.785,P=0.000; LSD-t=11.316,P=0.000; LSD-t=36.747,P=0.000; LSD-t=16.671,P=0.000; LSD-t=21.626,P=0.000); 马钱子总生物碱低、中、高剂量组的膝关节软骨损伤Mankin’s评分均高于塞来昔布组(LSD-t=70.210,P=0.000; LSD-t=31.420,P=0.000; LSD-t=56.902,P=0.000); 马钱子总生物碱中剂量组的膝关节软骨损伤Mankin’s评分低于马钱子总生物碱低、高剂量组(LSD-t=42.119,P=0.000; LSD-t=30.590,P=0.000)。④膝关节软骨损伤相关基因mRNA和蛋白表达检测结果。正常对照组、模型组、塞来昔布组及马钱子总生物碱中剂量组4组大鼠膝关节软骨组织中MMP-9、MMP-13和IL-1β的mRNA和蛋白相对表达量的组间差异均有统计学意义(mRNA相对表达量:0.312±0.048,0.763±0.062,0.361±0.037,0.379±0.042,F=2.851,P=0.016; 0.209±0.051,0.517±0.028,0.262±0.045,0.289±0.044,F=3.834,P=0.027; 0.116±0.037,0.336±0.024,0.147±0.037,0.173±0.035,F=4.523,P=0.033; 蛋白相对表达量:0.143±0.023,0.383±0.055,0.162±0.033,0.183±0.021,F=4.533,P=0.021; 0.267±0.024,0.524±0.021,0.290±0.002,0.302±0.040,F=5.124,P=0.018; 0.205±0.041,0.451±0.021,0.229±0.009,0.234±0.010,F=4.896,P=0.031)。。。
Abstract:
Objective:To observe the effects of total alkaloids(TA)extracted from nux-vomica for repairing cartilage injury in rats with knee osteoarthritis(KOA)and to explore its mechanism of action.Methods:Sixty Sprague-Dawley(SD)rats were randomly divided into normal control group,model group,celecoxib group,TA low-dose group,TA middle-dose group and TA high-dose group,10 cases in each group.The rats in model group,celecoxib group,TA low-dose group,middle-dose group and high-dose group were intervened by knee intra-articular injection of papain solution into bilateral hind limbs for inducing KOA.One week after the modeling,one rat was randomly selected from each group and were sacrificed,and their knee cartilages were harvested for observing the tissue morphology of knee cartilage to confirm whether the models were built successfully.After successful modeling,the rats in TA low-dose group,TA middle-dose group and TA high-dose group were intragastric administrated with nux-vomica TA suspension in daily dosages of 50 mg/kg,100 mg/kg and 150 mg/kg respectively,the ones in celecoxib group with celecoxib solution(celecoxib capsule powders were dissolved into deionized water)in daily dosage of 24 mg/kg,and the ones in normal control group and model group with normal saline(NS)in daily dosage of 1 mL.After 6-week drug intervention,all rats were sacrificed and their knee joints were harvested for observing knee cartilage tissue morphology,followed by evaluation on the degrees of knee cartilage injury by using Mankin’s scoring method.According to the result of evaluation on the degree of knee cartilage injury,the mRNA and protein expressions of genes related to knee cartilage injury were analyzed by using knee cartilage tissues of rats in the group with best repair effects among TA low-dose group,middle-dose group and high-dose group,and the ones of normal control group,model group and celecoxib group.The mRNA and protein expression levels of matrix metalloproteinase(MMP)-9,MMP-13,interleukin(IL)-1β,glycogen synthase kinase(GSK)-3β and β-catenin were detected by using real-time quantitative PCR(RT-qPCR)and Western-blot assays respectively.Results:One week after modeling,the knee cartilage presented with gelatinous appearance and smooth surface in normal rats,while dull color and rough surface in KOA model rats under direct vision.The knee cartilage tissue sections revealed that the knee cartilage tissues exhibited as complete structure,clear tidal line and evenly distributed chondrocytes in normal rats,while fuzzy structure,incomplete or missed tidal line as well as clustered and irregularly arranged chondrocytes in KOA model rats,which suggested that the models were successfully built.After 6-week drug intervention,the complete structure,clear tidal line and evenly distributed chondrocytes were found in knee cartilage tissues of rats of normal control group; while severely damaged articular cartilage structure,blurred tidal line as well as clustered chondrocytes were found in rats of model group; and discernible articular cartilage structure,decreased chondrocytes aggregation were found in rats of TA low-dose group,TA middle-dose group and TA high-dose group,and especially in TA middle-dose group; besides,more complete and clear articular cartilage structure and unconspicuous chondrocytes aggregation were found in rats of celecoxib group...

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更新日期/Last Update: 1900-01-01