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[1]王明喜,張麗霞,王長(zhǎng)平.馬錢子總生物堿修復(fù)膝骨關(guān)節(jié)炎大鼠軟骨損傷的效果觀察及作用機(jī)制研究[J].中醫(yī)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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馬錢子總生物堿修復(fù)膝骨關(guān)節(jié)炎大鼠軟骨損傷的效果觀察及作用機(jī)制研究()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第33卷
期數(shù):
2021年05期
頁(yè)碼:
11-18
欄目:
基礎(chǔ)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; 張麗霞; 王長(zhǎng)平
(安陽(yáng)市中醫(yī)院,河南 安陽(yáng) 455000)
Author(s):
WANG Mingxi; ZHANG Lixia; WANG Changping
Anyang Hospital of Traditional Chinese Medicine,Anyang 455000,Henan,China
關(guān)鍵詞:
骨關(guān)節(jié)炎; 膝;  馬錢子;  生物堿;  軟骨損傷;  Wnt/β-catenin信號(hào)通路;  大鼠;  動(dòng)物實(shí)驗(yàn)
Keywords:
osteoarthritis; knee;  semen strychni;  alkaloid;  cartilage injury;  Wnt/β-catenin signaling pathway;  rats;  animal experimentation
摘要:
目的:探討馬錢子總生物堿修復(fù)膝骨關(guān)節(jié)炎(knee osteoarthritis,KOA)大鼠軟骨損傷的效果及作用機(jī)制。方法:將60只SD大鼠隨機(jī)分為正常對(duì)照組、模型組、塞來(lái)昔布組和馬錢子總生物堿低、中、高劑量組,每組10只。模型組、塞來(lái)昔布組和馬錢子總生物堿低、中、高劑量組大鼠采用雙側(cè)后肢膝關(guān)節(jié)腔注射木瓜蛋白酶溶液建立KOA模型,造模完成1周后,從各組中隨機(jī)選取1只大鼠,處死后取膝關(guān)節(jié)軟骨,觀察膝關(guān)節(jié)軟骨組織形態(tài),判斷造模是否成功。造模成功后,馬錢子總生物堿低、中、高劑量組大鼠分別以馬錢子總生物堿混懸液進(jìn)行灌胃,每日劑量分別為50 mg·kg-1、100 mg·kg-1和150 mg·kg-1; 塞來(lái)昔布組大鼠以塞來(lái)昔布溶液(塞來(lái)昔布膠囊粉劑溶于去離子水中)進(jìn)行灌胃,每日劑量為24 mg·kg-1; 正常對(duì)照組和模型組大鼠以生理鹽水進(jìn)行灌胃,每日1 mL。藥物干預(yù)6周后,處死所有大鼠,取大鼠一側(cè)膝關(guān)節(jié),觀察膝關(guān)節(jié)軟骨組織形態(tài),并采用Mankin’s評(píng)分法評(píng)價(jià)膝關(guān)節(jié)軟骨損傷程度。根據(jù)膝關(guān)節(jié)軟骨損傷程度評(píng)價(jià)結(jié)果,選取馬錢子總生物堿低、中、高劑量組中軟骨修復(fù)效果最佳的一組以及正常對(duì)照組、模型組和塞來(lái)昔布組進(jìn)行膝關(guān)節(jié)軟骨損傷相關(guān)基因mRNA和蛋白的表達(dá)分析。分別采用實(shí)時(shí)定量PCR和蛋白印跡法檢測(cè)基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP)-9、MMP-13、白細(xì)胞介素(interleukin,IL)-1β、糖原合成酶激酶(glycogen synthase kinase,GSK)-3β和β-聯(lián)蛋白(β-catenin)的mRNA和蛋白表達(dá)水平。結(jié)果:①模型鑒定結(jié)果。造模完成1周后,直視下觀察,正常大鼠膝關(guān)節(jié)軟骨呈膠凍樣,表面光滑; KOA模型大鼠膝關(guān)節(jié)軟骨色澤暗淡,表面粗糙。膝關(guān)節(jié)軟骨組織切片顯示,正常大鼠膝關(guān)節(jié)軟骨組織結(jié)構(gòu)完整、潮線清晰,軟骨細(xì)胞均勻分布; KOA模型大鼠膝關(guān)節(jié)軟骨組織結(jié)構(gòu)模糊,潮線不完整或丟失,軟骨細(xì)胞聚集,排列紊亂,提示造模成功。②膝關(guān)節(jié)軟骨組織病理學(xué)檢查結(jié)果。藥物干預(yù)6周后,正常對(duì)照組大鼠膝關(guān)節(jié)軟骨組織結(jié)構(gòu)完整、潮線清晰,軟骨細(xì)胞均勻分布; 模型組大鼠膝關(guān)節(jié)軟骨結(jié)構(gòu)破壞嚴(yán)重,潮線模糊,軟骨細(xì)胞聚集; 馬錢子總生物堿低、中、高劑量組大鼠膝關(guān)節(jié)軟骨結(jié)構(gòu)可辨識(shí),軟骨細(xì)胞聚集現(xiàn)象較模型組減少,且馬錢子總生物堿中劑量組的大鼠軟骨改善效果最明顯; 塞來(lái)昔布組大鼠膝關(guān)節(jié)軟骨結(jié)構(gòu)較完整、清晰,軟骨細(xì)胞聚集現(xiàn)象不明顯。③膝關(guān)節(jié)軟骨損傷Mankin’s評(píng)分結(jié)果。6組大鼠膝關(guān)節(jié)軟骨損傷Mankin’s評(píng)分的組間差異有統(tǒng)計(jì)學(xué)意義[(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]。正常對(duì)照組和馬錢子總生物堿低、中、高劑量組及塞來(lái)昔布組的膝關(guān)節(jié)軟骨損傷Mankin’s評(píng)分均低于模型組(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); 馬錢子總生物堿低、中、高劑量組的膝關(guān)節(jié)軟骨損傷Mankin’s評(píng)分均高于塞來(lái)昔布組(LSD-t=70.210,P=0.000; LSD-t=31.420,P=0.000; LSD-t=56.902,P=0.000); 馬錢子總生物堿中劑量組的膝關(guān)節(jié)軟骨損傷Mankin’s評(píng)分低于馬錢子總生物堿低、高劑量組(LSD-t=42.119,P=0.000; LSD-t=30.590,P=0.000)。④膝關(guān)節(jié)軟骨損傷相關(guān)基因mRNA和蛋白表達(dá)檢測(cè)結(jié)果。正常對(duì)照組、模型組、塞來(lái)昔布組及馬錢子總生物堿中劑量組4組大鼠膝關(guān)節(jié)軟骨組織中MMP-9、MMP-13和IL-1β的mRNA和蛋白相對(duì)表達(dá)量的組間差異均有統(tǒng)計(jì)學(xué)意義(mRNA相對(duì)表達(dá)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; 蛋白相對(duì)表達(dá)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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備注/Memo

備注/Memo:
通訊作者:王明喜 E-mail:[email protected]
更新日期/Last Update: 1900-01-01