基于Wnt/β-catenin信号通路探讨去毒附子汤对骨关节炎大鼠的软骨保护作用  

Detoxicated Fuzi Decoction Ameliorates Cartilage Degradation via Wnt/β-catenin Signaling Pathway

作　　者：陈祖祥 葛彦志 周莉 王春雷[2] 陈俊杰 童培建 单乐天 刘福存 CHEN Zuxiang;GE Yanzhi;ZHOU Li(The First Clinical Medical College of Zhejiang Chinese Medical University,Hangzhou(310053),China)

机构地区：[1]浙江中医药大学第一临床医学院,杭州310053 [2]浙江省肿瘤医院 [3]海军军医大学附属长征医院

出　　处：《浙江中医药大学学报》2021年第6期571-581,共11页Journal of Zhejiang Chinese Medical University

基　　金：国家自然科学基金项目(81774331、82074464);浙江省基础公益研究计划项目(LY20H270014)。

摘　　要：[目的]基于Wnt/β-连环蛋白(Wnt/β-catenin)信号通路探讨去毒附子汤(detoxicated Fuzi decoction,FZT)对膝骨关节炎(knee osteoarthritis,KOA)的作用及相关机制。[方法]将30只雌性无特殊病原体(specific pathogen free,SPF)级SD大鼠随机分为正常组,模型组,FZT低、中、高剂量组,每组6只。模型组FZT低、中、高剂量组均采用碘乙酸关节腔注射造模法构建大鼠KOA模型。建模成功后FZT低、中、高剂量组分别以2.4、4.8、9.6g·kg^(-1)的FZT灌胃模型组和正常组以同剂量的0.9%氯化钠溶液灌胃连续4周。首次给药前和末次给药后对各组大鼠进行疼痛行为学检测。末次给药后1周取各组大鼠膝关节软骨组织进行番红O染色和Mankin评分。分离大鼠原代软骨细胞进行培养以不同浓度的FZT含药血清进行干预,采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT)法测定FZT含药血清对软骨细胞增殖活力的影响,选取最佳浓度。将软骨细胞随机分为NC组、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)组、TNF-α+FZT组,TNF-α+FZT组以10%的FZT含药血清干预,其余组使用空白培养基处理,以实时定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)检测2型胶原(collgen type 2,Col2)、10型胶原(collgen type 10,Col10)、基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)、带有血小板凝血酶敏感蛋白结构域的解聚素与金属蛋白酶4(adisintegrin and metalloproteinase with thrombospondin motifs 4 Adamts4)、带有血小板凝血酶敏感蛋白结构域的解聚素与金属蛋白酶5(adisintegrin and metalloproteinase with thrombospondin motifs 5,Adamts5)、卷曲关联蛋白(frizzled-related protein,FRZB)、Wnt、低密度脂蛋白受体相关蛋白5/6(low density lipoprotein receptor related proteins 5/6,LRP5/6)、糖原合酶激酶-3β(glycogen synthase kinase-3βGSK-3β)以及β-catenin的mRNA表�[Objective]To explore the mechanism of action of detoxicated Fuzi decoction(FZT)on knee osteoarthritis(KOA)rats based on Wnt/β-catenin signaling pathway.[Methods]Thirty female specific pathogen free(SPF)SD rats were randomly divided into normal group,model group,FZT low-dose group,FZT medium-dose group and FZT high-dose group,with 6 rats in each group.The KOA models in model group,FZT low-dose group,FZT medium-dose group and FZT high-dose group were all constructed by articular cavity injection of monoiodoacetate.FZT low-dose group,FZT mediumdose group and FZT high-dose group were given 2.4,4.8 and 9.6 g·kg^(-1) FZT and the same dose of 0.9% sodium chloride solution was given to model group and normal group for 4 weeks.Before the first administration and after the last administration,the rats in each group were tested for pain behavior.One week after the last administration,cartilage tissue of the knee joints of rats in each group was taken for safranine O staining and Mankin score.Primary chondrocytes of rats were taken and treated with different concentrations of FZT drug serum.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay was used to determine the effect of FZT drug serum on chondrocytes proliferative activity,and the optimal concentration was selected.Chondrocytes were randomly divided into NC group,tumor necrosis factor-α(TNF-α)group,TNF-α+FZT group.TNF-α+FZT group was treated with 10% FZT-containing serum and the remaining groups were treated with blank medium.Real-time quantitative polymerase chain reaction(Real-time qPCR)was used to detect mRNA expression of collagen type 2(Col2),collagen type 10(Col10),matrix metalloproteinase 13(MMP13),adisintegrin and metalloproteinase with thrombospondin motifs 4(Adamts4),adisintegrin and metalloproteinase with thrombospondin motifs 5(Adamts5),frizzled-related protein(FRZB),Wnt,low density lipoprotein receptor related proteins 5/6(LRP5/6),glycogen synthase kinase-3β(GSK-3β)and β-catenin,Western blot was used to detect protein express

关 键 词：骨性关节炎 WNT Β-CATENIN 软骨细胞 碘乙酸 去毒附子汤 含药血清 信号通路 大鼠 

分 类 号：R331[医药卫生—人体生理学]