| 陈祖祥,葛彦志,周莉,等.基于W m啡eaterin信号通路探讨去霉附子场对骨关节类大鼠的软骨保护作用[J].浙江中医药大学学报,2021,45(6):571-581. |
| 基于W m啡eaterin信号通路探讨去霉附子场对骨关节类大鼠的软骨保护作用 |
| Detoxicated Fuzi Decoction Ameliorates Cartilage Degradation via Wnt/β-catenin Signaling Pathway |
| DOI：10.16466/j.issn1005-5509.2021.06.002 |
| 中文关键词: 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 |
| 英文关键词: osteoarthritis Wnt β-catenin chondrocyte monoiodoacetate detoxicated Fuzi decoction drug containing serum signaling pathway rat |
| 基金项目: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) |
|
| 摘要点击次数: 2205 |
| 全文下载次数: 1036 |
| 中文摘要: |
| [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、9.6 g·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-3β，GSK-3β)以及β-catenin的mRNA表达情况，Western blot检测Wnt、LRP5/6、FRZB、GSK-3β以及β-catenin的蛋白表达情况。[结果]疼痛行为学数据显示，模型组大鼠的热痛和压痛阈值显著低于正常组(P＜0.01，P＜0.01)，FZT低、中、高剂量组大鼠的热痛和压痛阈值较模型组显著提高(均P＜0.01)。Mankin病理评分表明，模型组评分较正常组显著增高(P＜0.01)，FZT组评分较模型组显著降低(均P＜0.01)，且具有剂量依赖效应。MTT结果提示FZT含药血清能显著提高软骨细胞增殖活力。Real-time qPCR结果显示，与TNF-α组比较，FZT含药血清干预显著下调Col10、MMP13、Adamts4、Adamts5、Wnt、LRP5/6、GSK-3β及β-catenin的表达(均P＜0.01)，同时上调Col2的表达(P＜0.01)。Western blot结果显示，与TNF-α组比较，TNF-α+FZT组Wnt、LRP5/6、FRZB、GSK-3β以及β-catenin的蛋白表达减少(均P＜0.01)。[结论]FZT能显著改善KOA大鼠的疼痛行为学指标，修复软骨损伤，其作用机制可能与调控Wnt/β-catenin信号通路以及软骨细胞分解代谢有关。 |
| 英文摘要: |
| [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 medium-dose group and FZT high-dose group were given 2.4， 4.8 and 9.6g·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 expression of Wnt， LRP5/6， FRZB， GSK-3β， and β-catenin， and statistical analysis was performed. [Results] The pain behavior data showed that the threshold of thermal pain and tenderness in model group was significantly lower than that in normal group(P＜0.01，P＜0.01)， and the threshold of thermal pain and tenderness in FZT low， medium and high-dose groups was significantly higher than that in model group(all P＜0.01). Mankin pathological scores showed that model group was significantly higher than normal group(P＜0.01)， and FZT each dose group was significantly lower than model group(all P＜0.01)， with a dose-effect.MTT results showed that FZT containing serum significantly increased the proliferative activity of chondrocytes.Real-time qPCR results showed that compared with TNF-α group， 10% FZT containing serum significantly down-regulated Col10， MMP13， Adamts4， Adamts5， Wnt， LRP5/6， GSK-3β， β-catenin expression(all P＜0.01)， while up-regulated Col2 expression(P＜0.01). Western blot results showed that compared with TNF-α group， protein expressions of Wnt， LRP5/6， FRZB， GSK-3β and β-catenin were decreased in TNF-α+FZT group(All P＜0.01).[Conclusion] FZT can significantly improve the pain behavior index and repair cartilage damage in KOA rats， and its mechanism may be related to Wnt/β-catenin signaling pathway and the regulation of catabolism of chondrocytes. |
|
查看全文
查看/发表评论 下载PDF阅读器 |
| 关闭 |