杨梅素通过PI3K/AKT/NF-κB信号通路对骨性关节炎发展的影响

Effect of myricetin on the development of osteoarthritis through PI3K/AKT/NF-κB signaling pathway

目的:探讨杨梅素通过PI3K/AKT/NF-κB信号通路对骨性关节炎(OA)发展的缓解作用及其机制研究.方法:通过CCK-8法筛选出IL-1β诱导损伤的最适质量浓度;采用诱导损伤最适质量浓度的IL-1β构建体外骨性关节炎损伤模型,通过CCK-8测定不同浓度的杨梅素对损伤细胞存活率的影响,筛选杨梅素的最适作用浓度.细胞实验使用分离提取的软骨细胞,分为对照组、IL-1β模型组、杨梅素组;克隆形成实验检测各组细胞的增殖能力;流式细胞术检测各组细胞的凋亡能力;酶联免疫法检测各组细胞上清液中TNF-α、IL-1β、IL-6和IL-10的质量浓度;Western blot检测各种细胞中PI3K/AKT/NF-κB信号通路相关蛋白的表达情况.动物实验使用SD大鼠,分为假手术组、OA模型组、杨梅素组,并通过灌胃进行给药;Masson染色观察各组大鼠软骨组织的组织结构变化;ELISA检测各组大鼠关节液中TNF-α、IL-6和IL-10的质量浓度.结果:与质量浓度为0、1、5、50、100 ng/mL的IL-1β相比,当IL-1β的质量浓度为10 ng/mL时,细胞抑制率为48.36%(P<0.01),接近半数致死量,因此IL-1β诱导损伤的最适质量浓度确定为10 ng/mL.与浓度为5、20μmol/L的杨梅素相比,当杨梅素浓度为10μmol/L时,能明显提高损伤后的软骨细胞的活性(P<0.01),因此杨梅素的最适作用浓度选择为10μmol/L;杨梅素能提高软骨细胞的增殖能力(P<0.05),降低软骨细胞的凋亡能力;同时使TNF-α、IL-6的质量浓度均减少(P<0.05),IL-10的质量浓度增加(P<0.05);杨梅素也能使软骨细胞中PI3K、AKT、NF-κB P65、IKKαβ、IKBα蛋白的磷酸化程度均下调(P<0.05),IKBα蛋白表达上调;Masson结果显示:杨梅素使受损的软骨细胞形态变规则,软骨组织的胶原纤维排列变紧密,同时使大鼠关节液中TNF-α、IL-6的质量浓度均减少(P<0.05),IL-10质量浓度增加(P<0.05).结论:杨梅素可促进软骨细胞增殖,抑制软骨细胞的凋亡,抑制骨性关节炎炎症反应,抑制PI3K/AKT/NF-κB信号通路的激活,明显缓解关节软骨的退变,降低骨性关节炎的进展水平.

Objective:To investigate the mitigative effect of myricetin on the development of osteoarthritis(OA)and its mechanism through PI3K/AKT/NF-κB signaling pathway.Methods:The optimal mass concentration of IL-1βused for inducing injury was screened by CCK-8 method.The in vitro osteoarthritis injury model was induced by IL-1βtreatment at the optimal mass concentration.The effect of myricetin on the survival rate of injured cells at different concentrations was determined by CCK-8 method to screen the optimal concentration of myricetin.Isolated and extracted chondrocytes were used in cell experiments,which were divided into control group,IL-1βmodel group and myricetin group.Clone formation test was used to detect the proliferation ability of cells in each group.Flow cytometry was used to detect the apoptotic capacity of cells.Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of TNF-α,IL-1β,IL-6 and IL-10 in cell supernatant,Western blot to detect the expression of PI3K/AKT/NF-κB signaling pathway related proteins in cells.SD rats were used in animal experiments and divided into sham operation group,OA model group,and myricetin group.The animals were administered intragastrically.Masson staining was used to observe the tissue structure changes of cartilage tissue in rats,and ELISA was used to measure the mass concentration of TNF-α,IL-6 and IL-10 in the synovial fluid of rats.Results:The cytostatic rate was 48.36%when the mass concentration of IL-1βwas 10 ng/mL(P<0.01),which was close to half lethal dose.Therefore,the optimal mass concentration of IL-1βused for inducing injury was determined to be 10 ng/mL.When the concentration of myricetin was 10μmol/L,it significantly increased the activity of chondrocytes after injury(P<0.01)and was selected as the optimal concentration of myricetin.Myricetin increased the proliferative capacity of chondrocytes(P<0.05)and reduce the apoptotic capacity of chondrocytes.Also,the mass concentration of TNF-αand IL-6 was reduced(P<0.05),and that of IL-10 was increased(P<0.05).Myricetin down-regulated the phosphorylation of PI3K,AKT,NF-κB P65,IKKαβ,and IKBαproteins in chondrocytes(P<0.05)while up-regulated IKBαprotein expression(P<0.05).The results of Masson staining showed that myricetin regularized the morphology of damaged chondrocytes,and made the arrangement of collagen fibers in cartilage tissue become tight.The mass concentrations of TNF-αand IL-6 in the synovial fluid of rats was reduced(P<0.05),and the IL-10 content was increased(P<0.05).Conclusion:Myricetin promoted the proliferation and inhibited the apoptosis of chondrocytes.It inhibited the inflammatory response of osteoarthritis and the activation of PI3K/AKT/NF-κB signaling pathway,and significantly relieved the degeneration of articular cartilage,thus to reduce osteoarthritis progress.