摘要
目的 研究VX765对大鼠骨关节炎(osteoarthritis,OA)和软骨细胞炎症的影响及其作用机制.方法 分离4周龄SD大鼠膝关节软骨细胞,取第3代细胞采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测不同浓度(0、1、5、10、20、50、100 μmol/L)VX765对大鼠软骨细胞活性的影响;体外构建脂多糖(lipopoly-saccharides,LPS)诱导细胞炎症模型,将细胞分为对照组、LPS组、无明显细胞毒性VX765浓度1组和VX765浓度2组,行Western blot、实时荧光定量PCR和ELISA检测炎症因子TGF-β1、IL-6和TNF-α表达水平,Western blot和免疫荧光染色检测核因子E2相关的转录因子2(nuclear factor erythroid 2-related factor 2,Nrf2)和血红素氧化酶1(heme oxygenase 1,HO-1)表达.将32只SD大鼠随机分为假手术组(A组)、OA组(B组)、OA+VX765(50 mg/kg)组(C组)和OA+VX765(100 mg/kg)组(D组),每组8只.A组仅行膝关节前内侧切口,B～D组在A组基础上横切内侧副韧带和前交叉韧带,并切除内侧半月板;术后1周,C、D组分别灌胃给药50 mg/kg和100 mg/kg VX765,A、B组给予等量生理盐水灌胃.通过HE及番红固绿染色行组织病理学检查,并通过Mankin评分进行评估;采用免疫组织化学染色技术分析基质金属蛋白酶13(matrix metalloproteinase 13,MMP-13)和Ⅱ型胶原蛋白的表达.结果 CCK-8法检测示VX765浓度高于10 μmol/L后细胞活力显著降低(P＜0.05),选择无明显细胞毒性的4 μmol/L和8 μmol/L VX765进行后续实验.LPS诱导后细胞中的TGF-β1、IL-6和TNF-α表达显著高于对照组(P＜0.05),而在4 μmol/L和8 μmol/L VX765干预后表达较LPS组显著下降(P＜0.05).Western blot和免疫荧光染色检测示VX765显著上调Nrf2通路相关分子Nrf2和HO-1的蛋白表达(P＜0.05),激活Nrf2通路.大鼠膝关节组织病理学检查和免疫组织化学染色示,与B组相比,C、D组VX765处理改善了大鼠OA中关节结构损伤,减轻了炎症反应,下调MMP-13表达,增加Ⅱ型胶原蛋白的表达.结论 VX765可改善大鼠OA并减轻软骨细胞的炎症反应,其潜在机制可能是通过激活Nrf2通路.
Abstract
Objective To investigate the effects and underlying mechanisms of VX765 on osteoarthritis(OA)and chondrocytes inflammation in rats.Methods Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley(SD)rats.The third-generation cells were subjected to cell counting kit 8(CCK-8)analysis to assess the impact of various concentrations(0,1,5,10,20,50,100 μmol/L)ofVX765 on rat chondrocyte activity.An in vitro lipopolysaccharide(LPS)induced cell inflammation model was employed,dividing cells into control group,LPS group,VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity.Western blot,real-time fluorescence quantitative PCR,and ELISA were conducted to measure the expression levels of inflammatory factors—transforming growth factor β1(TGF-β1),interleukin 6(IL-6),and tumor necrosis factor α(TNF-α).Additionally,Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1).Thirty-two SD rats were randomly assigned to sham surgery group(group A),OA group(group B),OA+VX765(50 mg/kg)group(group C),and OA+VX765(100 mg/kg)group(group D),with 8 rats in each group.Group A underwent a sham operation with a medial incision,while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament,with removal of the medial meniscus.One week post-surgery,groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765,respectively,while groups A and B received an equivalent volume of saline.Histopathological examination using HE and safranin-fast green staining was performed,and Mankin scoring was utilized for evaluation.Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13(MMP-13)and collagen type Ⅱ.Results The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L(P＜0.05),so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments.Following LPS induction,the expressions of TGF-β1,IL-6,and TNF-α in cells significantly increased when compared with the control group(P＜0.05).However,intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group(P＜0.05).Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765(P＜0.05),indicating Nrf2 pathway activation.Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that,compared to group B,treatment with VX765 in groups C and D improved joint structural damage in rat OA,alleviated inflammatory reactions,downregulated MMP-13 expression,and increased collagen type Ⅱexpression.Conclusion VX765 can improve rat OA and reduce chondrocyte inflammation,possibly through the activation of the Nrf2 pathway.
维普
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