双氯芬酸钠对骨关节炎大鼠模型软骨细胞炎症和滑膜巨噬细胞极化的影响

Effect of diclofenac sodium on chondrocyte inflammation and synovial macrophage polarization in osteoarthritis rat model

目的探究双氯芬酸钠对骨关节炎(OA)大鼠模型软骨细胞炎症和滑膜巨噬细胞极化的影响。方法45只6周龄雄性Sprague-Dawley大鼠用于本研究,大鼠左膝关节的髌韧带关节内注射单碘乙酸(MIA)溶液诱导OA模型。根据实验目的将实验大鼠分为3组:对照组(关节内注射无菌0.9%氯化钠溶液作为实验对照,n=15),OA模型组(大鼠左膝关节的髌韧带关节内注射MIA诱导OA模型,n=15),双氯芬酸钠组(MIA注射后14 d,用双氯芬酸钠口服治疗8 d,n=15)。通过酶联免疫吸附试验(ELISA)试剂盒检测大鼠血清炎症因子肿瘤坏死因子(TNF-α)、白细胞介素(IL)-1β、IL-12和IL-10浓度。使用相应试剂盒测定大鼠血清丙二醛、还原型谷胱甘肽(GSH)和过氧化物歧化酶(SOD)浓度。通过蛋白印迹分析炎症介质相关蛋白的表达。通过免疫组织化学分析检测CD86和Arg1的蛋白表达。通过免疫细胞化学测定检查大鼠滑膜组织中CD86+和CD163+巨噬细胞的数量。组织学和免疫组织化学分析大鼠关节组织。通过膝弯曲试验和步态分析得分评估各组大鼠的关节疼痛。结果OA模型组TNF-α、IL-1β、IL-12浓度较对照组升高,IL-10浓度较对照组降低,差异均有统计学意义(P<0.05);双氯芬酸钠组TNF-α、IL-1β、IL-12较OA模型组降低,IL-10浓度较OA模型组升高,差异均有统计学意义(P<0.05)。OA模型组丙二醛浓度较对照组升高,GSH和SOD浓度较对照组降低,差异均有统计学意义(P<0.05);双氯芬酸钠组丙二醛浓度OA模型组降低,GSH和SOD浓度较OA模型组升高,差异均有统计学意义(P<0.05)。OA模型组iNOS、COX2、MMP-13和PGE2蛋白表达较对照组升高,差异均有统计学意义(P<0.05);双氯芬酸钠组iNOS、COX2、MMP-13和PGE2蛋白表达较OA模型组降低,差异均有统计学意义(P<0.05)。OA模型组CD163蛋白表达较OA模型组升高,Arg1蛋白表达较对照组降低,差异均有统计学意义(P<0.05);双氯芬酸钠组CD163蛋白表达较OA模型组降低,Arg1蛋白表达较OA模型组升高,差异均有统计学意义(P<0.05)。OA模型组CD86^(+)、CD163^(+)巨噬细胞的数量和M1/M2比值较对照组升高,差异均有统计学意义(P<0.05);双氯芬酸钠组CD86^(+)、CD163^(+)巨噬细胞的数量和M1/M2比值较OA模型组降低,差异均有统计学意义(P<0.05)。OA模型组的膝关节弯曲和步态评分较对照组升高,差异均有统计学意义(P<0.05);双氯芬酸钠组膝关节弯曲和步态评分较OA模型组降低,差异均有统计学意义(P<0.05)。结论双氯芬酸钠通过促进M2巨噬细胞极化减轻MIA诱导的OA大鼠的疼痛和软骨退化。双氯芬酸钠通过应用于膝关节腔后,M2巨噬细胞促进了膝关节的抗炎微环境,有助于结构改善和镇痛作用。

Objective To explore the effect of diclofenac sodium on chondrocyte inflammation and synovial macrophage polarization in a rat model of osteoarthritis.Methods Forty-five 6-week-old male Sprague-Dawley rats were used in this study.The OA model was induced by intra-articular injection of MIA solution into the patellar ligament of the left knee joint of the rat.According to the purpose of the experiment,the experimental rats were divided into 3 groups:control group(intra-articular injection of sterile 0.9%sodium chloride solutio as experimental control,n=15),OA model group(intra-articular injection of MIA into the patellar ligament of the left knee joint of rats to induce OA model,n=15),diclofenac sodium group(14 days after MIA injection,oral treatment with diclofenac sodium for 8 days,n=15).The serum inflammatory factors TNF-α,interleukin(IL)-1β,IL-12 and IL-10 concentrations were detected by enzyme-linked immunosorbent assay(ELISA)kit.The concentrations of rat serum malondialdehyde,reduced glutathione(GSH)and superoxide dismutase(SOD)were determined using the corresponding kits.The expression of inflammatory mediator-related proteins was analyzed by Western blotting.The protein expression of CD86 and Arg1 was detected by immunohistochemical analysis.The number of CD86+and CD163+macrophages in rat synovial tissue was examined by immunocytochemistry.Histological and immunohistochemical analysis of rat joint tissues.The joint pain of rats in each group was evaluated by knee bending test and gait analysis scores.Results The concentrations of TNF-α,IL-1βand IL-12 in the OA model group were higher than those in the control group,the concentration of IL-10 was lower than that in the control group,the differences were statistically significant(P<0.05);and the TNF-α,IL-1βand IL-12 in the diclofenac sodium group were lower than those in the OA model group,the concentration of IL-10 was higher than that in the OA model group,the differences were statistically significant(P<0.05).The MDA concentration in the OA model group was higher than that in the control group,the GSH and SOD concentrations were lower than those in the control group,the differences were statistically significant(P<0.05);the MDA concentration in the diclofenac sodium group was lower that in the OA model group,the GSH and SOD concentrations were higher than that those in the OA model group,the differences were statistically significant(P<0.05).The protein expressions of iNOS,COX2,MMP-13 and PGE2 in the OA model group were higher than those in the control group,the differences were statistically significant(P<0.05),and the protein expressions of iNOS,COX2,MMP-13 and PGE2 in the diclofenac sodium group were lower than those in the OA model group,the differences were statistically significant(P<0.05).The expression of CD163 protein in the OA model group was higher than that in the control group,the expression of Arg1 protein was lower than that in the control group,the differences were statistically significant(P<0.05);the expression of CD163 protein in the diclofenac sodium group was lower than that in the OA model group,the expression of Arg1 protein was higher than that in OA model group,the differences were statistically significant(P<0.05).The number of CD86+and CD163+macrophages and the M1/M2 ratio in the OA model group were higher than those in the control group,the differences were statistically significant(P<0.05);the number of CD86+and CD163+macrophages and the M1/M2 ratio in the diclofenac sodium group were lower than those in the OA model group,the differences were statistically significant(P<0.05).The knee flexion and gait scores of the OA model group were higher than those of the control group,the differences were statistically significant(P<0.05),and the knee flexion and gait scores of the diclofenac sodium group were lower than those of the OA model group,the differences were statistically significant(P<0.05).Conclusion Diclofenac sodium can reduce the pain and cartilage degradation in OA rats induced by MIA by promoting the polarization of M2 macrophages.After diclofenac sodium is applied to the knee joint cavity,M2 macrophages promote the anti-inflammatory microenvironment of the knee joint,which is helpful for structural improvement and analgesia.