天麻素通过Wnt/β-连环蛋白通路和核因子-κB通路对大鼠骨关节炎的治疗作用

Therapeutic effect of gastrodin on osteoarthritis in rats via Wnt/β-catenin pathway and nuclear factor-κB pathway

目的探讨天麻素对大鼠骨关节炎的治疗作用以及对Wnt/β-连环蛋白(β-catenin)通路和核因子-κB(NF-κB)通路的影响。方法30只购自河南实验动物中心的SD大鼠按照随机数字表法分为假手术组、关节炎组和天麻素组,每组大鼠10只。关节炎组和天麻素组大鼠采用改良Hulth法制备膝骨关节炎模型,假手术组手术过程同模型组但不形成骨关节炎模型。天麻素组大鼠腹腔注射天麻素50 mg/kg,假手术组和关节炎组大鼠腹腔注射等体积生理盐水。治疗8周,麻醉后,取3组大鼠膝关节软骨组织,进行后续研究。采用苏木精-伊红染色分析3组大鼠软骨组织病理学变化;采用酶联免疫吸附试验(ELISA)检测3组大鼠关节软骨组织白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)水平;采用丙二醛和超氧化物歧化酶试剂盒检测关节软骨丙二醛和超氧化物歧化酶表达水平;蛋白质免疫印迹分析3组大鼠关节软骨NF-κB、Toll样受体4(TLR4)和髓分化因子88(MyD88)、Ⅱ型胶原蛋白、基质金属蛋白酶13和蛋白聚糖的水平;采用Mankin’s评分分析3组大鼠关节软骨的损伤程度。组间计量数据比较采用单因素方差分析。结果天麻素组大鼠关节软骨组织IL-1β、IL-6和TNF-α水平[(33.84±6.37)、(21.64±3.98)、(58.61±8.83)pg/g]明显低于关节炎组大鼠[(74.97±9.81)、(45.63±7.87)、(140.68±20.79)pg/g],差异有统计学意义(t=9.742、8.261、11.780,P<0.05)。天麻素组大鼠关节软骨组织丙二醛水平[(1.33±0.14)mmol/g]明显低于关节炎组大鼠[(2.38±0.18)mmol/g],差异有统计学意义(t=14.720,P<0.05)。天麻素组大鼠关节软骨组织超氧化物歧化酶活性[(86.29±6.99)U/mg]明显低于关节炎组大鼠[(65.17±7.37)U/mg],差异有统计学意义(t=6.578,P<0.05)。天麻素组大鼠关节软骨组织NF-κB、MyD88、Wnt2和β-catenin水平(1.09±0.12、1.31±0.13、0.91±0.07、1.22±0.09)明显低于关节炎组大鼠(1.73±0.15、1.75±0.14、1.42±0.16、1.74±0.12),差异有统计学意义(t=9.742、8.261、9.090、11.330,P<0.05)。天麻素组大鼠关节软骨组织Ⅱ型胶原蛋白和蛋白聚糖水平(1.21±0.09、1.28±0.12)明显高于关节炎组(0.77±0.10、0.87±0.11),差异有统计学意义(t=10.240、7.971,P<0.05)。天麻素组大鼠关节Mankin’s评分[(3.75±1.00)分]明显低于关节炎组[(7.53±0.62)分],差异有统计学意义(t=10.130,P<0.05)。结论天麻素通过抑制Wnt/β-catenin通路和NF-κB通路,抑制大鼠骨关节炎症,延缓关节软骨退变,对骨关节炎达到治疗作用。

Objective To investigate the therapeutic effect of gastrodin on osteoarthritis in rats and the effects of gastrodin on Wnt/β-catenin pathway and nuclear factor-κB(NF-κB)pathway.Methods Totally,30 SD rats purchased from Henan Experimental Animal Center were divided into sham operation group,arthritis group and gastrodin group according to the random number table method,with 10 rats in each group.Knee osteoarthritis model was prepared by modified Hulth method in arthritis group and gastrodin group.The sham operation group underwent the same surgical procedure as the model group,but no osteoarthritis model was formed.Gastrodin group rats were intraperitoneally injected with gastrodin(50 mg/kg),and the animals in sham operation group and arthritis group were intraperitoneally injected with equal volume of normal saline.After 8 weeks of treatment and anesthesia,knee cartilage tissues of three groups were taken for follow-up study.Hematoxylin-eosin staining was used to analyze the histopathological changes of cartilage in the three groups.The levels of interleukin(IL)-1β,IL-6 and tumor necrosis factorα(TNFα)in articular cartilage of rats in the three groups were detected by enzymed-linked immunosorbent assay(ELISA).The expression levels of malondialdehyde and superoxide dismutase in articular cartilage were determined with malondialdehyde and superoxide dismutase kit.NF-κB,Toll-like receptor 4(TLR4)and the level of myeloid differentiation primary response protein 88(MyD88),typeⅡcollagen,matrix metalloproteinase 13 and proteoglycan were analyzed by Western blotting in the three groups.The injury degree of articular cartilage in the three groups was analyzed by Mankin’s score.One-way analysis of variance was used to compare the measurement data between groups.Results The levels of IL-1β,IL-6 and TNFαin articular cartilage of gastrodin group[(33.84±6.37),(21.64±3.98),(58.61±8.83)pg/g]were lower than those in model group[(74.97±9.81),(45.63±7.87),(140.68±20.79)pg/g,t=9.742,8.261,11.780,P<0.05].The level of malondialdehyde in articular cartilage of gastrodin group[(1.33±0.14)mmol/g]was significantly lower than that of model group[(2.38±0.18)mmol/g,t=14.720,P<0.05].The activity of superoxide dismutase in articular cartilage of gastrodin group[(86.29±6.99)U/mg]was significantly lower than that of model group[(65.17±7.37)U/mg,t=6.578,P<0.05].The levels of NF-κB,MyD88,Wnt2 andβ-catenin in articular cartilage of gastrodin group(1.09±0.12,1.31±0.13,0.91±0.07,1.22±0.09)were significantly lower than those of model group(1.73±0.15,1.75±0.14,1.42±0.16,1.74±0.12,t=9.742,8.261,9.090,11.330,P<0.05).The levels of typeⅡcollagen and proteoglycan in articular cartilage of gastrodin group(1.21±0.09,1.28±0.12)was significantly higher than in model group(0.77±0.10,0.87±0.11,t=10.240,7.971,P<0.05).The joint Mankin’s score of gastrodin group(3.75±1.00)was significantly lower than that of model group(7.53±0.62,t=10.130,P<0.05).Conclusion Gastrodin can inhibit osteoarthritis in rats by inhibiting Wnt/β-catenin pathway and NF-κB pathway,delay articular cartilage degeneration,and achieve therapeutic effect on osteoarthritis.