右归丸经IL-6/STAT3信号通路对膝骨关节炎模型大鼠软骨组织退变的调控机制

Regulatory Mechanism of Youguiwan on Articular Cartilage Degeneration via IL-6/STAT3 Signaling Pathway in Rats with Knee Osteoarthritis

目的:观察右归丸对膝骨关节炎(KOA)模型鼠软骨组织信号转导和转录激活因子3(STAT3)和白细胞介素-6(IL-6)表达水平的影响。方法:将大鼠随机分为假手术组、模型组、硫酸氨基葡萄糖组、右归丸高、中、低剂量组,每组10只。采用改良Hulth法制备大鼠KOA模型,假手术组和模型组给予等体积生理盐水灌胃,右归丸高、中、低剂量组分别给予右归丸4.8,2.4,1.2 g·kg^-1灌胃,硫酸氨基葡萄糖组给予硫酸氨基葡萄糖0.17 g·kg^-1灌胃,连续给药8周。干预结束24 h后股动脉采血处死各组大鼠,取鼠膝关节软骨,采用苏木素-伊红(HE)染色法观察各组软骨的病理改变,并进行Mankin评分;免疫组化法检测各组关节软骨组织中STAT3,超氧化物歧化酶3(SOD3)和Wnt抑制因子1(WIF1)的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测软骨组织中IL-6 mRNA的表达;蛋白免疫印迹法(Western blot)检测各组关节软骨组中WIF1蛋白的表达。结果:与假手术组比较,模型组大鼠软骨组织Makin评分明显升高,软骨组织STAT3在蛋白水平上的表达明显增加和IL-6 mRNA水平上的表达显著增加,WIF1在蛋白水平上的表达显著降低(P<0.01);模型组关节软骨边缘严重破坏,软骨细胞排列紊乱。与模型组比较,右归丸高剂量干预组大鼠软骨组织Makin评分,STAT3的在蛋白水平上的表达明显降低,右归丸各干预组IL-6 mRNA水平上的表达显著降低,WIF1在蛋白水平上的表达显著增加(P<0.05,P<0.01),软骨结构趋于正常,软骨细胞分布仅偶见不均,关节软骨表面欠光滑。结论:右归丸能显著改善KOA大鼠的关节软骨退变,抑制KOA中软骨组织的炎症反应,这可能与其抑制STAT3和IL-6的表达有关。

Objective:To observe the effect of Youguiwan on the levels of cartilage transducers and activators of transcription 3(STAT3)and interleukin-6(IL-6)in rats with knee osteoarthritis(KOA).Method:Sixty SD rats were randomly divided into six groups:sham control group,model group,glucosamine sulfate group and Youguiwan(high,middle and low-dose)groups.The modified Hulth method was used to prepare KOA models for 6 weeks.The shame control group and the model group were treated with normal saline,Youguiwan high,middle,low-dose groups received Youguiwan 4.8,2.4,1.2 g·kg^-1 by gavage respectively,and the glucosamine sulfate group was treated with glucosamine sulfate 0.17 g·kg^-1.The rats were administrated for 8 weeks according to the dose.After intervention,each group was put to death by femoral artery blood collection,and the keen cartilages of the rats were collected.The pathological changes were observed by htoxylin eosin(HE)staining method,and Mankin score was evaluated.The expressions of STAT3,superoxide dismutase3(SOD3)and Wnt inhibitory factor 1(WIF1)in articular cartilage were detected by immunohistochemistry.The expressions of IL-6 mRNA in articular cartilage were detected by quantitative real-time fluorescence polymerase chain reaction(Realtime PCR).The expression of WIF1 in articular cartilage was detected by Western blot.Result:Compared with the sham control group,the Makin score was obviously increased in the model group,the protein expression of STAT3 was increased significantly,the mRNA of IL-6 was raised significantly,but the protein expression of WIF1 was decreased significantly(P<0.01),articular cartilage was seriously damaged,and chondrocytes were arranged in disorder.Compared with the model group,the Makin score was declined obviously in the high-dose Youguiwan group,the protein expression of STAT3 was significantly reduced,the mRNA expression of IL-6 was significantly reduced in Youguiwan treatment group,while the protein expression of WIF1 was significantly increased(P<0.05,P<0.01),the cartilage structure returned to be normal,the chondrocytes distribution was uneven,and articular cartilage surface was not smooth.Conclusion:Youguiwan could significantly improve the articular cartilage degeneration of KOA rats,and inhibit the inflammation of chondrocytes,which may be related to the suppression of STAT3 and IL-6 expression.