薯蓣皂苷对膝骨性关节炎模型大鼠关节软骨的保护作用及机制研究

Protective effects and mechanisms of dioscin on articular cartilage in a rat model of knee osteoarthritis

目的观察薯蓣皂苷对膝骨性关节炎(KOA)模型大鼠关节软骨的保护作用,并探讨其可能机制。方法采用切断前交叉韧带离断法建立KOA大鼠模型,40只SD大鼠随机分为假手术组、KOA组及薯蓣皂苷低(50 mg/kg)、高剂量组(100 mg/kg),每组10只,假手术组、KOA组给予生理盐水灌胃,薯蓣皂苷低、高剂量组给予相应剂量薯蓣皂苷灌胃,每天1次。治疗8周后,观察各组大鼠刺激缩足反射潜伏期(TWL)、机械缩足反射阈值(MWT)、膝关节软骨组织病理变化,酶联免疫吸附试验(ELISA)检测各组大鼠关节液肿瘤坏死因子-α(TNF-α)、白介素-1β(IL-1β)、白介素-6(IL-6)含量,蛋白质印迹法(Westen blot)检测各组大鼠膝关节软骨组织Wnt3a、β-连环蛋白(β-catenin)、人基质金属蛋白酶13(MMP-13)、糖原合成酶激酶3β(GSK-3β)、Ⅱ型胶原蛋白(CollagenⅡ)蛋白表达水平。结果与假手术组比较,KOA组TWL[(9.43±0.31)s比(16.40±0.30)s,P<0.05]、MWT[(7.94±0.42)s比(14.38±0.32)s,P<0.05]明显降低;与KOA组比较,薯蓣皂苷低、高剂量组TWL[(11.25±0.65)s、(13.52±0.58)s比(9.43±0.31)s,P<0.05]、MWT[(10.29±0.90)s、(12.17±0.78)s比(7.94±0.42)s,P<0.05]明显升高;薯蓣皂苷低、高剂量组大鼠膝关节大体观察及组织病理改变较KOA组明显改善;与假手术组比较,KOA组关节液TNF-α[(168.96±6.12)pg/mL比(35.72±2.93)pg/mL,P<0.05]、IL-1β[(80.54±6.23)pg/mL比(22.17±1.21)pg/mL,P<0.05]、IL-6[(97.66±9.31)pg/mL比(19.93±2.42)pg/mL,P<0.05]明显升高,膝关节软骨组织内Wnt3a[(1.04±0.06)比(0.57±0.06),P<0.05]、β-catenin[(0.96±0.05)比(0.48±0.05),P<0.05]、MMP-13[(1.05±0.08)比(0.57±0.03),P<0.05]蛋白表达水平明显增加,GSK-3β[(0.51±0.05)比(1.13±0.08),P<0.05]、CollagenⅡ[(0.43±0.03)比(0.98±0.07),P<0.05]蛋白表达水平明显降低;与KOA组比较,薯蓣皂苷低、高剂量组TNF-α[(131.14±10.43)pg/mL、(106.77±8.28)pg/mL比(168.96±6.12)pg/mL,P<0.05]、IL-1β[(59.74±4.59)pg/mL、(37.93±4.14)pg/mL比(80.54±6.23)pg/mL,P<0.05]、IL-6[(78.03±5.85)pg/mL、(59.28±4.93)pg/mL比(97.66±9.31)pg/mL,P<0.05]及Wnt3a[(0.86±0.04)、(0.62±0.03)比(1.04±0.06),P<0.05]、β-catenin[(0.77±0.04)、(0.61±0.02)比(0.96±0.05),P<0.05]、MMP-13[(0.82±0.05)、(0.64±0.03)比(1.05±0.08),P<0.05]蛋白表达水平明显降低,GSK-3β[(0.70±0.06)、(0.89±0.07)比(0.51±0.05),P<0.05]、CollagenⅡ[(0.57±0.04)、(0.75±0.05)比(0.43±0.03),P<0.05]蛋白表达水平明显升高。结论薯蓣皂苷可能通过调控Wnt/β-catenin信号通路,抑制关节腔内炎症反应,维持软骨代谢平衡,从而减轻KOA模型大鼠疼痛症状和软骨退行性改变。

Objective To explore the protective effects of dioscin on articular cartilage in a rat model of knee osteoarthritis(KOA)and to elucidate its underlying mechanisms.Methods The KOA rat model was established using cutting the anterior cruciate ligament transection method.Forty SD rats were randomly divided into the sham operation,the KOA,the dioscin low-dose(50 mg/kg),and the dioscin high-dose(100 mg/kg)groups,with 10 rats in each group.The rats in the sham operation and KOA groups were administrated normal saline by gavage,while rats in the low-dose and high-dose groups were administrated the corresponding doses of dioscin,once daily.After 8 weeks of treatment,thermal withdrawal latency(TWL),mechanical withdrawal threshold(MWT),and histopathological changes of knee joint cartilage tissue were observed in each group.Enzyme-linked immunosorbent assay method was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)in the joint fluid of rats in each group.Western blot analysis was used to detect the protein expression levels of WNT3a antigen(Wnt3a),recombinant beta-catenin(β-catenin),human matrix metalloproteinase 13(MMP-13),and glycogen synthase kinase-3 beta(GSK-3β),collagenaseⅡ(CollagenⅡ)in the articular cartilage tissue of rats in each group.Results Compared with the sham operation group,the KOA group showed a significant decrease in TWL[(9.43±0.31)s vs.(16.40±0.30)s,P<0.05]and MWT[(7.94±0.42)s vs.(14.38±0.32)s,P<0.05].Compared with the KOA group,the low-dose and high-dose dioscin groups showed a significant increase in TWL[(11.25±0.65)s,(13.52±0.58)s vs.(9.43±0.31)s,P<0.05]and MWT[(10.29±0.90)s,(12.17±0.78)s vs.(7.94±0.42)s,P<0.05].The gross observation and histopathological changes of the rat knee joints in the low-dose and high-dose groups were significantly improved compared to the KOA group.Compared with the sham operation group,the KOA group showed a significant increase in the levels of TNF-α[(168.96±6.12)pg/mL vs.(35.72±2.93)pg/mL,P<0.05],IL-1β[(80.54±6.23)pg/mL vs.(22.17±1.21)pg/mL,P<0.05],and IL-6[(97.66±9.31)pg/mL vs.(19.93±2.42)pg/mL,P<0.05]in the joint fluid.The KOA group also showed increased protein expression levels of Wnt3a[(1.04±0.06)vs.(0.57±0.06),P<0.05],β-catenin[(0.96±0.05)vs.(0.48±0.05),P<0.05],and MMP-13[(1.05±0.08)vs.(0.57±0.03),P<0.05]and decreased levels of GSK-3β[(0.51±0.05)vs.(1.13±0.08),P<0.05]and CollagenⅡ[(0.43±0.03)vs.(0.98±0.07),P<0.05]in the knee joint cartilage tissue.Compared with the KOA group,the dioscin low and high dose groups showed a significant decrease in the levels of TNF-α[(131.14±10.43)pg/mL,(106.77±8.28)pg/mL vs.(168.96±6.12)pg/mL,P<0.05],IL-1β[(59.74±4.59)pg/mL,(37.93±4.14)pg/mL vs.(80.54±6.23)pg/mL,P<0.05],and IL-6[(78.03±5.85)pg/mL,(59.28±4.93)pg/mL vs.(97.66±9.31)pg/mL,P<0.05].Additionally,the protein expression levels of Wnt3a[(0.86±0.04),(0.62±0.03)vs.(1.04±0.06),P<0.05],β-catenin[(0.77±0.04),(0.61±0.02)vs.(0.96±0.05),P<0.05]and MMP-13[(0.82±0.05),(0.64±0.03)vs.(1.05±0.08),P<0.05]were significantly decreased,while the levels of GSK-3β[(0.70±0.06),(0.89±0.07)vs.(0.51±0.05),P<0.05]and CollagenⅡ[(0.57±0.04),(0.75±0.05)vs.(0.43±0.03),P<0.05]were significantly increased.Conclusion Dioscin may alleviate pain symptoms and cartilage degeneration in KOA model rats by regulating the Wnt/β-catenin signaling pathway,inhibiting inflammation in the joint cavity,and maintaining the balance of cartilage metabolism.