膝舒方防治阳虚寒凝型膝骨关节炎的实验研究

Experimental Study of Xishu Decoction on Knee Osteoarthritis with Syndrome of Yang Deficiency and Cold Coagulation

目的:观察中药膝舒方对阳虚寒凝型膝骨关节炎(KOA)兔的防治作用,探讨其作用机制。方法:采用木瓜蛋白酶注射的方式制备膝骨关节炎兔模型,薄荷油灌胃联合气候箱营造湿冷环境的方式诱导阳虚寒凝证型。实验兔随机分为5组(每组各12只):模型组、阳性组(塞来昔布100 mg/kg)、膝舒方高剂量组(生药16 g/kg)、膝舒方低剂量组(生药8 g/kg)、空白组。分别于给药前、给药3周后、给药6周后评价奎森(Lequesne)膝关节功能演算指数(Lequesne指数),并进行中医证候评分;给药6周后,制备膝关节软骨苏木精-伊红(HE)染色切片,光学电子显微镜下观察组织形态学病理并进行改良Mankin's病理评分;ELISA法检测血清促炎细胞因子白介素1β(IL-1β)、IL-6及抗炎细胞因子IL-4、IL-10水平;RT-qPCR法检测关节软骨基质金属蛋白酶1(MMP-1)、MMP-3、组织金属蛋白酶抑制因子1(TIMP-1)基因表达。结果:给药3周后或6周后,膝舒方高、低剂量组实验兔患肢肿胀和压痛反应显著减轻;膝关节活动范围及跛行现象均有所改善;Lequesne指数与同时段模型组相比均有显著降低,差异有统计学意义(P<0.05或P<0.01);中医证候评分与同时段模型组相比均有显著降低,差异有统计学意义(P<0.01)。给药6周后,与模型组相比,膝舒方高、低剂量组关节软骨的改良Mankin's评分明显降低,差异有统计学意义(P<0.01);血清IL-1β和IL-6水平均有显著降低,差异有统计学意义(P<0.01);IL-4和IL-10水平均有显著增高,差异有统计学意义(P<0.01);关节软骨MMP-1和MMP-3 mRNA相对表达量显著下调,差异有统计学意义(P<0.01),关节软骨TIMP-1 mRNA相对表达量则无明显变化。结论:膝舒方通过一定程度上纠正促炎/抗炎细胞因子及MMPs/TIMPs的失衡状态,以减轻阳虚寒凝型膝骨关节炎兔关节腔的炎症反应,纠正关节基质代谢紊乱,改善膝关节功能及软骨损伤。

Objective:To evaluate the effect of Xishu decoction on knee osteoarthritis(KOA)with syndrome of Yang deficiency and cold coagulation(KOA-YDCC)in rabbits and investigate its mechanisms.Methods:The KOA rabbits were established by papain injection,and the syndrome of Yang deficiency and cold coagulation was induced by peppermint oil gavage combined with a wet and cold environment created by climate box.The experimental rabbits were divided at random into 5 groups(12 cases in each group):model,positive control(celecoxib 1o0 mg/kg),Xishu decoction high and low dose(16 and 8 g/kg,respectively)and normal control groups.The Lequesne algofunctional index was evaluated before,3 weeks after and 6 weeks after of gavage administration,and the traditional Chinese medicine syndrome score was made.After 6 weeks of gavage administration,HEstained sections of knee cartilage were prepared;the histomorphology of knee cartilage was observed under light microscope,and modified Mankin's scores were evaluated.The expression levels of proinflammatory cytokines(IL-1βand IL-6)and anti-inflammatory cytokines(IL-4 and IL-10)in sera were measured by ELISA,and the relative gene expression of MMP-1,MMP-3 and TIMP-1 in articular cartilage of KOAYDCC rabbits was detected by RT-qPCR.Results:After 3 or 6 weeks of gavage administration,the swelling and pain response of the limbs were significantly reduced in Xishu decoction high and low dose groups;the range of motion of the knee joints and lameness were improved;the Lequesne index was significantly reduced(P<0.05 or P<0.01),and the traditional Chinese medicine syndrome scores were markedly decreased(P<o.O1)compared with those of the model group in the same period of time.After 6 weeks of gavage administration,compared with the model group,the modified Mankin's scores of knee cartilage in Xishu decoction high and low dose groups were significantly decreased(P<0.01);the levels of serum IL-1βand IL-6 were significantly decreased(P<0.O1);the levels of serum IL-4 and IL-10 were markedly increased(P<0.01);the relative gene expression of MMP-1 and MMP-3 in articular cartilage was significantly down-regulated(P<0.O1),while the relative gene expression of TIMP-1 showed no significant change.Conclusion:Xishu decoction treatment could alleviate the inflammatory reaction,correct the disorder of metabolism of the joint matrix,and improve the function of the knee joint and cartilage damage in rabbits with KOA-YDCC.