桑枝调控HIF-1α/VEGF/MMPs信号通路改善佐剂性关节炎大鼠骨破坏的研究

Study of Ramulus Mori on bone destruction in adjuvant-induced arthritis rats by regulating HIF-1α/VEGF/MMPs signaling pathway

目的基于HIF-1α/VEGF/MMPs信号通路探讨桑枝对佐剂性关节炎(adjuvant-induced arthritis,AIA)大鼠骨破坏的治疗作用及机制。方法将30只大鼠随机分成5组:正常组、模型组、阳性药(雷公藤多苷片10 mg/kg)组、桑枝水提物低剂量(3 g/kg)组、桑枝水提物高剂量(9 g/kg)组,每组6只。采用完全弗氏佐剂诱导AIA大鼠模型。造模后第1天开始灌胃给药,正常组、模型组给予10 mL/kg生理盐水,其余组给予相应药物,每天1次,连续30 d。给药前及给药后每3天进行称量、足趾肿胀度测定、关节炎指数计算;治疗结束后采用CT观察足骨破坏情况,HE染色法观察踝关节病理变化,免疫组化法检测踝关节滑膜组织低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白表达,Western blot检测膝关节滑膜组织HIF-1α、VEGF、MMP-3、MMP-9蛋白表达水平。结果与正常组相比,模型组体质量显著降低(P<0.05,P<0.01),足趾肿胀度、关节炎指数、骨破坏评分、组织学评分和HIF-1α、VEGF、MMP-3、MMP-9蛋白表达水平均显著升高(P<0.01)。与模型组相比,阳性药组、桑枝水提物低剂量组、桑枝水提物高剂量组体质量均显著升高(P<0.01),足趾肿胀度、关节炎指数、骨破坏评分、组织学评分和VEGF、MMP-3、MMP-9蛋白表达水平均显著降低(P<0.01)。与阳性药组相比,桑枝水提物高剂量组足趾肿胀度、关节炎指数(P<0.01),骨破坏评分、组织学评分和HIF-1α、VEGF、MMP-3、MMP-9蛋白表达水平均显著升高(P<0.05,P<0.01)。与桑枝水提物低剂量组相比,桑枝水提物高剂量组足趾肿胀度、关节炎指数、骨破坏评分、组织学评分和HIF-1α、VEGF、VEGF、MMP-9蛋白表达水平均显著升高(P<0.05,P<0.01)。结论桑枝可能通过调控HIF-1α/VEGF/MMPs信号通路,抑制血管新生、血管翳形成及细胞外基质的降解,从而发挥对AIA大鼠骨破坏的保护作用。

Objective To investigate the effect and mechanism of Ramulus Mori on bone destruction in adjuvant-induced arthritis(AIA)rats based on HIF-1α/VEGF/MMPs signaling pathway.Methods Thirty rats were randomly divided into five groups:normal group,model group,positive drug(10 mg/kg tripterygium wilfordii polyglycoside tablet)group,low-dose of Ramulus Mori water extract(3 g/kg)group and high-dose of Ramulus Mori water extract(9 g/kg)group,with six rats in each group.AIA rat model was induced by complete Freund’s adjuvant.Intragastric administration was started on the first day after modeling.The normal group and the model group were administrated with 10 mL/kg normal saline,while the other groups were given the corresponding medicine once per day for 30 d.The body weight,degree of toe swelling and arthritis index were measured each three days after modeling.The CT was used to evaluate the bone destruction of rat paws.The pathomorphological changes of the ankle joint in rats were observed by HE staining.The expression levels of hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),matrix metalloproteinase-3(MMP-3)and matrix metalloproteinase-9(MMP-9)in rat synovial tissue were detected by immunohistochemistry.Western blot was used to detect the protein expression levels of HIF-1α,VEGF,MMP-3 and MMP-9 in synovial tissue of knee joint.Results Compared with the normal group,the body weight of model group was significantly reduced(P<0.05,P<0.01),the degree of toe swelling,arthritis index,bone destruction score,histological score and the expression levels of HIF-1α,VEGF,MMP-3 and MMP-9 were significantly increased(P<0.01).Compared with the model group,the body weight of the positive drug group,the low-dose of Ramulus Mori water extract group and the high-dose of Ramulus Mori water extract group were significantly increased(P<0.01),and the degree of toe swelling,arthritis index,bone destruction score,histological score and the expression levels of VEGF,MMP-3 and MMP-9 were significantly decreased(P<0.01).Compared with the positive drug group,the degree of toe swelling,arthritis index(P<0.01),bone destruction score,histological score and the expression levels of HIF-1α,VEGF,MMP-3 and MMP-9 were significantly increased(P<0.05,P<0.01)in high-dose of Ramulus Mori water extract group.Compared with the low-dose of Ramulus Mori water extract group,the degree of toe swelling,arthritis index,bone destruction score,histological score and the expression levels of HIF-1α,VEGF and MMP-9 were significantly increased(P<0.05,P<0.01)in high-dose of Ramulus Mori water extract group.Conclusion Ramulus Mori may inhibit angiogenesis,pannus formation and extracellular matrix degradation by regulating HIF-1α/VEGF/MMPs signaling pathway,thus playing a protective role in bone destruction in AIA rats.