碘乙酸单钠诱导SD大鼠膝骨关节炎后HIF-1α、OPN、IL-1β、TNF-α、MMP-13和NGF蛋白表达及意义

Expression and significance of HIF-1α,OPN,IL-1β,TNF-α,MMP-13,and NGF proteins after MIA-induced KOA in SD rats

目的探讨碘乙酸单钠(monosodium iodoacetate,MIA)诱导SD大鼠膝骨关节炎(knee osteoarthritis,KOA)后HIF-1α、OPN、IL-1β、TNF-α、MMP-13和NGF蛋白表达及意义。方法采用SD大鼠(4周龄,80~120 g)为研究对象,随机分为假手术(Sham)组和模型(KOA)组。第0天,大鼠用戊巴比妥钠麻醉,模型组经右膝关节髌下韧带向关节腔内注射含2 mg MIA生理盐水50μL,Sham组大鼠给予50μL灭菌生理盐水。SD大鼠分别于第0、3、7、10、14天测量右膝关节直径和检测负重能力后,麻醉安乐死,切取软骨和分离滑膜组织。同时,原代培养假手术组和模型组滑膜组织,鉴定成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)的纯度和活性后,采用地塞米松处理FLS。通过Western Blot检测缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、骨桥蛋白(osteopontin,OPN)、白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)和神经生长因子(nerve growth factor,NGF)蛋白的表达。结果与假手术组比较,大鼠膝关节腔被注入MIA 3 d后,HIF-1α、OPN、IL-1β和TNF-α蛋白表达升高,呈不断增加趋势。大鼠膝关节腔横径,在MIA造模后第7天和第14天,显著大于假手术组。MIA注入大鼠膝关节腔第3天起,大鼠右后肢的负重能力持续在26%以下,显著低于假手术组。采用0.2%Ⅰ型胶原酶消化法,成功培养了FLS原代细胞。使用地塞米松对模型组大鼠体外FLS干预后,显著降低了促炎因子分泌、NGF和MMP-13蛋白的表达。结论HIF-1α、OPN、IL-1β、TNF-α、MMP-13和NGF蛋白在滑膜炎症、软骨降解及基质破坏和关节疼痛等方面起着重要作用,既是关节疼痛管理、炎症反应调节和抑制软骨降解和基质破坏的重要靶点,也是开发新型OA镇痛药物的蛋白靶点。

Objective To investigate the expression and significance of hypoxia-inducible factor-1α(HIF-1α),OPN,IL-1β,TNF-α,MMP-13,and NGF proteins in SD rats with knee osteoarthritis KOA induced by monosodium iodoacetate(MIA).Methods SD rats(4 weeks old,80~120 g)were randomly divided into sham operation(Sham)and model(KOA)groups.On day 0,the rats were anesthetized with sodium pentobarbital,and 50μL of normal saline containing 2 mg of MIA was injected into the joint cavity of the KOA group through the infrapatellar ligament of the right knee.In the sham group,50μL sterile saline was injected into the knee joint cavity.SD rats were euthanized by overdosed anesthesia on days 0,3,7,10,and 14 after measuring the diameter of the right knee joint and the weight-bearing capacity.Cartilage and synovial tissue were collected.Synovial tissues were primary cultured,and the purity and activity of fibroblast-like synoviocytes(FLSs)were assessed,and FLSs were treated with dexamethasone.Western Blot was used to detect HIF-1α,osteopontin(OPN),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-13(MMP-13),and nerve growth factor(NGF)proteins.Results Compared with the Sham group,HIF-1α,OPN,IL-1β,and TNF-α expression was increased at 3 days after injection of MIA into the knee cavity and showed an increasing trend.The transverse diameter of the knee cavity in the MIA group was significantly larger than that in the Sham group on days 7 and 14 after palpation.On day 3 after injection of MIA into the knee cavity of rats,the weight bearing capacity of the right hind limb continued to be<26%,which was significantly lower compared with the Sham group.Primary FLSs were successfully isolated by 0.2% type Ⅰ collagenase digestion.After treatment of FLSs from KOA rats with dexamethasone,secretion of proinflammatory factors and expression of MMP-13 and NGF proteins were decreased significantly.Conclusions HIF-1α,OPN,IL-1β,TNF-α,MMP-13,and NGF play major roles in cartilage and synovial inflammation,cartilage degradation,matrix destruction,and joint pain.They are important targets for joint pain management,regulation of inflammatory responses,and inhibition of cartilage degradation and matrix destruction,and the development of novel OA analgesics.