炎症外泌体对正常白兔膝关节功能和滑膜组织的影响及机制

Effects and mechanisms of inflammatory exosomes on knee joint function and synovial tissue in normal white rabbits

目的探讨炎症外泌体对正常白兔膝关节功能和滑膜组织的影响及其作用机制。方法取10只雄性新西兰大白兔,应用改良Hluth法建立单膝膝骨关节炎模型,提取模型兔关节冲洗液中外泌体。另取27只雄性新西兰大白兔,随机分为空白组、盐水组、外泌体组,每组9只。外泌体组于右膝关节腔注射提取的外泌体混悬液,盐水组注射生理盐水,均每周3次,连续注射2周后观察4周;空白组不予干预。记录各组白兔行为学及关节肿胀情况,计算膝骨性关节炎严重性Lequesne指数;HE染色观察滑膜组织病理形态,计算滑膜组织Krenn评分;ELISA法检测关节冲洗液中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)含量,Western blot法检测滑膜组织中Toll样受体(TLRs)/核因子-κB(NF-κB)通路中相关元件蛋白表达情况。结果干预后外泌体组白兔出现轻度跛行,关节腔周围轻度肿胀,Lequesne指数明显高于干预前及空白组、盐水组(P均<0.05)。滑膜HE染色见外泌体组白兔的滑膜组织及血管增生明显,大量炎性细胞浸润,Krenn评分明显高于空白组及盐水组(P均<0.05)。外泌体组关节冲洗液中TNF-α、IL-1β含量和滑膜组织中TLR2、TLR4、NF-κB p65、MyD88蛋白相对表达量均明显高于空白组及盐水组(P均<0.05)。结论炎症外泌体能够激活正常白兔滑膜组织中TLRs/NF-κB信号通路,诱导下游炎性因子释放,导致滑膜炎症形成。

Objective It is to observe the effect and mechanism of inflammatory exosomes on knee joint function and synovial tissue in normal white rabbits.Methods Ten male New Zealand large white rabbits were taken to establish the models of single knee osteoarthritis by modified Hluth method,and the exosomes were extracted from the modeled rabbit joint flushing fluid.Another 27 male New Zealand large white rabbits were taken and randomly divided into blank group,saline group and exosome group,with 9 rabbits in each group.The exosome group was injected with the extracted exosome suspension in the right knee joint cavity,and the saline group was injected with saline,both 3 times per week,continuously treated for 2 weeks and then observed for 4 weeks;the blank group was not treated.The behaviors and joint swelling of the white rabbits in each group were recorded,and the Lequesne index of knee osteoarthritis severity was calculated;the pathological morphology of synovial tissue was observed by HE staining,and the Krenn score of synovial tissue was calculated;the contents of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the joint flushing fluid were detected by ELISA,and the protein expressions of related components of Toll-like receptors(TLRs)/nuclear factor-κB(NF-κB)pathway in synovial tissues were detected by Western blot.Results After the intervention,the white rabbits in the exosome group showed mild lameness,with mild swelling around the joint cavity,and their Lequesne index were significantly higher than those before the intervention and in the blank group and saline group(all P<0.05).HE staining of synovium showed that there were obvious proliferation of synovial tissue and blood vessels in white rabbits of the exosome group,with a large number of inflammatory cells infiltrating,and their Krenn scores were significantly higher than those in the blank group and the saline group(all P<0.05).The contents of TNF-αand IL-1βin the joint flushing fluid and the relative expressions of TLR2,TLR4,NF-κB p65 and MyD88 proteins in the synovial tissue of the exosome group were significantly higher than those of the blank group and saline group(all P<0.05).Conclusion Inflammatory exosomes can activate the TLRs/NF-κB signaling pathway in normal synovium tissue,induce the release of downstream inflammatory factors and leading to synovial inflammation formation.