骨髓间充质干细胞来源的外泌体对弗氏完全佐剂诱导类风湿性关节炎大鼠的治疗作用

Treatment of BMSC-Exo on rats with rheumatoid arthritis induced by complete Freund's adjuvant

目的评价大鼠骨髓间充质干细胞(BMSC)来源的外泌体(Exo)在弗氏完全佐剂(CFA)诱导的类风湿性关节炎(RA)大鼠中的治疗作用。方法超速离心分离大鼠BMSC上清中的Exo(BMSC-Exo),利用纳米颗粒跟踪分析和Westernblotting法对BMSC-Exo表征进行鉴定。将20只雄性SD大鼠随机分为Sham组、CFA组、Exo组和Cel组,每组5只;除Sham组注射等体积生理盐水外,其余3组在大鼠左后肢足跖部皮下注射0.1 mLCFA,注射7 d后,Exo组大鼠尾静脉注射BMSC-Exo,Cel组灌胃Cel。采用足趾关节肿胀度、关节炎指数(AI)、热痛阈和脾脏指数评价BMSC-Exo对CFA大鼠的治疗效果;采用旷场实验和自动步态实验评价BMSC-Exo对CFA大鼠自主运动的影响;采用流式技术检测BMSC-Exo对CFA大鼠血清炎症因子水平的影响;采用Westernblotting法检测BMSC-Exo对CFA大鼠关节组织NF-κB通路蛋白表达的影响。结果与BMSC相比,BMSC-Exo特异性表达CD63和TSG101分子,但不表达CM130;BMSC-Exo的粒径分布在82.5 nm处达到峰值。与Sham组相比,CFA组大鼠足趾关节肿胀度、AI、脾脏指数高,热痛阈低,站立次数增加,运动距离及步长变短,步宽增加,摇摆时间变短及触地时间变长,血清炎症因子水平和关节组织NF-κB通路蛋白表达上调(P均<0.05)。与CFA组相比,Exo组、Cel组大鼠足趾关节肿胀度、AI、脾脏指数低,热痛阈高,站立次数减少,运动距离及步长变长,步宽降低,摇摆时间变长及触地时间缩短,血清炎症因子水平和关节组织NF-κB通路蛋白表达低(P均<0.05)。结论BMSC-Exo对CFA诱导的RA模型大鼠具有明显治疗作用,其机制可能与抑制NF-κB信号通路有关。

Objective To investigate effects of bone marrow mesenchymal stem cells-derived exosomes(BMSC-Exo)on rats with rheumatoid arthritis induced by complete Freund’s adjuvant.Methods Ultracentrifugation was used to separate Exo from BMSC in cell culture supernatant.Nanoparticle tracking analysis(NTA)and Western blotting were used to determine the characterization of BMSC-Exo.Twenty male SD rats were randomly divided into four groups with five rats in each group:Sham group,CFA group,Exo group,and Cel group;rats in the Sham group were injected with equal volume of saline,and rats in other three groups were injected with 0.1 mL CFA subcutaneously in the plantar surface of the left hind paw.After 7 d of injection,rats in the Exo group were injected with BMSC-Exo via tail vein,and rats in the Cel group were administrated with Cel.The joint swelling,arthritis index(AI),thermal pain threshold,and spleen index were applied to evaluate the therapeutic effect of BMSC-Exo on CFA rats.The effect of BMSC-Exo on voluntary movement of CFA rats was detected by open field and automatic gait tests.The effect of BMSC-Exo on serum inflammatory cytokines in CFA rats were detected by flow cytometry.The effect of BMSC-Exo on the expression of NF-κB pathway protein in joint tissues of CFA rats was detected by Western blotting.Results Compared with BMSC,BMSC-Exo specifically expressed CD63 and TSG101,but did not express CM130.The BMSC-Exo were homogeneous,with diameter distribution that peaked at 82.5 nm.Compared with the Sham group,rats in the CFA group had higher joint swelling,AI,and spleen index,lower thermal pain threshold,increased standing times,shorter distance and step length,increased step width,shorter swing time and longer touchdown time.The serum cytokines level and expression of NF-κB pathway protein in joint tissues were up-regulated(all P<0.05).Compared with the CFA group,rats in the Exo group and the Cel group had lower joint swelling,AI and spleen index,higher thermal pain threshold,decreased standing times,longer distance and step length,decreased step width,longer swing time and shorter touchdown time.The serum cytokines level and expression of NF-κB pathway protein in joint tissues were low(all P<0.05).Conclusion BMSC-Exo has a therapeutic effect on the CFA-induced RA rats model,and its mechanism may be related to the inhibition of NF-κB signaling pathway.