负载双氯芬酸钠的间充质干细胞外泌体对类风湿关节炎小鼠治疗的研究

Diclofenac sodium-loaded mesenchymal stem cells-derived exosomes for rheumatoid arthritis therapy in mice

背景类风湿关节炎(rheumatoid arthritis,RA)的临床干预措施以控制炎症为主,但一部分患者对常规治疗手段表现出低响应性,因此亟须探索新型治疗方式以满足治疗需求。目的探讨负载双氯芬酸钠(diclofenac sodium,DS)的间充质干细胞(mesenchymal stem cells,MSCs)来源的外泌体(exosomes,EXs)对类风湿关节炎的治疗作用。方法从小鼠的骨髓中获取MSCs并进行体外培养,将收集的MSCs上清液通过超速离心法提取外泌体,并利用超声法将DS装载进EXs以构建纳米药物DS@EXs。通过透射电镜观察EXs和DS@EXs的形态,纳米颗粒追踪分析EXs和DS@EXs的粒径和电势,免疫印迹鉴定EXs和DS@EXs的标志蛋白CD9和CD63。通过关节冰冻切片和免疫荧光实验分析DS@EXs在小鼠体内的靶向递送能力。利用关节炎评分和炎症因子[白细胞介素(interleukin,IL)-6、IL-1β和肿瘤坏死因子(tumor necrosis factor,TNF)]水平评价DS@EXs的抗炎效果,使用micro-CT评价DS@EXs对关节的保护。结果透射电镜展示EXs和DS@EXs的形态均呈茶托样,DS@EXs的粒径保持在100 nm,电势保持在-12 mV,与EXs相似。另外,EXs和DS@EXs均表达CD9和CD63。与普通药物递送载体相比(如脂质体),激光共聚焦成像可以观察到较多DIO标记的DS@EXs聚集于发炎的关节中(P<0.05)。DS@EXs组的关节炎评分显著低于PBS组(P<0.05)。与PBS组相比,DS@EXs组中小鼠血清中IL-6、IL-1β和TNF的水平分别下降了52.8%、63.8%和52.5%(P<0.05)。与PBS组相比,DS@EXs减弱了关节表面的骨侵蚀,其骨密度值由921 mg/cm3升高至1195 mg/cm3(P<0.05)。结论DS@EXs可以减弱类风湿关节炎小鼠的炎性反应并且缓解骨质的侵蚀。

Background Controlling inflammation is considered as a main clinic intervention in rheumatoid arthritis(RA).However,some patients show low responsiveness to conventional treatments,so it is urgent to explore some new therapies to meet the treatment needs.Objective To explore the therapeutic effect of diclofenac sodium(DS)-loaded mesenchymal stem cells(MSCs)derived exosomes(EXs)in RA.Methods MSCs were obtained from bone marrow of mice and cultured in vitro.EXs were obtained from supernatant by ultracentrifugation.Then,the nanodrug DS@EXs were constructed by loading DS into EXs by ultrasound.Morphological structure of EXs and DS@EXs were observed by transmission electron microscopy(TEM),the particle sizes and zeta potentials of EXs and DS@EXs were detected by nanoparticle tracking analysis.The markers(CD9 and CD63)of EXs and DS@EXs were verified by western blotting.The ability of targeted delivery of DS@EXs in inflamed joints in vivo were analyzed by frozen section of joint and immunofluorescence.The anti-inflammatory effect of DS@EXs was evaluated by arthritis score and the levels of inflammatory cytokines(interleukin-6,IL-6;interleukin-1β,IL-1β;tumor necrosis factor,TNF).The protective effect on joint was analyzed by micro-computed tomography(micro-CT).Results TEM revealed cup-like morphologies for EXs and DS@EXs.Diameter and zeta potential of DS@EXs was 100 nm and-12 mV,which were similar to EXs.In addition,the markers(CD9 and CD63)were both expressed on EXs and DS@EXs.Compared to ordinary drug delivery carrier(such as liposomes),confocal laser scanning microscope imaging showed more DS@EXs(labeled with DIO)accumulated in the inflamed joints(P<0.05).The arthritis score of the DS@EXs group was significantly lower than that of the PBS group(P<0.05).Compared to the PBS group,the levels of IL-6,IL-1,and TNF in serum of mice from the DS@EXs group decreased by 52.8%,63.8%and 52.5%,respectively(P<0.05).Compared to the PBS group,DS@EXs ameliorated bone erosion on the surface of joint,and the value of bone mineral density increased from 921 mg/cm3 to 1195 mg/cm3(P<0.05).Conclusion DS@EXs can attenuate inflammatory response and alleviate bone erosion.