摘要
目的 制备装载蛇床子素(Ost)的外泌体(EXOs),并考察其对类风湿关节炎大鼠的疗效。方法 采用超速离心法从巨噬细胞中提取外泌体,透射电镜检测其形貌,粒径仪检测其粒径,Western blot检测外泌体膜特征蛋白CD9和TSG101的表达。采用超声法将蛇床子素负载到外泌体中,紫外分光光度计法检测其载药量与包封率。MTT法检测其对HFLS-RA细胞与RAW 264.7细胞的抑制效果以考察体外药效与制剂安全性,胶原抗体诱导法建立胶原诱导性关节炎(CIA)大鼠模型,药效实验设对照组、模型组、Ost组、Ost-EXOs组、阳性药(地塞米松)组。以足肿胀度、免疫器官指数、滑膜组织病理学检查,酶联免疫吸附法(ELISA)检测血清炎症因子水平变化为指标进行药效学研究。结果 巨噬细胞源性外泌体平均粒径为(113.72±2.51)nm,载药后其粒径增长为(138.63±3.87)nm,透射电镜下外泌体形貌呈茶托状,符合外泌体形貌特征,Western blot结果显示外泌体特征蛋白CD9与TSG101在载药前后均正常表达。外泌体对蛇床子素包封率为(21.43±1.07)%、载药量为(1.79±0.46)%。体外细胞毒性实验结果表明与游离Ost相比,Ost-EXOs对HFLS-RA细胞抑制率更强,具备更好的体外药效,且Ost-EXOs具备良好的生物相容性。药效实验结果显示,与模型组相比,Ost组、Ost-EXOs组与阳性药组的足肿胀度与免疫器官指数均得到显著改善(P<0.05,P<0.01),血清中TNF-α、IL-1β和IL-6炎症因子水平显著降低(P<0.05,P<0.01),滑膜组织的病变均得到改善,同时与Ost组相比,Ost-EXOs组药效更佳(P<0.05)。结论 本研究成功制备了装载蛇床子素的外泌体,其对CIA模型大鼠具有良好的疗效,可有效解决蛇床子素生物利用度低、在体内代谢快等问题,为扩展此类中药的应用及类风湿关节炎等疾病的治疗提供了新思路。
Objective To prepare exosomes loaded with osthole and determine their therapeutic macrophages effect on rats with rheumatoid arthritis.Methods The exosomes were extracted from RAW 264.7 by ultracentrifugation.The morphology was detected by transmission electron microscope(TEM),the particle size was detected by particle size analyzer,and the expression of markers CD9 and TSG101 on the membrane of exosomes were detected by Western blot.Osthole was loaded into exosomes by the ultrasonic method,and its drug loading and encapsulation efficiency were detected by UV spectrophotometer.MTT assay was used to detect its inhibitory effect on HFLS-RA and RAW 264.7 cells to determine its efficacy and safety in vitro.The collagen-induced arthritis(CIA)rat model was established by collagen antibody induction.The pharmacodynamic experiments included a control group,a model group,an Ost group,an Ost-EXOs group and a positive drug(dexamethasone)group.The foot swelling,immune organ index,synovial histopathological examination,and the serum inflammatory factors were detected by enzyme-linked immunosorbent assay.Results The average particle size of exosomes derived from macrophages was(113.72±2.51)nm,and the particle size increased to(138.63±3.87)nm after the drug loading.The transmission electron microscope showed that the exosomes were saucer-shaped,which was consistent with the characteristics of exosomes.Western blot showed that exosomes characteristic proteins CD9 and TSG101 were normally expressed before and after the drug loading.The encapsulation efficiency of exosomes for osthole was(21.43±1.07)%,and the drug loading was(1.79±0.46)%.In vitro cytotoxicity test showed that Ost-EXOs had stronger inhibition rate on HFLS-RA cells and better efficacy in vitro than those of free Ost.Meanwhile,toxicity test results on RAW 264.7 cells showed that Ost-EXOs had good biocompatibility.The pharmacodynamic experiment showed that compared with the model group,the foot swelling and immune organ index were significantly improved(P<0.05,P<0.01)in the Ost group,Ost-EXOs group and positive drug group,while the levels of inflammatory factors such as TNF-α,IL-1βand IL-6 in the serum were significantly decreased(P<0.05,P<0.01).The synovial tissue lesions were improved.Each index in the Ost-EXOs group improved more than that in the Ost group(P<0.05).Conclusion Exosomes loaded with osthole are successfully prepared in this study,and have a good therapeutic effect on CIA rats.They can effectively solve the problem of low bioavailability and rapid metabolism of osthole in vivo,and provide a new idea for the treatment of rheumatoid arthritis with traditional Chinese medicine.
作者
周政署
华重钧
孙佳归
曹玉琪
文武龙
王锐
柳成刚
ZHOU Zheng-shu;HUA Chong-jun;SUN Jia-gui;CAO Yu-qi;WEN Wu-long;WANG Rui;LIU Cheng-gang(College of Basic Medical,Heilongjiang University of Traditional Chinese Medicine,Harbin 150040;College of Pharmacy,Heilongjiang University of Traditional Chinese Medicine,Harbin 150040)
出处
《中南药学》
2025年第5期1205-1210,共6页
Central South Pharmacy
基金
国家自然科学基金资助项目(No.82074271)。
