独活寄生汤含药血清细胞外囊泡对类风湿关节炎滑膜成纤维细胞的影响  

Effects of extracellular vesicles treated with Duhuo Jisheng Decoction on rheumatoid arthritis fibroblast-like synovial cells

作　　者：岳进茹 张育敏 刘静淑 步亚男 吴婧若 陈佳 王建茹[2] Yue Jinru;Zhang Yumin;Liu Jingshu;Bu Yanan;Wu Jingruo;Chen Jia;Wang Jianru(State Administration of Traditional Chinese Medicine Gene Expression Regulation Laboratory,School of Basic Medicine,Shanxi University of Chinese Medicine,Jinzhong 030606,Shanxi Province,China;School of Basic Medicine,Shanxi Medical University,Taiyuan 030012,Shanxi Province,China)

机构地区：[1]山西中医药大学,基础医学院,国家中医药管理局基因表达调控实验室,山西省晋中市030606 [2]山西医科大学基础医学院,山西省太原市030012

出　　处：《中国组织工程研究》2025年第23期4915-4923,共9页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(82274365),项目负责人:张育敏;山西省自然科学基金(20210302123301),项目负责人:王建茹;山西省中医药科研基金(2023ZYYA031),项目负责人:张育敏;国家中医药管理局基因表达调控实验室研究平台开放基金(2022JD-KF-13),项目负责人:张育敏;山西中医药大学“青年科学家培训计划”(2021PY-QN-01),项目负责人:张育敏。

摘　　要：背景:独活寄生汤是治疗类风湿关节炎的经典方剂,但具体作用机制尚不清楚,细胞外囊泡具有细胞间通讯及传递信号的强大功能,可能是该方剂发挥作用的信号载体。目的:探讨独活寄生汤含药血清细胞外囊泡对类风湿关节炎滑膜成纤维细胞增殖、迁移、侵袭及凋亡的影响。方法:肿瘤坏死因子α体外诱导构建类风湿关节炎滑膜成纤维细胞模型,实验分为5组:正常组、模型组、独活寄生汤含药血清组、独活寄生汤含药血清细胞外囊泡组、生理盐水血清细胞外囊泡组。采用CCK-8法筛选独活寄生汤含药血清及独活寄生汤含药血清细胞外囊泡作用的最佳浓度及时间;ELISA检测细胞上清炎症因子水平;划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力及迁移能力;Hoechst染色检测细胞凋亡情况;qRT-PCR及Western blot检测细胞凋亡相关基因及蛋白表达。结果与结论:(1)独活寄生汤含药血清作用的最佳体积分数为10%,独活寄生汤含药血清细胞外囊泡作用的最佳质量浓度为10 ng/mL,二者作用的最佳时间均为24 h;(2)与模型组相比,独活寄生汤含药血清、独活寄生汤含药血清细胞外囊泡及生理盐水血清细胞外囊泡均能抑制类风湿关节炎滑膜成纤维细胞的炎症因子表达(P<0.05)、划痕愈合(P<0.05)、迁移及侵袭(P<0.05),且独活寄生汤含药血清细胞外囊泡的抑制作用更为突出(P<0.05);(3)独活寄生汤含药血清及独活寄生汤含药血清细胞外囊泡均可促进类风湿关节炎滑膜成纤维细胞的凋亡;(4)与模型组相比,独活寄生汤含药血清、独活寄生汤含药血清细胞外囊泡及生理盐水血清细胞外囊泡均可增加促凋亡因子Caspase-3、Caspase-9、Bax表达(P<0.05),降低抑凋亡因子Bcl-2表达(P<0.05),且独活寄生汤含药血清细胞外囊泡对凋亡相关因子的调节作用更显著。上述实验结果表明独活寄生汤含药血清细胞外囊泡BACKGROUND:Duhuo Jisheng Decoction is a classic prescription for the treatment of rheumatoid arthritis,but its specific mechanism is not clear.Extracellular vesicles have the powerful function of inter-cell communication and signal transmission,and may be the signal carrier for the Decoction.OBJECTIVE:To explore the effects of serum extracellular vesicles treated with Duhuo Jisheng Decoction on the proliferation,migration,invasion,and apoptosis of rheumatoid arthritis fibroblast-like synovial cells.METHODS:The rheumatoid arthritis fibroblast-like synovial cell model was established by co-culturing with tumor necrosis factor-αin vitro.The experiment was divided into five groups:normal group,model group,serum treated with Duhuo Jisheng Decoction group,extracellular vesicles treated with Duhuo Jisheng Decoction group,and extracellular vesicles treated with normal saline group.The optimal concentration and time of drug-containing serum and extracellular vesicles were screened by CCK-8 assay.Expression of inflammatory cytokines in the supernatants of cells in each group was detected by ELISA.The migration ability of rheumatoid arthritis fibroblast-like synovial cells was detected by scratch assay.The invasive ability of cells was measured by Transwell Invasion assay.Hoechst staining was adoped to detect cell apoptosis.The expression levels of apoptosis-related genes and proteins were detected by qRT-PCR and western blot assay.RESULTS AND CONCLUSION:(1)The optimal volume fraction of serum treated with Duhuo Jisheng Decoction was 10%and optimal mass concentration of extracellular vesicles treated with Duhuo Jisheng Decoction was 10 ng/mL;the optimal time for the interaction between the two was 24 hours.(2)Compared with the model group,serum treated with Duhuo Jisheng Decoction,extracellular vesicles treated with Duhuo Jisheng Decoction,and extracellular vesicles treated with normal saline could suppress the expression of inflammatory factors of rheumatoid arthritis fibroblast-like synovial cells(P<0.05),scratch healing

关 键 词：独活寄生汤 类风湿关节炎 滑膜成纤维细胞 细胞外囊泡 增殖 迁移 侵袭 凋亡 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R593.22