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

doi:10.12307/2025.081

Abstract

Abstract: 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-α in 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 (P < 0.05), migration and invasion (P < 0.05). Moreover, the inhibition of extracellular vesicles treated with Duhuo Jisheng Decoction was more significant (P < 0.05). (3) Drug-containing serum and extracellular vesicles treated with Duhuo Jisheng Decoction promoted the apoptosis of rheumatoid arthritis fibroblast-like synovial cells. (4) 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 increase the expression of proapoptotic factors Caspase-3, Caspase-9, and Bax (P < 0.05) and decrease the expression of antiapoptotic factor Bcl-2 (P < 0.05). Moreover, extracellular vesicles treated with Duhuo Jisheng Decoction had a more significant regulatory effect on apoptosis-related factors. Above findings indicate that extracellular vesicles treated with Duhuo Jisheng Decoction can inhibit the excessive proliferation, migration, and invasion of rheumatoid arthritis fibroblast-like synovial cells and promote their apoptosis.

Key words: ">Duhuo Jisheng Decoction, rheumatoid arthritis, synovial fibroblasts, extracellular vesicles, proliferation, migration, invasion, apoptosis

CLC Number:

Yue Jinru, Zhang Yumin, Liu Jingshu, Bu Yanan, Wu Jingruo, Chen Jia, Wang Jianru. Effects of extracellular vesicles treated with Duhuo Jisheng Decoction on rheumatoid arthritis fibroblast-like synovial cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(23): 4915-4926.

Figures/Tables 2

2.1 独活寄生汤含药血清干预的最佳体积分数及时间如图1所示，干预24 h各组的细胞抑制率均高于12 h及48 h；体积分数10%独活寄生汤含药血清对RA-FLS抑制作用高于体积分数5%，20%独活寄生汤含药血清(P < 0.05)，故选取体积分数10%独活寄生汤含药血清和干预24 h进行后续实验。 2.2 独活寄生汤含药血清细胞外囊泡干预的最佳质量浓度及时间如图1所示，干预24 h各组的细胞抑制率均高于12 h及48 h；20，40 ng/mL独活寄生汤含药血清细胞外囊泡干预24 h后对细胞的抑制作用增强(P < 0.05)，其中20 ng/mL独活寄生汤含药血清细胞外囊泡对RA-FLS的抑制作用最高(P < 0.05)，故选取10 ng/mL独活寄生汤含药血清细胞外囊泡和干预24 h进行后续实验。 2.3 ELISA检测RA-FLS上清中炎症因子水平如图2所示，与正常组相比，模型组细胞上清中促炎因子白细胞介素1β及白细胞介素6水平升高(P < 0.05)，而抑炎因子白细胞介素10水平降低(P < 0.05)；与模型组相比，含药血清组、含药血清细胞外囊泡组白细胞介素1β及白细胞介素6水平降低(P < 0.05)，而白细胞介素10水平升高(P < 0.05)；与含药血清细胞外囊泡组相比，含药血清组白细胞介素6水平升高(P < 0.05)，生理盐水血清细胞外囊泡组白细胞介素1β、白细胞介素6水平升高(P < 0.05)，含药血清组及生理盐水血清细胞外囊泡组白细胞介素10水平降低(P < 0.05)。 2.4 各组RA-FLS整体迁移能力如图3所示，与正常组相比，模型组细胞划痕愈合率明显升高(P < 0.05)；与模型组相比，含药血清组及含药血清细胞外囊泡组细胞划痕愈合率降低(P < 0.05)；与含药血清细胞外囊泡组相比，含药血清组和生理盐水血清细胞外囊泡组细胞划痕愈合率升高(P < 0.05)。 2.5 各组RA-FLS个体迁移能力及侵袭能力如图4所示，与正常组相比，模型组迁移细胞数及侵袭细胞数显著增加(P < 0.05)；与模型组相比，含药血清组、含药血清细胞外囊泡组及生理盐水血清细胞外囊泡组迁移细胞数及侵袭细胞数减少(P < 0.001)；与含药血清细胞外囊泡组相比，含药血清组侵袭细胞数增多(P < 0.05)，生理盐水血清细胞外囊泡组迁移细胞数及侵袭细胞数均增多(P < 0.05)。"

2.6 各组RA-FLS凋亡情况如图5所示，正常组镜下可见散在高亮荧光细胞，模型组凋亡细胞几乎不可见；与模型组相比，含药血清组及含药血清细胞外囊泡组凋亡细胞数量明显增多，可见大片圆状或固缩状、团块状结构，且含药血清细胞外囊泡组细胞荧光更为明亮，生理盐水血清细胞外囊泡组视野中偶尔可见个别高亮凋亡细胞，说明含药血清细胞外囊泡组及含药血清组促凋亡的效果高于生理盐水血清细胞外囊泡组。 2.7 各组RA-FLS凋亡相关基因表达如图6所示，与正常组相比，模型组促凋亡基因Caspase-3、Caspase-9及Bax表达降低(P < 0.05)，抑凋亡基因Bcl-2表达升高(P < 0.05)；与模型组相比，含药血清组、含药血清细胞外囊泡组及生理盐水血清细胞外囊泡组Caspase-3、Caspase-9及Bax表达升高(P < 0.05)，含药血清组及含药血清细胞外囊泡组Bcl-2表达降低(P < 0.05)；与含药血清细胞外囊泡组相比，含药血清组Caspase-3及Caspase-9表达降低(P < 0.05)，生理盐水血清细胞外囊泡组Caspase-3、Caspase-9及Bax表达降低(P < 0.05)，含药血清组和生理盐水血清细胞外囊泡组Bcl-2表达升高(P < 0.05)。 2.8 Western blot检测各组RA-FLS凋亡相关蛋白表达如图7所示，与正常组相比，模型组促凋亡蛋白Caspase-3、Caspase-9及Bax表达降低(P < 0.05)，抑凋亡蛋白Bcl-2表达升高(P < 0.05)；与模型组相比，含药血清组及含药血清细胞外囊泡组Caspase-3、Caspase-9和Bax表达升高(P < 0.05)；与含药血清细胞外囊泡组相比，生理盐水血清细胞外囊泡组Caspase-3、Caspase-9、Bax表达降低(P < 0.05)，Bcl-2表达升高(P < 0.05)。在凋亡相关蛋白的表达上，含药血清细胞外囊泡组的促凋亡作用优于生理盐水血清细胞外囊泡组，验证了凋亡基因表达的结果。"

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