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.

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.