| Background:Rheumatoid arthritis(RA)is an autoimmune disease characterized by persistent synovial hyperplasia and progressive joint destruction.Synovial fibroblasts(FLSs)are the main target cells involved in RA synovitis.They can secrete a variety of inflammatory factors and chemokines,drive joint inflammation and mediate immune response.Histone acetylation modification is a reversible epigenetic regulation mode that occurs on the lysine residue of histone.It regulates and participates in the pathogenesis of RA through histone deacetylase(HDAC)in many ways.Our research team found that human umbilical cord mesenchymal stem cells derived exosomes(h UCMSC-Exos)can improve joint symptoms of collagen-induced arthritis(CIA)rats,inhibit synovial hyperplasia and promote apoptosis of RA FLSs,and reduce the expression level of HDAC1 protein in RA FLSs.HDAC can regulate many signal molecules and inflammatory factors by NF-κB pathway,and plays a role in inhibiting inflammatory cells and reducing inflammation.It is noteworthy that the resistance of FLSs to apoptotic signals is the main cause of synovial hyperplasia in RA,many studies have found that HDAC inhibitor(HDACi)can inhibit the activation of NF-κB and induce apoptosis of cancer cells,so whether h UCMSC-Exos can promote the apoptosis of RA FLSs by affecting HDAC1/NF-κB pathway still needs further experimental confirmation.Objective:To investigate the effect of h UCMSC-Exos on the expression level of HDAC in different subtypes of RA FLSs,and clarify whether h UCMSC-Exos can affect the level of apoptosis and the secretion of inflammatory cytokines in RA FLSs by regulating the HDAC/NF-κB pathway,providing a theoretical basis for the treatment of RA with h UCMSC-Exos from the perspective of epigenetic histone acetylation.Methods:1.Sample preparationIsolation and identification of h UCMSC-Exos: the umbilical cord tissue of full-term newborns was collected and the primary h UCMSC was obtained by tissue adherent method.The cultured h UCMSC was subcultured to P3-P7,"starved" for 36-48 hours,and the supernatant was collected.The h UCMSC-Exos was obtained by differential ultracentrifugation.Its morphology was observed by transmission electron microscope,its particle size was determined by nanoparticle tracking analysis technology,its concentration was determined by BCA method,and its surface molecular markers were identified by Western blot.2.Compare the effect of different intervention groups on the expression of HDAC in RA FLSs(1)To clarify the effect of h UCMSC-Exos on the expression of HDAC1-3 in RA FLSs,the experiment was divided into two groups: RA FLSs group(blank control group)and h UCMSC-Exos group.Detection method: RT-q PCR was used to detect the expression level of HDAC1,HDAC2 and HDAC3 m RNA in FLSs of two groups.(2)To further investigate the differences in the effects of h UCMSC and h UCMSC-Exos on HDAC1 in RA FLSs,the experiment was divided into five groups: blank control group,h UCMSC group,h UCMSC-Exos group,Trichostatin A(TSA)group,HDAC1 Inhibitor(Pyroxamide)group.Detection methods:(1)RT-q PCR was used to detect the effect of h UCMSC-Exos on HDAC1 m RNA expression in FLSs;(2)Western blot was used to detect the effect of h UCMSC-Exos on HDAC1 protein(10197-1-AP)expression in FLSs.3.Explore the effect of h UCMSC-Exos on apoptosis and secretion of pro-inflammatory cytokines in RA FLSs based on HDAC1/NF-κB pathway(1)To clarify the effect of h UCMSC-Exos on apoptosis and secretion of pro-inflammatory cytokines in RA FLSs through the HDAC1/NF-κB pathway,the experiment was divided into five groups: blank control group,h UCMSC group,h UCMSC-Exos group,TSA group,HDAC1 Inhibitor group.Detection method:(1)The effect of h UCMSC-Exos on apoptosis of FLSs was detected by flow cytometry;(2)The effect of h UCMSC-Exos on concentration of TNF-α、IL-6、IL-1β、IL-8 of FLSs was detected by ELISA(enzyme linked immunosorbent assay);(3)Western blot was used to detect the expression level of NF-κB related proteins in the effect of h UCMSC-Exos on FLSs: NF-κB p65(10745-1-AP),phosphorylated NF-κB p65(Ser 536)(AP0475).(2)To further validate the intervention NF-κB can affect the regulatory effect of h UCMSC-Exos on RA FLSs,the experiment was divided into four groups: blank control group,NF-κB Inhibitor(PDTC)group,h UCMSC-Exos group,PDTC+h UCMSC-Exos co-intervention group.Detection method:(1)Effect of h UCMSC-Exos on apoptosis of FLSs after interference with NF-κB detected by flow cytometry;(2)Detection of TNF-α、IL-6、IL-1β、IL-8 secretion by h UCMSC-Exos on FLSs after intervention in NF-κB by ELISA(enzyme linked immunosorbent assay).4.Statistical method: SPSS26.0 software and Graph Pad Prism 8.0 mapping software were used for statistical analysis of data.The measurement data conforming to normal distribution and homogeneity of variance were compared between multiple groups using one-way analysis of variance,and the independent sample t-test was used for comparison between the two groups,which is expressed by mean ± standard deviation(X±S).Measurement data that do not conform to the normal distribution are tested using Kruskal-Wallis test,which is expressed as median(quartile spacing).P < 0.05 indicates that the difference is statistically significant.Results:1.Sample identificationIsolation and identification of h UCMSC-Exos: Spindle cells were observed climbing out of the edge of the primary umbilical cord tissue after 10-12 days of culture.The supernatant was collected and the exosomes were isolated by ultracentrifugation at a concentration of 1.835ug/ul.Under transmission electron microscopy,it was found that h UCMSC-Exos was a tea tray shaped membranous vesicle;Nanoparticle tracking analysis showed that the peak particle size of h UCMSC-Exos was about 132 nm;Western blot showed that h UCMSC-Exos expressed Alix,CD9,and CD63,which met the criteria for exocrine identification.2.h UCMSC-Exos can inhibit the abnormal expression of HDAC1 in RA FLSs(1)Expression of HDAC1-3 m RNA in RA FLSs: Compared with the blank control group,the expression level of HDAC1 and HDAC2 m RNA in the h UCMSC-Exos group decreased,while the expression level of HDAC3 increased.The difference of HDAC1 was statistically significant(P < 0.05).(2)Expression of HDAC1 m RNA in RA FLSs: Compared with the blank control group,the expression level of HDAC1 m RNA in h UCMSC group,h UCMSC-Exos group,TSA group and HDAC1 Inhibitor group decreased(P < 0.05),and the effect of h UCMSC-Exos was stronger than that of h UCMSC and HDAC1 Inhibitor(P < 0.05).(3)Expression of HDAC1 protein in RA FLSs: Compared with the blank control group,the expression level of HDAC1 protein in h UCMSC group,h UCMSC-Exos group,TSA group and HDAC1 Inhibitor group decreased(P < 0.05),and the effect of h UCMSC-Exos was stronger than that of h UCMSC and HDAC1 Inhibitor(P < 0.05).3.Explore the effect of h UCMSC-Exos on apoptosis and cytokine secretion of RA FLSs based on HDAC1/NF-κB pathway(1)Effects on apoptosis of RA FLSs: Compared with the blank control group,the apoptosis rate of RA FLSs in each group increased(P < 0.05);Compared with the h UCMSC group and the HDAC1 Inhibitor group,h UCMSC-Exos had a stronger proapoptotic effect(P < 0.05).(2)Effects on the secretion of pro-inflammatory cytokines by RA FLSs: Compared with the blank control group,the secretion of TNF-α、IL-6、IL-1β、IL-8 in the supernatant of RA FLSs in each group decreased(P < 0.05);The effect of h UCMSC-Exos on TNF-α and IL-8 was stronger than that of h UCMSC(P < 0.05);The effect of h UCMSC-Exos on TNF-α,IL-1β and IL-8 was stronger than that of TSA(P < 0.05);The effect of h UCMSC-Exos on IL-1β and IL-8 was stronger than that of HDAC1 Inhibitor(P < 0.05).(3)Effects on NF-κB in RA FLSs: Compared with the blank control group,the activation of NF-κB in RA FLSs in each group was significantly inhibited(P < 0.05),and the inhibitory effect of h UCMSC-Exos was stronger than that of h UCMSC and HDAC1 Inhibitor(P < 0.05).(4)Effects of h UCMSC-Exos on apoptosis and secretion of proinflammatory cytokines in RA FLSs after intervention in NF-κB: Compared with the blank control group,the apoptosis rate of RA FLSs in PDTC group,h UCMSC-Exos group and PDTC+h UCMSC-Exos co-intervention group increased(P < 0.05).PDTC could reverse the promoting effect of h UCMSC-Exos on the apoptosis of RA FLSs(P < 0.05).Compared with the blank control group,each groups decreased the secretion of TNF-α、IL-6、IL-1β、IL-8 in the supernatant of RA FLSs(P < 0.05),and PDTC could reverse the effect of h UCMSC-Exos on the secretion of IL-8 in RA FLSs(P < 0.05).Conclusion:h UCMSC-Exos which are similar to maternal cells,can effectively inhibit the abnormal expression of HDAC1 in RA FLSs;h UCMSC-Exos can inhibit HDAC1/NF-κB pathway affect the apoptosis of RA FLSs and reduces the secretion of inflammatory factors. |
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