摘要
Background: Daphnetin is an active ingredient that is extracted from Jiangxi flowers(Marginata). We had applied it in to treat fibroblast-like synoviocytes(FLSs) from collagen-induced arthritic(CIA) rats resulted in the modulation of the expression of Th17 and related cytokines.Objective: To investigate whether Daphnetin combined with 5-aza-Dc and a Bcl-2-miRNAinterference plasmid(MRBcl-2) induces apoptosis in CIA-FLSs and to explore the mechanisms potentially underlying the activity of this combination as a treatment for RA. Methods: The relative mRNA expression levels were detected using RT-PCR. The methylation status was measured using methylation-specific PCR. The rate of apoptosis was detected in synovial cells using the Annexin V/PI double-labelling method. The protein expression levels were detected using cell-based ELISA. Transmission electron microscopy was used to observe changes in the ultrastructure of CIA-FLSs.Results: We explored the effects of a combined approach including Daphnetin, 5-aza-Dc, and MR-Bcl-2. We show that this combination down-regulated the relative mRNA and protein expression levels of Bcl-2 and up-regulated the relative mRNA and protein expression levels of Bax, Bid, Caspase-3, Caspase-8, and Caspase-9. Daphnetin cooperated with 5-aza-Dc to demethylate the proapoptotic gene Bid, which increased in expression, to promote synovial cell apoptosis.Conclusion: Daphnetin cooperated with 5-aza-Dc and MR-Bcl-2 to induce apoptosis in CIA-FLSs. This effect potentially induced apoptosis-related molecular mechanisms via the down-regulation of Bcl-2 and the up-regulation of Bax and Bid. It also activated the apoptotic channels involving Caspase-3, Caspase-8, and Caspase-9 to induce apoptosis in CIAFLSs.
Background: Daphnetin is an active ingredient that is extracted from Jiangxi flowers(Marginata). We had applied it in to treat fibroblast-like synoviocytes(FLSs) from collagen-induced arthritic(CIA) rats resulted in the modulation of the expression of Th17 and related cytokines.Objective: To investigate whether Daphnetin combined with 5-aza-Dc and a Bcl-2-miRNAinterference plasmid(MRBcl-2) induces apoptosis in CIA-FLSs and to explore the mechanisms potentially underlying the activity of this combination as a treatment for RA. Methods: The relative mRNA expression levels were detected using RT-PCR. The methylation status was measured using methylation-specific PCR. The rate of apoptosis was detected in synovial cells using the Annexin V/PI double-labelling method. The protein expression levels were detected using cell-based ELISA. Transmission electron microscopy was used to observe changes in the ultrastructure of CIA-FLSs.Results: We explored the effects of a combined approach including Daphnetin, 5-aza-Dc, and MR-Bcl-2. We show that this combination down-regulated the relative mRNA and protein expression levels of Bcl-2 and up-regulated the relative mRNA and protein expression levels of Bax, Bid, Caspase-3, Caspase-8, and Caspase-9. Daphnetin cooperated with 5-aza-Dc to demethylate the proapoptotic gene Bid, which increased in expression, to promote synovial cell apoptosis.Conclusion: Daphnetin cooperated with 5-aza-Dc and MR-Bcl-2 to induce apoptosis in CIA-FLSs. This effect potentially induced apoptosis-related molecular mechanisms via the down-regulation of Bcl-2 and the up-regulation of Bax and Bid. It also activated the apoptotic channels involving Caspase-3, Caspase-8, and Caspase-9 to induce apoptosis in CIAFLSs.
引文