Apoptosis Of Rheumatoid Arthritis Fibroblast-Like Synoviocytes Induced By Radix Paeoniae Rubra801

Objective:To explore the effect of Radix paeoniae rubra801(P801) on the apoptosis of fibroblast like synoviosytes (FLS) from patients with rheumatoid arthritis (RA).Methods:RA FLS were primary culture from articular genu synovia of RA patients. Cell proliferation determined by MTT assay. According to the results of MTT, the cells will divided into the high dose of P801group (64μg/ml), low dose of P801group (4μg/ml) and solvent control group. The cell morphology observed by the optical inverted microscope; the apoptotic rate detected by Annexin V-FITC/PI double labeling staining; the relative expressions of Fas/Fasl mRNA were measured by reverse transcription polymerase chain reaction (RT-PCR) and real-time fluorescence quantitative polymerase chain reaction(FQ-PCR); the expression and positioning of P65and P50were detected by immunohistochemistry assay; the protein levels of P65and P50were measured by Western blotting from nuclear extraction.Results:Cells gradually purified after repeated adherent cultured from3to5passages. In the absence of P801stimulation, the vast majority of RA FLS displayed long spindle shape; a few of them performed the star-shape, polygon and so on. All of their cytoplasm was bright and uniform and the positions of their nuclear located in center of the cells. Though the morphological changes were not observed in group of lower dose of P801, the cells stimulated by higher dose of P801changed significantly with irregular or round shape; cell density reduced significantly, cell volume reduced and nuclei condensation. P<0.05。MTT results showed that P801has the ability to inhibit RA FLS proliferation with concentration dependence manner (P<0.05). Flow cytometry results showed that P801can induce RA FLS apoptosis. The apoptotic rate in group of lower dose of P801does not increased significantly when compared with control group (P>0.05), but it was increased significantly in higher dose group (P<0.05).Fasl mRNA was not detected in the RA FLS by reverse transcription PCR, but Fas mRNA did. relative expressions of Fas mRNA increased significantly in higher dose of P801group when compared with control group (P<0.05), but not changed significantly in lower dose group. Immunohistochemistry results showed that the protein of P65and P50mainly concentrated in RA FLS’s plasma, but the nuclear amount increased when P801stimulus for24h. Western blotting results pointed out that P65and P50were detected in RA FLS nucleoprotein extract, and were increased significantly in group of higher dose P801compared with control group.Conclusion:P801has the function to induce apoptosis of RA FLS, and simultaneous with over-expressions of Fas gene and intranuclear transfer of NF-κB.