Celastrol-introduced Apoptosis, Cell Cycle Arrest And DNA Damage In Rheumatoid Arthritis Fibroblast-like Synovial

ObjectiveCelastrol could induce apoptosis, cell cycle arrest and DNA damage in rheumatoid arthritis fibroblast-like synovial. The effects were studied on molecular level in vitro, the underline mechanism and the relationship of the three effects were explored for providing the experiment data and theoretical bases to the therapies of rheumatoid arthritisMethod1. Celastrol-introduced apoptosis in rheumatoid arthritis fibroblast-like synovialFLS was treated with different dose of Celastrol (0,1,2,5μM) and incubated for24h. Cell proliferation and viability were assessed by the MTT assay and cytotoxicity was detected by LDH assay. The cell morphology of FLS was observed through optical microscopy following the Celastrol treatment. Apoptotic cell death was evaluated by fluorescence microscopy after staining with DAPI. The mitochondrial membrane potential (ΔΨm) was monitored by the fluorescent probe JC-1staining. The extent of apoptosis was measured by Annexin V-FITC/PI apoptosis detection kit as described by the manufacture’s instruction. SDS-PAGE and Western blotting were performed to evaluate the protein levels of Bcl-2, Bax, Caspase-3,9, PARP and Fas. 2. Celastrol-introduced cell cycle arrest and DNA damage in rheumatoid arthritis fibroblast-like synovialFLS was treated with different dose of Celastrol (0,1,2,5μM) and incubated for24h. Cell cycle was analyzed using a flow cytometer, and the percentages of cells distribution in G0/G1, G2/M, and S phases were measured and results were analyzed with Mod Fit LT3.0. The levels of DNA damage was valued by comet assay and the comet were visualized under a Laser Scanning Confoca! Microscope and took photos. To observe the distribution and expression of yH2AX in FLS, immunofluorescence was carried out and the red points were visualized under a Laser Scanning Confocal Microscope and took photos. SDS-PAGE and Western blotting were performed to evaluate the protein levels of Chk1、γH2AX、pCdc-25、pCdc-2、Cdc-2and Cyclin-b1.Results1. The viabilities of cells was decreased after the treatment with Celastrol at concentrations of0.1,1,2,5and10μM for24h, and the IC50was4.9μM. The LDH release ratio of cells was increased in a dose-dependent manner. FLS treated with Celastrol were seen to have detached from the dish, with cell rounding, cytoplasmic blebbing, and irregularity in shape through optical microscopy. In addition, FLS were observed through fluorescence microscopy following stain with DAPI and the result has revealed the occurrence of condensed chromatin, shrunken, crimpled and condensed gray-blue nuclei on Celastrol-treated cells. In JC-1assay, after Celastrol treatment with different concentration, the green fluorescence was much stronger than control group suggesting the reduction of ΔΨm. After treatment with1-5μM Celastrol the percentage of annexin V-FITC binding FLS significantly increased versus control group. In FLS treated with Celastrol, increased levels of Bax, cleavage ofCaspase-3, Caspase-9and PARP, and decreased levels of Bcl-2and Fas were observed.2. G2/M phase population of FLS treated with1-5μM Celastrol was increased from6.86to37.37%and FLS was arrest in G2/M phase in a concentration-dependent manner. In comet assay. The DNA damage was expressed as the population of DNA in the comet tail. The result implicated that the DNA damage was increased in Celastrol treated cells, especially in2and5μM groups. The aggregation of red points and enhancement of fluorescence intensity was observed in FLS by immuno-fluorescence. In FLS treated with Celastrol, increased levels of Chk1、γH2AX、pCdc-25、pCdc-2, and decreased levels of Cdc-2、Cyclin-b1were observed.Conclusion1. Celastrol could inhibit the proliferation of FLS.2. Celastrol could induce the apoptosis and reverse the apoptosis resistance of FL S.3. FLS arrest in G2phase was induced by Celastrol.4. DNA damage of FLS was occurred following the treatment with Celastrol.5. Celastrol induced the DNA damage of FLS, which lead to G2phase arrest, and the process of apoptosis was initiated in FLS who failed in DNA repair and retreated the cell cycle, and the proliferation of FLS was suppressed.