Effect And Related Mechanism Of Mitomycin C On Apoptosis Of Fibroblast-like Synoviocytes In Rheumatoid Arthritis

Objective:To investigate the effect of mitomycin C on proliferation and apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis in vitro, and to explore the related mechanism of apoptosis.Methods:1. Isolate the synovial tissues in patients with rheumatoid arthritis and cultivate primary cells. The morphology of FLS was observed by phase-contrast microscopy. 2. Cells were exposed to increasing concentrations of MMC(0, 10, 25, 50, or 100 mg/L) for different periods(0, 6, 12, 24 and 48 h). Cell viability was determined by using the CCK-8 assay. After treatment with 50 mg/L MMC for different durations(0, 6, 12, 24 and 48 h), cell apoptosis of RA FLS was measured by using AV/PI double staining and Td T-mediated d UTP nick and labeling(TUNEL) staining. The production of intracellular ROS was detected using the specific fluorogenic probe DCFH-DA via flow cytometry after treatment with 50 mg/L MMC for different durations(0, 1, 3, 6, 12, and 24 h), and mitochondrial apoptotic associated proteins were examined by Western blotting.Results:1. Effect of MMC on the proliferation of RA FLS The CCK-8 results showed that MMC significantly inhibited the proliferation of FLS in a dose- and time-dependent manner. Among them, a concentration of 50 mg/L MMC for 24 h could reduce cell viability to approximately 50%. 2. MMC induces apoptosis in RA FLS AV/PI double staining showed that following treatment with 50 mg/L MMC for 6, 12, 24, or 48 h, the percentage of apoptotic cells was found to gradually increase, the apoptosis rate significantly increased after treatment with MMC for over 12 h compared with control group. Furthermore, apoptosis was confirmed by TUNEL staining. After exposure to 50 mg/L MMC for 6, 12, 24, or 48 h, the frequency of TUNEL-positive cells was also increased. 3. Effect of MMC on the production of intracellular ROS After treatment with 50 mg/L MMC for different periods(0, 1, 3, 6, 12, and 24 h), the results revealed that the production of intracellular ROS was significantly increased compared with control group. 4. Effect of MMC on the mitochondrial function and the expression of apoptosis-related proteins in RA FLS To investigate whether MMC-induced apoptosis is associated with mitochondrial dysfunction, the change of ΔΨm was assessed using JC-1 fluorescence staining and visualised via fluorescence microscopy. After exposure to MMC, the results revealed that the ΔΨm was collapsed. Moreover, Western blot analysis showed that MMC treatment increased the expression level of Bax protein, decreased the expression level of Bcl-2protein and activated Caspase-9, Caspase-3 and PARP in a time-dependent manner in RA FLS after treatment with 50 mg/L MMC. We next examined the release of Cytochrome c from the mitochondria into the cytosol. The results revealed that the level of cytosolic cytochrome c had significantly increased after exposure to MMC.Conclusion1. MMC can inhibit cell proliferation of RA FLS in a dose- and time-dependent manner in vitro. 2. MMC can induce apoptosis of RA FLS. 3. The mechanism underlying this MMC-induced apoptosis may be through the mitochondrial signaling pathway...