Investigate The Mechanism Of Mitomycin C In The Promotion Of Fibroblast Apoptosis From The Endoplasmic Reticulum Stress Pathway

Objective:To Investigate The Mechanism Of Mitomycin C In The Promotion Of FibroblastApoptosis In Vitro From The Endoplasmic Reticulum Stress Pathway.Methods:After being cultured and passaged in vitro, the3rd to10th generation of thelogarithmic growth phase fibroblasts were selected in our Experiment. The culturedfibroblasts were exposed for1h with0.001mg/ml,0.005mg/ml,0.01mg/ml,0.05mg/ml,0.1mg/ml,0.2mg/ml,0.3mg/ml,0.4mg/ml,0.5mg/ml mitomycin C,respectively. The Survival rate and the MMC median effective dose (ED50) weredetected by CCK-8assay. The fibroblasts were divided into8groups：the controlgroup, caspase-8inhibitor pretreated group, caspase-9inhibitor pretreated group,MMC group, caspase-8inhibitor+MMC group, caspase-9inhibitor+MMC group,caspase-8inhibitor and caspase-9inhibitor+MMC group. After being treated underdifferent conditions, the apoptotic cell death was analyzed by Hoechst33342stainand Flow cytometry.The expression of chop, caspase-12, caspase-3, Bcl-2, Bax wereanalyzed by Western blot.Results:The expression of Caspase-12, CHOP, BAX was significantly increased whileBCL-2expression was decreased in MMC group. The same thing happened incaspase-8inhibitor pretreated+MMC group, caspase-9inhibitor pretreated+MMCgroup and caspase-8inhibitor pretreated+caspase-9inhibitor pretreated+MMC group. But there was no significant difference between these four groups(P>0.05).The cell survival rate of MMC group was lower than that of the pretreatedgroups(P<0.05). After being treated with MMC, the survival rate of combinedinhibitors pretreated fibroblasts was higher than single inhibitor pretreated ones, andthe difference was significant (P<0.05).Conclusion:Fibroblast Apoptosis still exists after the Blockade of mitochondrial and deathreceptor pathway, suggesting that other apoptotic pathways may be involved in theprocess of fibroblast apoptosis. A certain concentration of MMC can induceexpression of apoptosis protein such as CHOP、 Caspase-12、 Bax throughEndoplasmic Reticulum Stress Pathway, thereby increasing the expression ofapoptosis execution protein Caspase-3，thus inducing apoptosis of fibroblasts.