| Background:Keloid are the result of abnormal wound healing and have the characteristics of invading surrounding normal tissues like tumors.They tend to occur in chest,shoulder,ear and other parts and will not disappear spontaneously over time.Keloid brings great pain to patients both psychologically and physically.Keloid has become a difficult problem in plastic surgery because it is easy to recur after surgical resection.Melatonin is a neurohormone that comes from the pineal gland and is well known for its sleep-improving effects.However,in recent years,a large number of domestic and foreign literatures show that melatonin also has anti-aging,anti-tumor,anti-fibrosis,anti-inflammation,improve hypoxia,regulation of angiogenesis and other functions.Inparticular,the antitumor effect has been demonstrated in vivo and in vitro experiments on many types of tumors.Melatonin can promoting cell apoptosis,regulating cell autophagy,cell cycle arrest,inhibiting tumor cell invasion,radiochemotherapy sensitization and so on.At present,there is a lack of relevant studies on the effect of melatonin on keloid fibroblasts at home and abroad.Therefore,this study aims to explore the effects of melatonin on the biological function of keloid fibroblasts and explore the related mechanism of inducing apoptosis.to provide a new direction for the treatment of keloid.Hope to provide a new direction for the treatment of keloid.Objective:1.To explore the effects of melatonin on proliferation,apoptosis,cell cycle,migration and invasion of human keloid fibroblasts.2.It was confirmed that the mechanism of melatonin promoting apoptosis of human keloid fibroblasts is accomplished through the ROS-mediated mitochondrial apoptosis pathway.Methods:1.First,the primary human keloid fibroblasts were cultured by the tissue block method,and identified by vimentin immunohistochemical staining,and then the appropriate concentration and duration of action of melatonin were screened by CCK-8 assay,and the effect of different concentrations of melatonin on the cell proliferation were detected.Flow cytometry,cell scratch assay,transwell migration and invasion assay were used to detect the effects of different concentrations of melatonin on cell apoptosis,cell cycle,cell migration and invasion.2.Use fluorescence microscopy and flow cytometry to qualitatively and quantitatively detect changes in mitochondrial membrane potential,qualitative and quantitative analysis of ROS levels by laser confocal microscopy and flow cytometry,and the effect of NAC pretreatment on the cell apoptosis rate was detected by flow cytometry annexin V-FITC/PI double staining method.Moreover,the mRNA and protein expressions of mitochondrial apoptosis pathway-related factors Bax/Bcl-2,Cyto-c,and Caspase-3 were detected by RT-qPCR and Western Blot.Results:1.Vimentin immunohistochemical staining was positive,and the cultured cells were fibroblasts.In addition,CCK-8 assay,flow cytometry,cell scratch assay and transwell migration and invasion assay showed that melatonin significantly inhibited the proliferation of human keloid fibroblasts,promoted apoptosis,blocked the cells in G1 phase,and weakened the migration and invasion of human keloid fibroblasts in a concentration-dependent manner.2.By detecting the changes of mitochondrial membrane potential and intracellular ROS level,it was found that melatonin significantly reduced the mitochondrial membrane potential and increased the level of intracellular ROS,and with the increase of melatonin concentration,the effect was more obvious;However,NAC pretreatment for 2 h reversed the melatonin-induced decrease in mitochondrial membrane potential and the increase in intracellular ROS levels;Flow cytometry annexin V-FITC/PI double staining method showed that the pro-apoptotic effect of melatonin was significantly inhibited after NAC pretreatment;In addition,RT-qPCR and Western Blot experiments also showed that the expressions of Bax/Bcl-2,Cyto-c,Casepase-3 mRNA and protein were significantly up-regulated after melatonin intervention;Whileafter NAC pretreatment,the mRNA and protein expressions of Bcl-2,Cyto-C and Casepase-3 were significantly down-regulated.Conclusion:1.Melatonin can significantly inhibit the proliferation and apoptosis of human keloid fibroblasts,block the cell cycle in G1 phase,and weaken the invasion and migration of cells,in a concentration-dependent manner.2.Melatonin can induce apoptosis of human keloid fibroblasts through the ROS-mediated mitochondrial apoptosis pathway. |
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