| BackgroundKeloid is a dermal fibroproliferative disease characterized by abnormal proliferation of fibroblasts and overproduction of extracellular matrix(ECM).It can not only affect the appearance,but also cause pain,itching,and other symptoms,which would become a cosmetic and psychological burden for patients.However,there is no satisfactory treatment for keloid as the key factor responsible for the disease has not been identified.Warburg effect is first found in tumors and refers to enhanced glycolysis under aerobic conditions,which takes an important part in the occurrence and development of tumors.It was found that the glycolysis in keloid fibroblasts(KFs)was enhanced and there might be a Warburg effect like tumors in KFs.This study aims to demonstrate whether there is a Warburg effect in KFs,to study the effects of the Warburg effect in KFs and related mechanisms,and to explore the pathogenesis and new treatment of keloids in terms of energy metabolism.Objective1.The aim of this study is to demonstrate whether there is a Warburg effect in KFs and find out whether Warburg effect is a special way of energy metabolism in keloid or a common metabolic feature of other scars.2.This study aims to investigate the relationship between the Warburg effect and the biological activity,the function of KFs,and to explore whether inhibition of Warburg effect can provide new hope for the treatment of keloid.Methods1.Fibroblasts were isolated from keloid samples,hypertrophic scar samples,atrophic scar samples,proliferative stage scar samples,and normal skin samples.Study whether there is a Warburg effect in keloid fibroblasts(KFs),hypertrophic scars fibroblasts(HSFs),atrophic scar fibroblasts(ASFs),and proliferative stage scar fibroblasts(PSSFs).1.1.The same amounts of cells in each group were cultured under the same conditions.Then,the glucose consumption and the lactate production were detected using a glucose assay kit and a lactic acid assay kit.1.2.Cells in each group were cultured in the same condition,and total RNA and total protein were extracted.The mRNA and protein expression levels of glycolysis key enzyme genes in each group were detected by qPCR and Western-blot.1.3.The same amounts of KFs and NFs were cultured and treated with 2-DG at different concentrations.After 48h,the cell proliferation of KFs and NFs was detected with a Cell Counting Kit-8(CCK-8).2.KFs were isolated and cultured with different concentrations of oxamate(0,20,40,80 mmol/1)in vitro.After the Warburg effect was inhibited,we detected the biological activity and function of KFs,and explored the related mechanism.2.1.The product of glycolysis(the production of lactate)in KFs was detected after the treatment of oxamate to reflect the inhibition effects of oxamate on the Warburg effect in KFs.2.2.After the inhibition of the Warburg effect,a scratch wound assay was used to detect the cell migration ability of KFs.Then,total RNA was extracted and the mRNA expression levels of type I collagen,type III collagen,TGF-?1,VEGF,and ?-SMA were detected using qPCR.Besides,the apoptosis rate in KFs was detected using flow cytometry and the Annexin V PE apoptosis kit.What's more,a CCK-8 was used to detect the cell proliferation in KFs.2.3.The cell cycle distribution in KFs was detected using flow cytometry and the Cell Cycle Analysis Kit.Then,total protein was extracted and the protein expression levels of proteins related to cell cycles were detected using Western-blot.Results1.The detection of Warburg effect in KFs,HSFs,ASFs,and PSSFs:1.1.Glucose consumption and lactate production in KFs were significantly higher than that in NFs.Glucose consumption and lactate production in HSFs,ASFs,and PSSFs were not significantly different from those in NFs.1.2.The mRNA and protein expression levels of hexokinase 2(HK2),pyruvate kinase isoform M2(PKM2),and lactate dehydrogenase A(LDHA)in KFs were significantly higher than those in NFs.The mRNA and protein expression levels of glycolysis key enzyme genes in HSFs,ASFs,and PSSFs were not significantly different from those in NFs.1.3.After the treatment of 2-DG,the cell proliferation of KFs decreased more pronounced than that of NFs.2.The effect of inhibition of Warburg effect on the biological activity,function of KFs and related mechanism:2.1.The lactate production in KFs decreased gradually when treated with increasing concentrations of oxamate.Oxamate could inhibit Warburg effect in KFs in a dose-dependent manner.2.2.Effect of inhibition of Warburg effect on biological activity and function of KFs:2.2.1.The migration ability of KFs decreased gradually when treated with increasing concentrations of oxamate for 24h and 48h.Oxamate could inhibit the migration ability of KFs in a dose-dependent manner.2.2.2.Compared with the control group,the mRNA expression levels of type I collagen in KFs treated with 20 mmol/1 and 40 mmol/1 oxamate decreased significantly.And the mRNA expression levels of ?-SMA in KFs treated with 40 mmol/1 oxamate also decreased significantly.2.2.3.The apoptosis rate in KFs treated with oxamate increased significantly compared with the control group.2.2.4.The cell proliferation of KFs decreased gradually when treated with increasing concentrations of oxamate for 24h and 48h.Cell proliferation decreased more pronounced when treated with oxamate for 48h.Oxamate could inhibit the cell proliferation of KFs in a dose-and time-dependent manner.2.3.Study on the Molecular Mechanism of the decrease of the cell proliferation of KFs after inhibition of Warburg effect:2.3.1.When treated with increasing concentrations of oxamate,cells in G0/G1 phase increased significantly,and cells in S phase and G2/M phase decreased correspondingly.Oxamate can induce G0/G1 arrest in KFs in a dose-dependent manner.2.3.2.When treated with oxamate,the protein expression levels of cell cycle related protein-Cyclin D1 decreased significantly and so does its upstream regulators-Akt and GSK3?(phospho S9).Conclusion1.There is both aerobic glycolysis and oxidative phosphorylation in KFs,and there is no similar phenomenon in HSFs,ASFs,or PSSFs.The Warburg effect in KFs is a special feature,which not exists in other types of scars or in the proliferative stage of scars.2.Inhibition of the Warburg effect in KFs could significantly suppress cell proliferation,migration ability,and collagen production,and could induce G0/G1 arrest through Akt-GSK3?-Cyclin D1 pathway in KFs.The biological activity and function of KFs were closely associated with the Warburg effect.And inhibition of the Warburg effect in keloid can provide new hope for the treatment of keloid. |
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