Characteristics And Mechanism Of Glucose Metabolism Of Keloid Fibroblasts Under Hypoxia

ObjectThe keloid disorder is a type of pathological scar featured by uncontrolled proliferation of dermal fibroblasts and excessive deposition of extracellular matrix(ECM).It is characterized with outgrowth beyond its origin and frequent recurrence after surgery.The pathogenesis has not been fully elucidated.Our previous studies found that keloid fibroblasts underwent glucose metabolic reprogramming and exhibited exaerobic glycolysis.The glucose metabolism mode and related mechanisms of keloids in hypoxic microenvironment remain unclear.To clarify the pathogenesis of the keloid,the study explored the glycolysis level,mitochondrial function,cellular function and signaling pathways in keloid fibroblasts(KFb)and normal skin fibroblasts(NFb)under normoxic and hypoxic(3%)conditions.Additionally,the effects of PI3K/AKT signaling pathways on KFb glucose metabolism and cell function were also studied.Finally,the interaction between HIFla and PI3K/AKT pathway was explored.Methods(1)The optimal hypoxic concentration to promote the proliferation of KFb was determined with cell counting under microscope.(2)Expressions and enzyme activities of GLUT1,HK2,PFKFB3,PGK1,ENO1,PKM2 and LDHA in the glycolytic pathway were investigated using qRT-PCR,Western Blot and enzyme activity kits.In addition,the number of mitochondria and mitochondrial membrane potential(MMP)were identified by flow cytometry.Detection of mitochondrial complex I-IV enzyme activities were completed with enzyme activity kits.The ultrastructure of mitochondria was observed under electron microscope.Furthermore,glycolysis and mitochondrial respiration in live cells were measured with Seahorse XFe96 Analyzer in real time.(3)Reactive oxygen species(ROS)and mitochondrial ROS(mitochondrial ROS,mitoROS)content were detected with flow cytometry.NOX,SOD and GSH-Px activities were identified with enzyme activity kits.Western Blot was used to detect the expression of LC3Ⅰ/Ⅱ and p62,numbers of red and green spots of autophagosomes and autolysosomes were observed by laser confocal microscope,and the ultrastructure of autophagosomes and autophagolysosomes were observed by transmission electron microscope.(4)The proliferation,apoptosis,migration and invasion of KFb and NFb under normoxic and hypoxic(3%)conditions were established using cell counting,flow cytometry and transwell chamber,respectively.Expressions of TGFβ1,Col Ⅰ and Col Ⅲ were detected with qRT-PCR and Western Blot.(5)Phosphorylation levels of PI3K/AKT pathway and the expression of HIF1α in KFb and NFb under normoxic and hypoxic(3%)conditions were determined with Western Blot.(6)The concentrations of PI3K/AKT signaling pathway inhibitor LY294002 were determined by cell proliferation experiment.(7)After KFb were treated with LY294002(0,5 and 25μM),expressions of PGK1,ENO1,HK2,PFKFB3,PKM2,LDHA and GLUT1 were established using Western Blot.Mitochondrial numbers and MMP were detected using flow cytometry.Ultrastructure of mitochondria was observed with electron microscope.Seahorse energy metabolism meter monitored glycolysis and mitochondrial respiration.(8)After treatment with LY294002(0,5 and 25μM),the contents of ROS and mitoROS in KFb were detected by flow cytometry.(9)After intervened with inhibitor LY294002(0,5 and 25 μM),the proliferation,migration,invasion and apoptosis of KFb under normoxic and hypoxic(3%)conditions were determined with cell counting,transwell chamber and flow cytometry,respectively.(10)KFb were treated with LY294002(0 and 25μM)and HIF1α inhibitor LW6(0 and 25μM),respectively.The protein expression levels of HIF1α,PI3K,p-PI3K,AKT and p-AKT were detected by Western Blot.Results(1)Compared with other oxygen concentrations,3%low oxygen concentration had the most obvious effect on promoting KFb proliferation.(2)Under normoxic condition,the mRNA levels,protein expressions and enzyme activities of the enzymes GLUT1,HK2,PFKFB3,PGK1,ENO1,PKM2 and LDHA of KFb were higher than those of NFb.Under hypoxia(3%),the mRNA levels,protein expressions and enzyme activities in KFb and NFb were significantly increased compared with those under normoxia.Under normoxia,the number of mitochondria,MMP and mitochondrial complex activities of KFb were lower than those of NFb.After 3%hypoxia treatment,the number of mitochondria and MMP increased while the complex activities decreased.Observation of the mitochondrial ultrastructure under electron microscope revealed that the mitochondria in KFb were swollen,vacuolated,and ridge disappeared under normoxia,while the mitochondrial ultrastructure in NFb was normal.After hypoxia(3%)treatment,the mitochondrial ultrastructure in NFb and KFb remained previous conditions,respectively.(3)Under normoxic condition,Seahorse XFe96 Analyzer showed that the extracellular acidification rate(ECAR)including non-glycolytic acidification,glycolysis,glycolytic capacity and glycolytic reserve in KFb was higher than those in NFb;while oxygen consumption rate(OCR)including the non-mitochondrial respiration,basal respiration,maximal respiration and spare capacity in KFb was lower than those of NFb.After KFb were treated with hypoxia(3%),the various ECAR indicators(not including glycolytic reserve)significantly increased,while the OCR indicators(not including H+leak)remarkedly decreased.(4)Under normoxic condition,the levels of ROS and mitoROS in KFb were higher than those in NFb.The activities of the main enzymes NOX,SOD and GSH-Px of KFb were higher than those of NFb.Under hypoxia(3%),NFb and KFb were in a state of oxidative stress,NOX activity and mitoROS production were increased,while the main antioxidant enzymes SOD and GSH-pX activity were also enhanced.Under normoxic condition,the LC3Ⅱ/Ⅰ ratio of KFb was higher than that of NFb,and the expression level of p62 was lower than that of NFb.In addition,the numbers of red and green spots,autophagosomes and autophagolysosomes in KFb were higher than those of NFb;after the intervention of hypoxia(3%),the ratio of LC3Ⅱ/Ⅰ,the number of red and green spots,autophagosomes and autophagolysosomes were significantly increased,while the expression of p62 was significantly reduced.(5)Under normoxic condition,the proliferation,migration,invasion and collagen synthesis capacity of KFb were higher than those of NFb,and the level of apoptosis was lower than that of NFb.After hypoxia(3%)treatment,the proliferation,migration,invasion and collagen synthesis capabilities of KFb and NFb were enhanced.Apoptosis of them was significantly inhibited.KFb was more responsive to hypoxia(3%)than NFb.(6)Under normoxic condition,HIF1α was almost undetectable in KFb and NFb,and the phosphorylation levels of PI3K and AKT in KFb were significantly higher than those in NFb.After 3%hypoxia treatment,the expressions of HIF1α,PI3K and AKT phosphorylation levels in KFb and NFb were significantly increased compared with those under normoxia.(7)When the PI3K/AKT pathway was inactivated,the protein expressions of glycolytic enzymes GLUT1,HK2,PFKFB3,PGK1,ENO1,PKM2 and LDHA decreased,while the number of mitochondria and MMP increased.Seahorse Metabolic Analyzer showed that the key parameters of ECAR decreased significantly and those of OCR increased after inhibiting the PI3K/AKT pathway.(8)When the PI3K/AKT pathway was surpressed,the contents of ROS and mitoROS significantly increased.(9)When the PI3K/AKT pathway was inhibited,the proliferation,migration and invasion ability of KFb decreased,while the level of apoptosis increased under normoxia and hypoxia(3%).(10)When the PI3K/AKT pathway was inhibited,the expression of HIF1α and the phosphorylation levels of PI3K and AKT and decreased.When the HIF1α pathway was repressed,both the expression of HIF1α and the phosphorylation levels of PI3K and AKT decreased.ConclusionsBased on the above research results,it confirmed that KFb exhibited glucose metabolic reprogramming.Under normoxic condition,glycolysis was enhanced,mitochondrial functions and ultrastructure were abnormal.In-depth studies found that hypoxic microenvironment increased glycolysis level and inhibited mitochondrial functions.Furthermore,hypoxia promoted proliferation,migration,invasion and collagen synthesis,but inhibited apoptosis.Finally,the PI3K/AKT pathway was proven to participate in regulation of glucose metabolism of KFb.When the pathway was inhibited,the glycolysis capacity was significantly reduced and the mitochondrial functions increased.Accordingly,proliferation was inhibited and apoptosis was enhanced.Our findings contribute to clarify the pathological mechanism of the effect of glucose metabolism on the function of keloid fibroblasts and the promotion of keloid hyperplasia.These findings may provide a certain theoretical basis for keloid treatments by targeting glucose metabolism.