Autophagy Inhibits 2-DG Induced Apoptosis Of Endothelial Cells And Autophagy Activity In Infantile Hemangioma

BackgroundInfantile hemangiomas(IH)is the most common benign tumor in infants and young children,fetuses and infants,IH is mostly found in the superficial areas of the head and neck,interfere with the patient's appearance and proliferation phase of rapid growth,lead to various complications,such as: ulcers,bleeding,airway obstruction,heart failure,serious can endanger life and extinction can also be a series of beautiful sequelae,so often cause psychological disorders of children and their families.The main characteristic of IH is that there are a large number of abnormal proliferation of vascular endothelial cells(cells endothelial,ECs).Therefore,inhibiting the proliferation and differentiation of ECs has a very important clinical significance for the prevention and treatment of IH.Although the majority of IH may be self-resolution,it is generally accepted that early diagnosis,reasonable intervention,suppression of growth,and Promote regression.Infantile hemangiomas(IH)is the most common benign tumor in infants and young children,fetuses and infants,IH is mostly found in the superficial areas of the head and neck,hinder the appearance of patients,proliferative phase of rapid growth,lead to various complications,such as: ulcers,bleeding,respiratory tract obstruction,heart failure,severe cases can be life-threatening,extinction can also be a series of beautiful sequelae,because this often causes psychological obstacles of children and their families.The main characteristic of IH is that there are a large number of abnormal proliferation of vascular endothelial cells(cells endothelial,ECs),so it is very important for the prevention and treatment of IH to inhibit the proliferation of ECs.Although most of the IH can be dissipated by itself,it is generally accepted that early diagnosis,reasonable intervention,suppression of growth,and the promotion of regression.2-DG,as a classical inhibitor of glucose fermentation,has aroused widespread concern and a large number of clinical researchers since 1950.Previous studies have confirmed that 2-DG can inhibit the proliferation and differentiation of many kinds of tumor cells(lung cancer,breast cancer,and bone cancer).Our previous research found that 2-DG had a significant inhibitory effect on HUVECs cells in vitro,and the IH transplanted tumor was inhibited in nude mice.As for the specific mechanism of action,the literature reported,that 2-DG may compete with mannose,inhibite N-linked glycosylation of protein,resulting in aggregation of unfolded protein in endoplasmic reticulum,resulting in endoplasmic reticulum stress(ERS)and cell apoptosis.Other studies have found that 2-DG can induce autophagy and autophagy is often regarded as a kind of protective effect of cells under stress.So what is the role of 2-DG induced autophagy in HUVECs? Many studies found that there is a close relationship between autophagy and cancer,aging,cardiovascular disease and other neurodegenerative diseases,metabolic diseases such as,in particular tumor.However,the role in the different stages of the specific tumor are not the same.Autophagy has two sides,which can promote cell survival and induce cell death.Although the autophagy activity of most tumor cells is down regulated,there are still some tumor cell autophagy activities,such as colorectal cancer,lung cancer and cervical cancer Hela cells,etc..Some studies have confirmed that autophagy can protect the survival of tumor cells in harsh environments.Recent studies have found that autophagy exists in human IH specimens,and co expression of proliferating hemangioma autophagy related molecules of Beclin1 and Survivin,presumably in the proliferating IH,autophagy promotes survival of endothelial cell.Due to IH is a benign tumor of the abnormal proliferation of vascular endothelial cells,to explore the expression activity of autophagy in the development of IH and the role of,autophagy have an important significance in prevention and treatment of IH through providing new targets,and providing 2-DG with ideas of more efficiency in the treatment of IH.ObjectiveThe experiment is carried out to explore the mechanism of 2-DG,in vitro experiments HUVECs were divided into control group,different concentrations of 2-DG group,2-DG combined mannose mannose group,positive control group(TM group)and 2-DG combined with 3-MA group,detection of HUVECs proliferation,apoptosis and autophagy,and then explore the specific mechanisms of apoptosis of endothelial cell induced by 2-DG,apoptosis and autophagy of internal relations.Collectting each phase of IH clinical specimens,HE staining was used to observe the proliferation and regression of IH vascular endothelial cell proliferation and morphology,the difference apoptosis rate between two groups through TUNEL apoptosis detection were compared,and apoptosis level between two groups was assessed,using immunohistochemical observation between the two groups of ATG5 expression levels and immunofluorescence observation between the two groups of LC3-II expression levels to detecte and analyse the difference and change of autophagic activity in proliferation and regression hemangioma.Methods1.MTS analysis.HUVECs were cultured in 96 well culture plate,divided into eight groups: experimental group 1 was the control,the working concentrations of 2-DG in the experimental group 2 and group 3 and 4 groups were 0.05 m M,0.5 m M and 5 m M,5,6,7 group were added D-mannose based on 2,3,4 groups,the concentration of D-mannose was 0.5 m M.The 8 group was Tm group(ERS inducer),the concentration of it was 1ug/ml,and the OD values were detected by MTS assay.2.Flow cytometry to explore the effect of 2-DG on HUVECs apoptosis.The cells were divided into Control,2-DG 2-DG,10 m M 1m M + D-mannose 2-DG,10 m M 2m M + 3-MA 2m M,3-MA group,10 m M,the apoptosis of HUVEC cells was detected by FITC-Annexin V /PI double staining method.3.The expression of GRP78 protein was detected by blot Western assay.Cells were divided into control,2-DG 10 mm,2-DG 5mm,2-DG 10 mm + 1 mm D-mannose,2-DG 5 mm + 1 mm D-mannose,tunicamycin(1ug / ml).After 24 h,the cells were harvested,Western blot method was used to detect the protein expression level and GAPDH as the internal control,to calculate the relative optical density of target protein.4.The expression of Beclin1 and Atg5 protein was detected by blot Western assay.Cells were divided into control,10 mm 2-DG,5mm 2-DG,2-DG 10 mm + 2mm 3-m A,2-DG 5mm and 2mm 3-m A,2mm 3-m A,after 24 h,cells were collected.Western blot method was used to detect the protein expression level and GAPDH as the internal control,to calculate the relative optical density of target protein.5.HE staining.Infantile hemangioma resection specimens were collected,paraffin imbedding.The tissue structures of proliferation and involuting IH were observed by optical microscope after HE staining.6.TUNEL apoptosis detection.After TUNEL staining,the apoptosis rate was compared between the two groups according to the number of apoptotic cells,and evaluated.7.Immunohistochemistry of Atg5.The proliferation and regression phase of IH sections were stained by immunohistochemical staining,and the expression levels of Atg5 in the two groups were analyzed.8.Immunofluorescence labeling method.IH sections of proliferation and involuting phase were labeled by immunofluorescence,and the expression levels of LC3-II in the two groups were analyzed.Results The cell proliferation activity of 2-DG group was significantly decreased compared with the control group,with a significant difference(p <0.01),and this inhibition was dose-dependent;Proliferation activity of 2-DG joint mannose group increased compared with 2-DG group compared with a significant difference(p <0.01),Apoptosis rate of 2-DG group increased significantly compared with the control group with a significant difference(p <0.01),and apoptosis rate of 2-DG joint mannose group decreased significantly compared with 2-DG group(p < 0.01);GRP78 protein level of 2-DG group increased,compared with the control group with significant difference(p <0.01),after adding mannose,GRP78 protein level reduced significantly,compared with 2-DG group(p < 0.01);Beclin1 and Atg5 protein levels of 2-DG group increased,as compared with the control group,with a significant difference(p <0.01).After 3-MA pretreatment,2-DG-induced apoptosis rate increased,Beclin1 and Atg5 protein expression levels also decreased significantly,compared with 2-DG group(p <0.01).TUNEL method found that IH cells apoptosis rate of involuting IH was significantly higher than that of proliferating IH with statistical difference(P < 0.01);Immunohistochemistry showed that the expression level of ATG5 in the proliferating IH group was higher than those in the involuting hemangioma(P < 0.01);Immunofluorescence indicated that in the proliferation phase of IH group LC3-II expression activity was higher than that of involuting phase.Conclusion In vitro experiments we found that 2-DG can significantly inhibit the proliferation of HUVECs and induce HUVECs apoptosis and autophagy;endoplasmic reticulum stress may mediate apoptosis induced by 2-DG;2-DG can induce autophagy,and the autophagy can inhibit cell apoptosis.The detection of clinical samples showed that: apoptosis cells in the involuting phase of IH increased more than those in the proliferation hemangioma;the autophagy activity of proliferation phase is stronger than that of involuting phase,and down-regulation of autophagy may promote the conversion of IH from the period of proliferation.to the involuting phase.