The Role Of Cx43 In The Regulation Of Radiation Injury Of Vascular Endothelial Cells And Its Mechanism

BackgroundEpidemiological studies showed that the incidence of cardiovascular disease increased significantly among cancer patients receiving radiotherapy or survivors of radiation accidents.Vascular endothelial cells are the key cells mediating radiation-induced cardiovascular diseases.The death of vascular endothelial cells and the secretion of cytokines are the main acute effects of radiation injury.These phenomena change the internal environment of the irradiated organism and cause chronic inflammation in the whole body,leading to chronic diseases,such as atherosclerosis and chronic fibrosis,which seriously affect the prognosis and quality of life of radiotherapy patients and accident survivors.Therefore,understanding the molecular mechanism of vascular endothelial cells injury induced by X-ray is of great significance for reducing the side effects of radiotherapy,maximizing the therapeutic and prognostic effects,and improving the quality of life of radiation accidents survivors.Cx43 is a member of the gap junction protein family,which is responsible for small molecules and signal transduction between cells.A large number of studies have shown that Cx43 plays an important role in cardiovascular injury.Changes in the expression and distribution of Cx43 in hypertrophic cardiomyopathy,heart failure and ischemic heart diseases;Cx43 also plays an important role in vascular permeability,endothelial cell differentiation and vascular repair,hemodynamics and proliferation and migration of vascular smooth muscle cells.However,the role of Cx43 in X-ray-induced vascular endothelial cell injury and its mechanism have not been reported.ObjectiveThe aim of this study is to investigate the role and mechanism of Cx43 in X-ray-induced injury of human vascular endothelial cells.To analysis the expression,distribution and phosphorylation of Cx43 in human umbilical vein endothelial cells(HUVEC)induced by X-ray and the role of Cx43 in the proliferation,apoptosis and pyroptosis of irradiated HUVECs.By investigating the relationship between Cx43 and Panxl or calcium signaling pathway,the possible mechanism of Cx43 regulating pyroptosis was explored,which provided new ideas and potential targets for the study of prevention and treatment methods of X-ray induced injury.Method1.The effect of X-ray on Cx43 expression,distribution and phosphorylation.Western blot and Immunofluorescence staining were used to detect the expression,distribution and phosphorylation of Cx43 at different time(6 h,12 h,24 h,48 h and 72 h)after exposure to different dose X-rays(0 Gy,5 Gy,10 Gy and 20 Gy).2.Effects of Cx43 on proliferation and apoptosis of X-irradiated HUVECs.After Cx43 was silenced by siRNA technology and Cx43 was overexpressed by transfected plasmid,CCK-8 and clone formation assay were used to detect the proliferation of HUVECs 72 h after 10 Gy X-ray irrradiation and the survival rate of HUVECs 14 days after 2.5 Gy X-ray irradiation,respectively;PI-AnnexinV apoptosis kit were used to detect cell apoptosis at 48 h,72 h and 96 h after irradiated to different dose X-ray(0 Gy,5 Gy,10 Gy and 20 Gy).Western blot was used to detect the expression of caspase-3 and Cx43 protein at 72 h after irradiated to different dose X-ray(0 Gy,5 Gy,10 Gy and 20 Gy).3.X-ray induced pyroptosis of HUVECs.At 6?48 h after 0?20 Gy X-ray irradiated,Western blot was used to detect the expression and activation of caspase-1;72 h after 10 Gy X-ray irradiation.PI-FLICA staining was used to detect the pyroptosis rate of HUVECs by flow cytometry and confocal laser microscopy;transmission electron microscopy was used to detect the morphological changes of cells;LDH was used to detect cell rupture and cytotoxicity;and the above indicators were used to illustrate the pryoptosis of HUVECs.4.Cx43 regulates pyroptosis of irradiated HUVECs.After Cx43 was silenced by siRNA technology and Cx43 was overexpressed by transfected plasmid,72 h after 10 Gy X-ray irradiation.PI-FLICA staining was used to detect the pyroptosis rate of HUVECs by flow-cytometry and confocal laser microscopy;transmission electron microscopy was used to detect the morphological changes of cells;LDH was used to detect cell rupture and cytotoxicity;12 h after 10 Gy X-ray irradiation,Western blot was used to detect the expression and activation of caspase-1;and the above indicators were used to illustrate the role of Cx43 in regulation of pryoptosis ·5.Cx43 regulates pyroptosis by regulating cleaved Panx1.Western blot was used to detect cleaved Panxl expression in 10 Gy X-ray irradiated HUVECs at 0?72 h and at 12 h after 0-10 Gy in irradiated HUVECs.After Cx43 was silenced by siRNA technology and Cx43 was overexpressed by transfected plasmid,Western blot was used to detect cleaved Panxl expression at 12 h after 10 Gy X-ray irradiated.After Panxl and Cx43 were silenced by siRNA technology,at 72 h after 10 Gy X-ray irradiation,PI-FLICA staining was used to detect the pyroptosis rate of HUVECs by flow cytometry and confocal laser microscopy;transmission electron microscopy was used to detect the morphological changes of cells;LDH was used to detect cell rupture and cytotoxicity;12 h after 10 Gy X-ray irradiation,Western blot was used to detect the expression and activation of caspase-1;and the above indicators were used to illustrate Cx43 regulates pyroptosis by regulating cleaved Panx1.6.Cx43 regulates pyroptosis by regulating intracellular calcium signaling.Fluo-4/AM staining was used to detect intracellular calcium by flow cytometry and laser confocal microscopy in 10 Gy X-ray irradiated HUVECs at 0?72 h and at 12 h after 0?20 Gy in irradiated HUVECs.After Cx43 was silenced by siRNA technology and Cx43 was overexpressed by transfected plasmid.Fluo-4/AM staining was used to detect the effect of Cx43 on intracellular calcium 12 h after 10 Gy X-ray irradiated.While Cx43 was silenced by siRNA,intracellular calcium was inhibited by inhibitors BAPTA/AM,2-APB and Nifedipine,respectively;at 72 h after 10 Gy X-ray irradiation,PI-FLICA staining was used to detect the pyroptosis rate of HUVECs by flow cytometry and confocal laser microscopy;transmission electron microscopy was used to detect the morphological changes of cells;LDH was used to detect cell rupture and cytotoxicity;12 h after 10 Gy X-ray irradiation,Western blot was used to detect the expression and activation of caspase-1;and the above indicators were used to illustrate Cx43 regulates pyroptosis by regulating intracellular calcium signaling.Result1.X-ray induced changes of Cx43 in expression,distribution and phosphorylation in irradiated HUVECs.The expression of Cx43 decreased at 6?72 h after 10 Gy X-ray irradiated and the expression of Cx43 decreased in a dose dependent manner at 12 h.The distribution of Cx43 was transferred from intercellular to nucleus and perinuclear area at 24 h after irradiated to 5 Gy,10 Gy,20 Gy.The phosphorylation level at Ser368 site increased at 48 h after irradiation and increased in a dose dependent manner.2.Effects of Cx43 on proliferation and apoptosis of irradiated HUVECs.(1)Cx43 promotes the proliferation of irradiated HUVEC cells.At 72 h after X-ray irradiation,the proliferation of HUVECs decreased with the increase of irradiation dose.The proliferation of Cx43 overexpression group was significantly higher than that of vector group at 72 h after 10 Gy X-ray irradiated and the survival rate of HUVECs increased at 14 d after 2.5 Gy irradiated;the proliferation at 72 h after 10 Gy X-ray irradiated and 14 d survival rate of 2.5 Gy X-ray irradated were significantly lower in Cx43 silenced group than that of scramble group.(2)Cx43 inhibited apoptosis of irradiated HUVECs.The apoptotic rate of HUVECs increased from 48?96 h after 10 Gy X-ray irradiated and at 72 h within 0?20 Gy in a dose-dependent manner.In the range of 0-20 Gy,the expression of Cx43 decreased and cleaved caspase-3 expression level increased with the increase of dose.The percentage of early apoptotic and late apoptotic HUVECs in Cx43 silenced group was significantly higher than that in scramble group;the percentage of early apoptotic and late apoptotic HUVECs in Cx43 overexpression group was significantly lower than that in vector group;the expression of cleaved caspase-3 in Cx43 silenced group was higher than that in scramble group,while that in overexpression group was lower than that in vector group.3.X-ray induced HUVECs pyroptosis.The expression of cleaved caspase-1 increased 6-48 h after 10 Gy X-ray irradiated and increased with the irradiation dose at 12 h.At 72 h after X-ray irradiation,FLICA staining increased in 10 Gy group,the proportion of PI-active caspase-1 staining increased,the morphological characteristics of cell rupture appeared and the level of LDH in extracellular fluid increased.4.Cx43 inhibited HUVECs pyroptosis.At 72 h after 10 Gy X-ray irradiated,Cx43 overexpression group compared with vector group,the percentage of pyroptosis cells decreased significantly;FLICA staining decreased;LDH level in extracellular fluid decreased;at 12 h after 10 Gy X-ray irradiation,the expression level of cleaved caspase-1 decreased in the Cx43 over-expressing group compared with the vector group.On the contrary,72 h after 10 Gy X-ray irradiated,Cx43 knockdown group compared with scramble group,the percentage of pyroptosis cells increased significantly;FLICA staining increased;LDH level in extracellular fluid increased;and 12 h after 10 Gy X-ray irradiation,the expression level of cleaved caspase-1 increased in the Cx43 knockdown group compared with the scramble group.5.Cx43 downregulated HUVECs pyroptosis by inhibiting Panx1 cleavage.At 12?72 h after 10 Gy X-ray irradiated,the expression of cleaved Panxl increased and within 0-10 Gy showed a dose effect relationship.At 72 h after 10 Gy X-ray irradiated,Panx1 knockdown group compared with scramble group,the percentage of pyroptosis cells decreased;the level of LDH in extracellular fluid decreased;and at 12 h after 10 Gy X-ray irradiated the expression of cleaved caspase-1 decreased.At 12 h after 10 Gy X-ray irradiated,cleaved Panx1 decreased after overexpression of Cx43,while Cx43 silence increased cleaved Panx1 expression.When Panx1 and Cx43 were both silenced,72 h after 10 Gy X-ray irradiated,the percentage of pyroptosis cells decreased,FLICA staining decreased,LDH level in extracellular fluid decreased,and cleaved caspase-1 expression decreased 12 h after 10 Gy X-ray irradiation compared with single Cx43 knockdown group.6.Cx43 downregulated HUVECs pyroptosis by reducing intracellular calcium.At 0-12 h after 10 Gy X-ray irradiated of HUVECs,intracellular calcium increased significantly and reached its maximum at 12 h.At 12 h,intracellular calcium increased in a dose dependent manner(0 Gy,2.5 Gy,5 Gy,10 Gy and 20 Gy).At 12 h after 10 Gy X-ray irradiated,Cx43 overexpression inhibited intracellular calcium increase and silence increased intracellular calcium.At 72 h after 10 Gy X-ray irradiation,calcium inhibitors BAPTA/AM,2-APB,Nifedipine group effectively inhibited intracellular calcium increase,while the percentage of pyroptosis cells decreased significantly,the level of LDH in extracellular fluid decreased.At 12 h after 10 Gy X-ray irradiation,calcium inhibitors BAPTA/AM,2-APB,nifedipine group compared with DMSO control group,the expression level of cleaved caspase-1 decreased.Cx43 silenced and calcium inhibitors were used simultaneously,all three calcium inhibitors could alleviate the increase of pyroptosis caused by Cx43 knockdown.At 72 h after 10 Gy X-ray irradiation,compared with Cx43 alone silencing group,Cx43 silencing + calcium inhibitor group decreased the percentage of pyroptosis cells,LDH level in extracellular fluid and at 12 hours after 10 Gy X-ray irradiation cleaved caspase-1 expression level of HUVEC cells.Conclusion1.X-ray results in the decrease of Cx43 expression and the change of Cx43 distribution through phosphorylation of Ser368 in irradiated HUVECs.2.Cx43 improves the proliferation of HUVECs after X-ray irradiation,inhibit cell apoptosis.Cx43 regulates apoptosis by inhibiting the activation of caspase-3.3.X-ray induces the pyroptosis of HUVECs.4.Cx43 reduces pyroptosis of irradiated HUVECs by inhibiting the activation of caspase-1,the key molecule of pyroptosis,or by downregulating the cleavage of Panxl.5.Cx43 reduces pyroptosis of irradiated HUVECs by inhibiting intracellular calcium singnaling.