The Role Of ERS Involved In Endothelial Cells Inflammatory Injury Via Cx43 Hemichannel-Mediated Activation Of P2X7 Receptor And Its Mechanism

Objective:Atherosclerosis is the basis for the pathogenesis of multitudinous cardiovascular diseases.It has been found that endothelial cells inflammatory injury being an essential trigger for atherosclerosis over the years.Studies have shown that endoplasmic reticulum stress（ERS）is in connection with cellular inflammatory injury,but the mechanism has not been fully clarified.Connexin 43（Cx43）hemichannel is involved in the process of endothelial injury,and P2X7 receptor has been widely studied as a potential target for various inflammatory diseases.Therefore,this study aims to investigate the role and regulatory mechanism of endoplasmic reticulum stress,Cx43 hemichannel and P2X7 receptor in endothelial cells inflammatory injury.Methods:HUVECs were stimulated with 800μmol/L H₂O₂for 4 h to construct endothelial cells inflammatory injury model;ERS was inhibited by 500μmol/L TUDCA treated for 1 h in HUVECs;Cx43 hemichannel activity was inhibited by100μmol/L Gap19 treated for 24 h in HUVECs;HUVECs were treated with 20μmol/L A-740003 for 1 h to inhibit the expression of P2X7 receptor;HUVECs were treated with 100μmol/L CT9 for 24 h to activate Cx43 hemichannel activity.According to the above treatment,the corresponding groups were carried out,the detection methods and indicators were as follows:monocyte-endothelial adhesion was observed under the inverted fluorescence microscope;NO release level was detected by NO assay kit;the expression levels of inflammatory injury-associated proteins（ICAM-1,VCAM-1,NLRP3 and Cleaved-caspase-1）,ERS marker proteins（GRP78,IRE1αand XBP1）,Cx43 protein and P2X7 receptor were examined by Western blot;the activity of Cx43 hemichannel was assessed by Et Br uptake assay;ATP release level was detected by ATP assay kit;the formation of NLRP3inflammasomes was assessed by immunofluorescence experiment.Results:1.H₂O₂ stimulated HUVECs,the results showed that the monocyte-endothelial adhesion was improved;NO release was declined;the expression levels of inflammatory injury-associated proteins ICAM-1,VCAM-1,NLRP3 and Cleaved-caspase-1 were elevated;and the formation of NLRP3 inflammasomes was increased.Indicating that the model was successfully constructed.2.To explore the function of ERS in H₂O₂-induced endothelial cells inflammatory injury.The expression of ERS marker proteins GRP78,IRE1αand XBP1 in HUVECs were all increased after H₂O₂ treatment;HUVECs were treated with TUDCA and H₂O₂successively,the results indicated that TUDCA inhibited the increase of ERS marker proteins expression levels caused by H₂O₂,the elevation of monocyte-endothelial adhesion,the decrease of NO release level,the upregulation of inflammatory injury-associated proteins and the increase of the formation of NLRP3 inflammasomes.Demonstrating that ERS was involved in the process of endothelial cells inflammatory injury and inhibiting ERS could reduce endothelial cells inflammatory injury.3.To verify ERS regulating Cx43 hemichannel activity.In the model,the expression of Cx43 was increased,the activity of Cx43 hemichannel was elevated and the extracellular ATP release was augmented;after TUDCA inhibited ERS,the expression of Cx43 protein was downregulated,the Cx43 hemichannel activity was declined and the ATP release was decreased.Suggesting that Cx43 hemichannel was closely related to endothelial cells inflammatory injury and the activity of Cx43hemichannel was decreased by inhibiting ERS.4.To explore the role of Cx43 hemichannel in H₂O₂-induced endothelial cells inflammatory injury.HUVECs were pretreated with Gap19 and then stimulated by H₂O₂,the results revealed that compared with H₂O₂ group,the cells in Gap19+H₂O₂group showed decreased activity of Cx43 hemichannel,declined monocyte-endothelial adhesion,elevated NO release,decreased inflammatory injury-associated proteins expression level and decreased NLRP3 inflammasomes formation.Showing that inhibiting Cx43 hemichannel activity alleviated endothelial cells inflammatory injury.5.To verify ERS regulating Cx43 hemichannel activity and further affecting the expression of P2X7 receptor.The expression of P2X7 receptor was upregulated after H₂O₂ treatment in HUVECs;the P2X7 receptor was downregulated after TUDCA inhibited ERS.The release of ATP was reduced and the P2X7 receptor was downregulated after Gap19 inhibited Cx43 hemichannel activity.Indicating that the inhibition of ERS and Cx43 hemichannel activity both downregulated the expression of P2X7 receptor.6.To explore the effect of P2X7 receptor in H₂O₂-induced endothelial cells inflammatory injury.HUVECs were treated with A-740003 and H₂O₂successively,the results indicated that compared with DMSO+H₂O₂group,HUVECs in A-740003+H₂O₂ group showed decreased P2X7 receptor expression,declined monocyte-endothelial adhesion,increased NO release,reduced expression of inflammatory injury-associated proteins and decreased NLRP3 inflammasomes formation.Suggesting that inhibiting P2X7 receptor alleviated endothelial cells inflammatory injury.7.To confirm ERS activating P2X7 receptor through Cx43 hemichannel leading to endothelial cells inflammatory injury.HUVECs were treated with TUDCA,CT9 and H₂O₂ successively,the expression of P2X7 receptor was elevated;the monocyte-endothelial adhesion was increased,the NO release was reduced,the expression of inflammatory injury-associated proteins were increased and the NLRP3 inflammasomes formation was augmented.Demonstrating that activating Cx43 hemichannel could reverse the effect of inhibiting ERS on endothelial cells inflammatory injury.Conclusions:1.ERS is involved in H₂O₂-induced endothelial cells inflammatory injury.2.ERS may cause endothelial cells inflammatory injury through the activation of P2X7 receptor via Cx43 hemichannel.