| Background Coronary atherosclerosis is the main cause of acute coronary syndrome(ACS),leading to the occurrence of most cardiovascular events.It is a chronic inflammatory disease of arterial wall.Dysfunction of vascular endothelial cells plays a key role in the early stage of atherosclerosis,and excessive lipid and inflammatory response are considered as the main factors of plaque formation.The involvement of arterial lesions begins with endothelial dysfunction,followed by extensive lipid deposition within the endometrium,adhesion and aggregation of phagocytes and platelets,and the release of inflammatory factors leading to inflammatory responses.PECAM-1 is highly expressed on the surface of endothelial cells,especially at the junction of vascular endothelial cells.Studies have suggested that PECAM-1 plays a role in the pathogenesis of atherosclerosis,and as a mechanoreceptor on the surface of endothelial cells to regulate changes in cellular functions.Reactive oxygen species(ROS)production by oxidative stress is an important cause of many vascular diseases.ROS are mainly produced by NADPH oxidase,which has low activity in blood vessels under physiological conditions.However,NADPH oxidase content can be increased under the action of LPS and other stimulants,inducing changes in endothelial cell function,causing inflammatory reactions,leading to pathological changes and destroying vascular homeostasis.PARP-1 is a highly conserved DNA-binding ribozyme,which is mainly activated when oxidative stress causes DNA damage.PARP-1 can regulate the expression of various key inflammatory factors,including intercellular adhesion molecule 1(ICAM-1)and vascular cell adhesion molecule 1(VCAM-1).PARP-1 inhibitors can protect endothelial dysfunction,reduce inflammation and atherosclerotic plaque size.Objective To investigate the role of pecam-1 expressed by vascular endothelial cells in the AS plaques,and to verify the role of NOX/ROS in the PECAM-1 upregulation of PARP-1.Methods 1.In order to verify whether PECAM-1 regulates the expression of NOX4,PARP-1 and inflammatory factors in the absence of LPS stimulation,endothelial cells were divided into control group and PECAM-1 siRNA group without any intervention from the control group.PECAM-1 siRNA group was transfected with PECAM-1 small interfering RNA and cultured in CO2 incubator for 24 h.2.In order to verify whether PECAM-1 regulates the expression of PARP-1 and inflammatory factors under LPS stimulation,endothelial cells were divided into control group and LPS group without any intervention.LPS group was given 0.5 ug/ml LPS stimulation and cultured in CO2 incubator for 24 h.3.To verify whether PECAM-1 expressed PARP-1 and inflammatory factors without the presence of NOX4,the endothelial cells were divided into the control group and the apocynin group,and the control group did not make any intervention.4.In order to verify whether PARP-1 promotes the expression of downstream inflammatory factors,endothelial cells were divided into control group,LPS group and DPQ group.The control group received no intervention,and the LPS group was given 0.5 ug/ml LPS stimulation.5.Verify whether inhibition of PECAM-1 can reduce the LPS-induced inflammatory response(reduce the expression of PARP-1 and inflammatory signals).Endothelial cells were divided into the control group and the PECAM-1 siRNA +LPS group.No intervention was performed in the control group.PECAM-1 siRNA +LPS group was transfected with PECAM-1 small interfering RNA.Results Flow cytometry analysis showed that DCFDA fluorescent probes entering the cells in the LPS group were 54.9%,and DCFDA fluorescent probes entering the cells in the control group were 33.4%.Compared with the two groups,the ROS expression in the LPS group was significantly higher;DCFDA fluorescent probes entering the cells in the PECAM-1 siRNA were 24.0%,and DCFDA fluorescent probes entering the cells in the control group were 20.7%.There was no significant difference between the two groups in the expression of ROS.The results of cellular immunofluorescence showed that the expression of NOX4 protein was higher in LPS group than in control group.There was no significant difference in NOX4 expression between PECAM-1 siRNA group and control group.Western blot and ELISA results showed that the expression of PARP-1 protein was significantly higher in the LPS group(0.430 ± 0.019)compared with the control group(0.232 ± 0.030)(P<0.05);the expression of inflammatory factor VCAM-1 was significantly higher in the LPS group(1768 ± 39.75)and the control group(829.7 ± 18.67),(P<0.05).The expression of inflammatory factor ICAM-1 was significantly higher in the LPS group(14.05 ± 1.15)compared with the control group(3.28 ± 0.31).Compared with the control group(1352 ± 109.8),the expression of MCP-1 in the LPS group(1909 ± 32.16)was significantly higher,and the difference was statistically significant(P<0.05).There was no significant difference in the expression of PARP-1 protein between the Apocynin group(0.093 ± 0.006)and the control group(0.088 ± 0.005)(P>0.05).There was no significant difference in the expression of VCAM-1 in Apocynin group(650.8 ± 64.74)between control group(591.0 ± 26.57)and the expression of ICAM-1 in Apocynin group(4.007 ± 0.95)between control group(2.893 ± 0.51)(P>0.05).There was no significant difference in the expression of PARP-1 between PECAM-1 siRNA group(0.065 ± 0.011)and control group(0.065 ± 0.013)(P>0.05).There was no significant difference in the expression of VCAM-1 and ICAM-1 between PECAM-1 siRNA group(786.2 ± 54.8,3.283 ± 0.31)and control group(728.4 ± 25.59,2.73 ± 0.32)(P>0.05).There was no significant difference in the expression of PARP-1 protein between PECAM-1 siRNA+LPS group(0.105 ± 0.008)and control group(0.108 ± 0.008)(P>0.05).There was no significant difference in the expression of VCAM-1 between PECAM-1 siRNA+LPS group(709.50 ± 67.89)and control group(664.70 ± 37.84),There was no significant difference in the expression of ICAM-1 between PECAM-1 siRNA+LPS group(3.44 ± 0.34)and the control group(3.54 ± 1.14)(P>0.05).The DPQ group(962.6 ± 23)was significantly reduced compared with the LPS group(1768 ± 39.75).The expression of ICAM-1 was significantly decreased in the DPQ group(669.9 ± 20.82)compared with the LPS group(1181 ± 34.70),and the expression of MCP-1 was significantly decreased in the DPQ group(1519 ± 32.16)compared with the LPS group(1909 ± 32.16),and the difference was statistically significant(P<0.05).Compared with the control group(0.220 ± 0.015)and the Apocynin group(0.247 ± 0.028),the expression of PECAM-1 was higher in the LPS group(0.407 ± 0.023).Conclusion 1.LPS can increase the expression of reactive oxygen species in vascular endothelial cells,promote the expression of DNA repair enzyme PARP-1 and the production of inflammatory cytokines,suggesting that oxidative stress plays an important role in the pathogenesis of AS to promote atherosclerosis.2.NOX/ROS participates in the regulation of vascular endothelial cell function by PECAM-1/PARP-1,which may be an important mechanism of action of LPS-induced atherosclerosis. |
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