| Background Paraquat is a herbicide,which accumulates mainly in the lungs after being taken,touched,inhaled or mistakenly taken into the human body.Paraquat can cause alveolar epithelial cell damage,alveolar hemorrhage,edema and inflammatory cell infiltration,acute lung injury in the early stage,and irreversible pulmonary fibrosis in the late stage.In our previous study,we found that the number of inflammatory cells in peripheral blood of patients with early PQ poisoning increased,especially the number of white blood cells,indicating that the body had inflammation.Leukocyte mainly plays a role by adhering to vascular endothelial cells,thus passing through the blood vessel to the injured site,but how PQ plays a role in this process is still unclear.Objective In this study,human umbilical vein endothelial cells(HUVECs)were used as models to investigate whether PQ could induce leukocyte-vascular endothelial cell adhesion and the mechanism of PQ in leukocyte-vascular endothelial cell adhesion,and how does antioxidant acetylcysteine(NAC)play a role in the adhesion of vascular endothelial cells to monocytes.To provide experimental basis for formulating prevention and control measures and Strategies of PQ exposure hazards.Research methods HUVEC cells in good condition were used for PQ dose and time trend exposure.The doses were 125,250,500,1000,2000 ?M,and the exposure time was 12 h,24h,36 h,respectively.The most suitable dose and time were selected for treatment..Carry out the following experiments:(1)To study the adhesion and mechanism of vascular endothelial cells and monocytes induced by PQ,the number of human acute monocytic leukemia cell line(THP-1)cells adhering to HUVEC cells was detected by cell adhesion assay.Westernblotting was used to detect the expression of intercellular adhesion molecule-1(ICAM-1)and MAPKs/NF-?B signaling pathway protein in HUVEC cells by semi-quantitative reverse transcription-polymerase chain.The expression of ICAM-1 gene was detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).The total active oxygen level in HUVEC cells was detected by the chemical fluorescent reagent Dichlorofluorescein diacetate(DCFH-DA).The THP-1 cells in good condition were evenly divided into 5 groups and cultured in the plate.When the cell density was as long as 80-90%,the cells were added with 125,250,500,1000 ?M PQ for 36 h,and the control group was added with the same amount of PBS.The expression of CD11 a protein on the surface adhesion molecule of THP-1 cells was detected by immunoblotting.(2)In order to study the inhibitory effect of antioxidant NAC on PQ-induced vascular endothelial cell and monocyte adhesion and the mechanism of action,HUVEC cells with good state were evenly divided into 6 groups into culture plates,which were control group,NAC group,PQ group,PQ+ low dose NAC group,PQ+ medium dose NAC group,PQ+ high dose NAC group.The number of THP-1 cells adhering to HUVEC cells was detected by cell adhesion assay.The expression levels of ICAM-1and MAPKs/NF-?B signaling pathway proteins were detected by Westernblotting.The total active oxygen level in HUVEC cells was detected by chemical fluorescent reagent DCFH-DA.(3)In order to study the role of MAPKs/NF-?B signaling pathway in the adhesion of vascular endothelial cells to monocytes induced by PQ,the well-precised HUVEC cells were evenly divided into 4 groups,which were control group,MAPKs/NF-?B signaling pathway inhibitor group(P38 inhibitor SB-203580,ERK1/2 inhibitor U-0126,JNK inhibitor SP600125,NF-?B inhibitor BAY11-7085),PQ group,PQ+MAPKs/NF-?B signaling pathway inhibitor group.The adhesion of THP-1 cells to HUVEC cells was detected by cell adhesion assay.The expression levels of ICAM-1 and MAPKs/NF-?Bsignaling pathway proteins were detected by Westernblotting.Result 1.PQ induced an increase in the number of THP-1 cells adhered to HUVEC cells,and an increase in the expression of adhesion molecule ICAM-1 on the surface of HUVEC cells and CD11 a,a marker of adhesion molecules on the surface of THP-1cells: as paraquat exposure concentration and time increased,the number of fluorescent label THP-1 adhering to human umbilical vein endothelial cells is also gradually increasing,compared with the control group,the difference was statistically significant(P<0.01).With the increase of paraquat exposure concentration or time,the expression level of ICAM-1 gene and protein in human umbilical vein endothelial cells increased gradually,and the expression level of surface adhesion molecule marker CD11 a protein in THP-1 cells gradually increased.The difference was statistically significant compared with the control group(P<0.01).2.Paraquat induced the increase of total reactive oxygen species in human umbilical vein endothelial cells: with the increase of paraquat exposure concentration,the fluorescence intensity of human umbilical vein endothelial cells added with DCFH-DA fluorescent dye increased gradually,indicating that the total reactive oxygen species in the cells increased.3.Paraquat induced activation of MAPK/NF-?B signaling pathway: compared with the control group,the phosphorylation level of MAPK/NF-?B signaling pathway protein P38,ERK,JNK,P65 in human umbilical vein endothelial cells exposed to paraquat was higher than that of the control group.The difference was statistically significant(P<0.01).4.NAC can reduce the number of THP-1 cells and the expression of cell surface adhesion molecules induced by paraquat-induced adhesion to human umbilical vein endothelial cells: compared with the paraquat-treated group,the number of fluorescently labeled THP-1 cells was significantly reduced in the NAC-treated group,the difference was statistically significant(P<0.01);and the greater the concentration of NAC,thesmaller the number of labeled THP-1 cells.Compared with the paraquat-treated group,the expression of ICAM-1 protein in human umbilical vein endothelial cells was significantly lower than that in the NAC-treated group(P<0.01).5.NAC could decrease the level of total reactive oxygen species in human umbilical vein endothelial cells induced by paraquat: compared with paraquat exposure group,the fluorescence intensity of human umbilical vein endothelial cells in NAC intervention group was significantly lower than that in paraquat exposed group,and the higher the NAC concentration was,the weaker the fluorescence intensity was.6.NAC inhibited the activation of MAPK/NF-?B signaling pathway induced by paraquat: compared with paraquat exposure group,the phosphorylation of MAPK/NF-?B signaling pathway protein P38,ERK,JNK,P65 in human umbilical vein endothelial cells treated with NAC decreased.The difference was statistically significant(P<0.01).7.MAPK/NF-?B signaling pathway inhibitors reduced paraquat-induced monocyte adhesion and increased ICAM1 expression: the number of monocyte adhesion in the MAPK/NF-?B signaling pathway inhibitor group compared with the paraquat-treated group was reduced,the expression level of adhesion molecule ICAM1 was decreased,and the difference was statistically significant(P<0.01).Conclusion 1.Paraquat can activate intracellular ROS production by oxidative stress,increase the expression of ICAM-1 and CD11 a on the surface of human umbilical vein endothelial cells and human peripheral blood mononuclear macrophages,and induce increased adhesion of vascular endothelial cells and mononuclear cells.2.Antioxidant NAC can reduce the expression of ICAM-1 on the surface of human umbilical vein endothelial cells by reducing ROS expression,and finally reduce paraquat-induced adhesion between human umbilical vein endothelial cells and monocytes.3.MAPK/NF-?B signaling pathway plays an important role in PQ-induced adhesion ofhuman umbilical vein endothelial cells to monocytes and ICAM-1 expression. |
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