Impact Of Cooking Oil Fumes-derived PM_2.5 On Blood Vessel Formation Through A ROS-mediated NLRP3 Inflammasome Pathway

Objective To explore the molecular mechanism of oxidative stress in human umbilical vein endothelial cells?HUVECs?induced by cooking oil fumes-derived fine particulate matter(COFs-derived PM_2.5),and to observe the role of ROS mediated NLRP3 inflammasome on blood vessel formation induced by COFs-derived PM_2.5.5 in HUVECs.To provide more convincing evidence for further study of umbilical cord vascular injury triggered by COFs-derived PM_2.5.Methods 1.According to our previous method,the cooking oil fume PM_2.5.5 was collected,desiccant method was used to measure the quality of PM_2.5,and then using dimethyl sulfoxide?DMSO?to dissolved PM_2.5.5 making 100 mg/ml stock solution.2.HUVECs were treated with different concentration?0?g/ml,25?g/ml,50?g/ml,100?g/ml,150?g/ml and 200?g/ml?COFs-derived PM_2.5.5 for 12h,24h and 36 h,and incubated with or without NAC.The effect of COFs-derived PM_2.5.5 on viability of HUVECs was assessed by 3-?4,5-dimethylthiazol-2-yl?-2,5-diphenyl tetrazolium bromide?MTT?assay.3.HUVECs were exposure to different concentration COFs-derived PM_2.5?0?g/ml,25?g/ml,50?g/ml,and 100?g/ml?for different time?12h,24h and 36 h?and incubated with or without NAC.Intracellular ROS level was measured with DCFH-DA assay,and mitochondrial ROS level was measured with MitoSOX?assay after the treatment.4.HUVECs were treated with different concentration COFs-derived PM_2.5.5 for different time and incubated with or without NAC.Proteins involved in NLRP3inflammasomes signaling pathway and VEGF was assessed by western blot.The mRNA involved in NLRP3 inflammasome signaling pathway and VEGF was detected by RT-PCR.5.HUVECs were treatment with different concentration COFs-derived PM_2.5.5 for different time and incubated with or without NAC.The effect of COFs-derived PM_2.5on tube formation in HUVECs were detected by tube formation assay.6.HUVECs were treatment with different concentration COFs-derived PM_2.5.5 for different time and incubated with or without NAC.Then apoptosis of the cells was measured by flow cytometry using annexin V-FITC/propidium iodide?PI?staining.Results 1.There existed dose-response and time-response relationship between COFs-derived PM_2.5.5 exposure and viability of HUVECs.However,the cell viability was effectively rescued by incubated with NAC.2.COFs-derived PM_2.5.5 can significantly increase the expression of intracellular ROS level and mitochondrial ROS level.In addition,mitochondrial ROS fluorescence intensity was decrease with the increase of time,co-treatment with NAC can significantly reduced the intracellular ROS and mitochondrial ROS level.3.Compared with the control group,exposure to COFs-derived PM_2.5.5 could affect the mRNA expression of NLRP3 inflammasome signaling pathway and VEGF in HUVECs.the mRNA level of NLRP3,caspase-1,and IL-1?increased by treated COFs-derived PM_2.5.However,co-treatment by NAC significantly reduced those mRNA level.The mRNA expression of VEGF was decreased after treated by COFs-derived PM_2.5.In addition,co-treatment by NAC could increased the VEGF mRNA level.4.With the increase of the concentration of COFs-derived PM_2.5,the protein expression of NLRP3,caspase-1 and IL-1?were increased.However,the level of NLRP3,caspase-1,and IL-1?significantly reduced after co-treatment with NAC.the protein expression of VEGF was decreased by COFs-derived PM_2.5.5 treated,while co-treatment with NAC can enhance the protein level of VEGF.5.Exposure to COFs-derived PM_2.5.5 could affect the tubes formation of HUVECs in vitro,while the application of NAC can increase the tubes formation of HUVECs.6.Exposure to COFs-derived PM_2.5.5 could induce apoptosis of HUVECs,and the application of NAC can reduce the apoptosis rate.Conclusions COFs-derived PM_2.5.5 could significantly affect the viability of HUVECs,induce excessive ROS which could lead to oxidative stress,induce apoptosis,and affect the angiogenesis of HUVECs cells.The activation of NLRP3 inflammasome play an important role in the damage of HUVECs cells induced by COFs-derived PM_2.5.The damages of HUVECs induce by COFs-derived PM_2.5.5 could be rescued by the co-treatment of NAC.In summary,this study proves the partial mechanisms of COF-derived PM_2.5.5 on damages of human umbilical vein endothelial cells.It is revealed that NLRP3 inflammasome play a key role in the damage of HUVECs induce by COF-derived PM_2.5.