The Effects Of Blood Vessel Formation Through The VEGF/VEGFR2/MEK1/2/ERK1/2/mTOR Pathway Induced By Cooking Oil Fumes-derived PM_2.5 In HUVECs

Purpose In order to explore the effect of exposure of COFs-derived PM_2.5 on the tube formation in HUVECs.we prepared a model of injury HUVECs blood vessels within exposure of COFs-derived PM_2.5,and observe the influence of tube formation induced by COFs-derived PM_2.5;and explore related molecular signal mechanism of the effect of COFs-derived PM_2.5 on the tube formation in HUVECs.Further study related molecular and cellular mechanisms of COFs-derived PM_2.5 in adverse pregnancy outcomes through injury of umbilical cord blood vessels,to provide more convincing evidence for the safety evaluation and risk assessment of the adverse pregnancy outcome caused by COFs-derived PM_2.5,and to provide potential preventive strategies and research ideas for further prevention and treatment of COFs-derived PM_2.5exposure leading to adverse pregnancy outcomes.Methods1 According to previous research methods,PM_2.5 sampling instrument was used to collect PM_2.5 from COFs,desiccant method was used to measure the quality of PM_2.5,and then dissolved with DMSO,and diluted concentration of COFs-derived PM_2.5 to100mg/m L stock solution;2 100mg/m L stock solution of COFs-derived PM_2.5 was diluted to different concentration dyeing poison with cell culture solution which was freshly prepared,HUVECs were treated with different concentrations of COFs-derived PM_2.5（0,12.5,25,50,75,100,200μg/m L）for 12,24,36 hours.The method of methyl thiazolyl tetrazolium（MTT）was used to test the effect of COFs-derived PM_2.5 on survival rate of HUVECs;3 HUVECs were treated with 0（solvent control,1‰DMSO）and 100μg/m L COFs-derived PM_2.5 for 24 hours,HUVECs morphology change was observed under light microscope;4 HUVECs were treated with different concentrations of COFs-derived PM_2.5（0,25,50,75,100μg/m L）for different times（6,8,11h）.The method of tubule experimental was used to detect the effect of exposure of COFs-derived PM_2.5 on the tube formation in HUVECs.The tubes formation in HUVECs were observed under inverted microscope;5 HUVECs were treated with different concentrations of COFs-derived PM_2.5（0,25,50,75,100μg/m L）for 24 hours.The method of enzyme-linked immune sorbent assay（ELISA）was used to detect the level of VEGF and b FGF;6 HUVECs were treated with different concentrations of COFs-derived PM_2.5（0,25,50,75,100μg/m L）for 24 hours.The method of Reverse Transcription Polymerase Chain Reaction（RT-PCR）was used to test the level of VEGF/MEK1/2/ERK1/2/m TOR m RNA expression;6 HUVECs were treated with different concentrations of COFs-derived PM_2.5（0,25,50,75,100μg/m L）for 24 hours.The method of Western blot was used to test the level of VEGF/VEGFR2/MEK1/2/ERK1/2/m TOR proteins expression.Results1 There was a dose-response and time-response relationship in cell survival rate with the increase of the concentration and time exposure of COFs-derived PM_2.5.When the concentration of COFs-derived PM_2.5 was 50,75,100,200μg/m L,exposure time was24 and 36 hours,the cell survival rate was significantly decreased,and there was statistically significant differences when comparing to the control group（P<0.01）.When the concentration of COFs-derived PM_2.5 was 50,75,100,200μg/m L,exposure time was 12 hours,the cell survival rate was relatively high;When the concentration of COFs-derived PM_2.5 was 50,75,100,200μg/m L,exposure time was 12 hours,the cell survival rate was relatively low;while the concentration of COFs-derived PM_2.5 was100μg/m L,exposure time was 24 hours,cell survival rate was close to 50%.According to the results of MTT test,the experiments virus concentration was determined as（0,25,50,75,100 μg/m L）and the exposure time was was determined as24 hours for follow-up experiments.2 HUVECs were treated with 0（solvent control,1‰ DMSO）and 100μg/m L COFs-derived PM_2.5 for 24 hours,which morphology change was observed under light microscope.Under light microscope（100 ×）,when compared to the control group,exposed to 100 μg/m L of COFs-derived PM_2.5,the number of HUVECs was small,the shape of cell can not see clearly,and field of vision was fuzzy under light microscope.Under light microscope（400 ×）,when compared to the control group,exposed to 100μg/m L of COFs-derived PM_2.5,the number of cells were small,incomplete and out of shape,the boundaries fuzzied,cell body swelling deformation.3 The tube formation of HUVECs at different concentrations,it was not conducive to HUVECs tube formation was cell concentration too high or too low.When HUVECs at concentration was 1 × 105 in well,tube formation is better.The effect of exposure of different concentrations of COFs-derived PM_2.5（0,25,50,75,100μg/m L）for different times（6,8,11h）on the tube formation in HUVECs.There was a dose-response and time-response relationship in HUVECs tube formation with the increase of the concentration and time of COFs-derived PM_2.5 exposure as compared with the control group,while the concentration of COFs-derived PM_2.5 was 100μg/m L,the number of tube formation were significantly decreased（P<0.05）;When exposure time was 6hours,the number of tube formation were most,and there was statistically significant differences when comparing to the control group（P<0.01）.However,the decomposition of tubules rate with the increase of the concentration and time of COFs-derived PM_2.5 exposure was increased.When exposure time was 8 hours,the cell tube decomposition,exposure time was 11 hours,almost all tubular disintegration.The decomposition of cell tubules significantly increased as comparing with the control group（P<0.05）.4 The effects of exposure of different concentration of COFs-derived PM_2.5（0,25,50,75,100μg/m L,24h）on expression of VEGF and b FGF in HUVECs.the level of of VEGF and b FGF were similar.Both the level of VEGF and b FGF were reduced,and there were statistically significant decreased when comparing to the control group（P<0.05）.5 The effects of exposure of different concentration of COFs-derived PM_2.5（0,25,50,75,100μg/m L,24h）on expression of VEGF,MEK1/2,ERK1/2,m TOR m RNA in HUVECs.The VEGF levels in all the exposure groups were significant decreased when compared to the solvent control group（P <0.05）;The expression of MEK1/2,ERK1/2 and m TOR were significant decreased,but only 75μg/m L and100μg/m L exposure group were statistically significant decreased when compared to the control group（P <0.05）.6 The effects of exposure of different concentration of COFs-derived PM_2.5（0,25,50,75,100μg/m L,24h）on expression of VEGF/VEGFR2,MEK1/2,ERK1/2,m TOR total proteins and phosphorylated protein.VEGF/VEGFR2,MEK1/2,ERK1/2,m TOR phosphorylation proteins ratio of total protein had a descending trend with the increase of the concentration of COFs-derived PM_2.5,there was a dose-response relationship and the differences were statistically significant as compared with the control group（P<0.05）.Conclusions Exposure of COFs-derived PM_2.5 within different concentration and could generate obvious cytotoxicity and effect cells tube formation in HUVECs,VEGF/VEGFR2/MEK1/2/ERK1/2/m TOR signal pathway involved in the process of cells tube formation induced by COFs-derived PM_2.5 in HUVECs.and injured the function of umbilical cord blood vessels,might affect the transportation of nutrition and oxygen,which might result in adverse pregnancy outcomes occur.