The Role Of ROS Mediated PI3K/AKT/mTOR Pathway In Autophagy Induced By Cooking Oil Fumes-derived PM_2.5 In HUVECs

Purpose In order to explore the effects of exposure of cooking oil fumes-derived fine particulate matter(PM_2.5) on oxidative damage and autophagy in human umbilical vein endothelial cells（HUVECs）, and observe related molecular mechanism of autophagy induced by PM_2.5 of cooking oil fumes（COFs） in HUVECs, to preliminarily study related molecular and cellular mechanisms of COFs-derived PM_2.5 in adverse pregnancy outcomes through injury of umbilical cord blood vessels, and further provide new ideas and new targets for prevention and treatment of adverse pregnancy outcomes induced by PM_2.5. Methods 1 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, which was dissolved with DMSO, and adjust concentration of COFs-derived PM_2.5 to 100mg/m L stock solution. 2 100mg/m L stock solution of COFs-derived PM_2.5 was diluted to different virus concentration with cell culture fluid which was freshly prepared, to deal with HUVECs 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; and the method of DCFH-DA staining was undertaked to detected the level of active oxygen（ROS）. 3 HUVECs were treated with 0（solvent control, 1‰DMSO）, 100μg/m L PM_2.5、100μg/m L PM_2.5+10mmol/L NAC, serum-free 50 n M Rapamycin for 24 hours, autophagosome was observed under transmission electron microscope（TEM）, distributed situation of autophagy related proteins in HUVECs was detected by the method of cell immunofluorescence. 4 After HUVECs were treated with different concentrations of COFs-derived PM_2.5 at different times, the method of western blot was used to test autophagy related proteins expression level of LC3, Beclin1 and proteins expression level of PI3K、AKT、m TOR. Results 1 There was a dose-response and time-response relationship in cell survival rate with the increase of the concentration and time of COFs-derived PM_2.5 exposure. Only when the concentration of COFs-derived PM_2.5 was 50、100、200μg/m L, the cell survival rate was significantly decreased, and there was a significant statistical differences（P<0.01）, as compared with the control group. After cells were treated with COFs-derived PM_2.5 for 36 hours and the concentration reached to 200μg/m L, cell survival rate was very low, while the concentration was 100μg/m L, cell survival rate was close to 50%. According to the results of MTT test, follow-up experiments virus concentration was determined as 0、25、50、100μg/m L, and the exposure time was was determined as 24 hours for its convenience to experiment operations. 2 Under fluorescence microscope, cell green fluorescence of 100μg/ml PM_2.5 exposed group in 12 hours was the strongest than all other exposed group, and NAC can inhibit the production of green fluorescenced. Through quantitative analysis, ROS fluorescence intensity had a strengthening trend with the increase of the concentration, and presented a certain dose-response relationship; while ROS fluorescence intensity had a decreasing trend with the increase of time. Compared with the control group, ROS fluorescence intensity had no significant change with the increase of the concentration for 36 hours. 3 Under transmission electron microscope（TEM）, autophagosomes with double membrane structure containing the cell contents could be observed in 100μg/m L PM_2.5 and Rapamycin group, and these membranous autophagosome had a phenomenon of accumulation, the structure of mitochondria was dim and cracked; when NAC and 100 μg/m L PM_2.5 were treated toghter, the number of autophagosome decreased significantly and had no accumulation, mitochondria structure could be clearly seen, as compared with only PM_2.5 exposure group. Under laser scanning confocal microscope, compared with the control group, proteins expression level of LC3, Beclin1 were increased in Rapamycin and PM_2.5 exposure group, LC3 distributed around the nucleus in cytoplasm uniformly, and gathered into a green punctiform, and Beclin1 piled up in the nucleus of peripheral and arranged as a tubular; When NAC and 100 μg/m L PM_2.5 were treated toghter, the two protein expression levels decreased significantly as compared with only PM_2.5 exposure group. 4 As compared with the control group, COFs-derived PM_2.5 increased the ratio of LC3II/LC3 I and the proteins expression level of LC3 II, Beclin1, and there was a dose-response and time-response relationship with the increase of the concentration and time of COFs-derived PM_2.5 exposure, while only in 100μg/m L exposure group and exposure for 36 h, compared with the control group, the differences were statistically significant（P<0.05）. PI3 K, AKT, 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（P<0.05） as compared with the control group. When NAC and 100μg/m L PM_2.5 were treated toghter, PI3 K, AKT, m TOR phosphorylation proteins ratio of total protein was increased significantly as compared with only PM_2.5 exposure group（P<0.05）. Conclusions COFs-derived PM_2.5 could produce obvious cytotoxicity and oxidative damage in HUVECs, and it could induce HUVECs autophagy. ROS mediated PI3K/AKT/m TOR signal pathway involved in the process of autophagy induced by COFs-derived PM_2.5, while NAC could play a protective role in the process of autophagy, therefore, we speculated that NAC may prevent the serious consequences induced by the damage of umbilical cord blood vessels. The next step we will explore the damage effect of cell apoptosis in endothelial cells and study the relationship between autophagy and apoptosis, further investigate the related molecular mechanism of adverse pregnancy outcomes occur in cell level.