Vitamin D₃ Inhibits Apoptosis Of Human Umbilical Vein Endothelial Cells Induced By Cooking Oil Fume Derived Fine Particulate Matter By Regulating P53/Bax/caspase Signaling Pathway

Objective To establish the cell model of cooking oil fume derived fine particulate matter(COFsPM_2.5)exposure and vitamin D3 intervention,explore the role of vitamin D3 in the apoptosis of human umbilical vein vascular endothelial cells(HUVECs)damage by COFs-PM_2.5 exposure,and to further explore its potential molecular mechanism.So as to provide new evidence for further research on vitamin D3 inhibiting umbilical cord blood vessel damage induced by COFs-PM_2.5.Methods 1.Simulate the kitchen cooking environment,collected COFs-PM_2.5 through PM_2.5 sampler,calculated the quality of COFs-PM_2.5 by weight gain method,and diluted to 100 mg/ml stock solution with dimethyl sulfoxide(DMSO);2.Placed the COFs-PM_2.5 filter membrane in a flask,added appropriate amount of absolute ethanol,oscillated in ultrasonic vibrator for 12 hours and then observed by transmission electron microscope(JEM1400 Japan);3.HUVECs were incubated with different doses of COFs-PM_2.5(0,25,50,100,150,and 200 ?g/m L)and VD3(0,0.1,1,10,100,and 1000 nmol/L)in 96-well culture plate for 12,24 and 36 h.The effects of COFs-PM_2.5 and VD3 on the viability of HUVECs were determined by MTT assay,and finalized the concentration and incubation time of COFs-PM_2.5 and VD3;4.The cells were exposed to different reagents(1‰ DMSO+1‰ absolute ethylalcohol,1000nmol/l VD3,100?g/ml PM_2.5,or 1000nmol/l VD3+100?g/ml PM_2.5)for 24 h,respectively.The proteins involved in the p53/Bax/caspase signaling pathway were measured with western blot,while RTPCR was used to assess the expression of m RNA;5.The cells were exposed to different reagents(0,1000nmol/l VD3,100?g/ml PM_2.5,or 1000nmol/l VD3+100?g/ml PM_2.5)for 24 h,flow cytometry was used to detect the effects of different treatments on the apoptosis level of HUVECs.Results 1.The COFs-PM_2.5 observed by transmission electron microscope,and the result shown that the rang of COFs-PM_2.5 from 200 nm to 2.5?m,and most of the shapes are irregular.2.The results of MTT show that with the increase of concentration and time of COFsPM_2.5,the viability of HUVECs gradually decreases in dose-and time-dependent manners;while the viability of HUVECs significant increase after the co-incubation of VD3,and with the increase of VD3 concentration,cells viability also increased significantly.3.Compared with the control group,both western blot and RT-PCR showed that the expression level of p53 and Bax in HUVECs increased significantly in COFs-PM_2.5 exposure group,while the level of bcl-2 decreased.which could be effectively rescued by the co-incubation of VD3.4.Compared with the control group,the proteins expression level of caspase-3,7,9 decreased significantly in COFs-PM_2.5 exposure group,while the level of cleavedcaspase-3,7,9 increased,and the level of cleaved/caspase-3,7,9 increased,while co-incubation with VD3 rescued these adverse effects.In addition,the result of RTPCR shown that the m RNA level of caspase-3,7,9 increased after exposed to COFs-PM_2.5,while co-incubated HUVECs with VD3 evidently up-regulated the m RNA level of caspase-3,7,9.5.Exposed of COFs-PM_2.5 could significantly promote the apoptosis of HUVECs,while co-incubated with VD3 could effectively reduce the apoptosis of HUVECs.Conclusions This study proved that COFs-PM_2.5 could promote the apoptosis of HUVECs,while VD3 could inhibit the effect of COFs-PM_2.5 on cells apoptosis,and expounded its potential molecular mechanism,revealed the role of p53/Bax/caspase signal pathway in VD3 inhibited COFs-PM_2.5-induced apoptosis.Provided new evidence for the study of VD3 inhibited COFs-PM_2.5 on umbilical cord vascular damage,and further expanded the clinical application of VD3.