Protective Effects Of VD₃ On Tube Formation Of Huvecs In Vitro By Inhibiting Activation Of ROS/NLRP3/VEGF Signaling Pathway Induced By Cooking Oil Fume Derived PM_2.5

Objective To observe the roles of VD₃ in the tube formation of HUVECs damaged by COFs-derived PM_2.5 in vitro and explore the molecular mechanism of VD₃ on COFs-derived PM_2.5 induced umbilical cord injury.To provide further evidence for VD₃ inhibition of COFs-derived PM_2.5 on umbilical cord vascular injury,and expand the clinical application range of VD₃.Methods 1.The samples of COFs-derived PM_2.5 were collected according to the procedures described in our group’s previous studies.The PM_2.5 was diluted by dimethyl sulfoxide（DMSO）to 100 mg/m L.2.HUVECs were incubated with different concentratons of COFs-derived PM_2.5（0,25μg/m L,50μg/m L,100μg/m L,150μg/m L,and 200 μg/m L）for 12 h,24h and 36 h,respectively.The effects of COFs-derived PM_2.5 on HUVECs viability were determined by MTT assay.3.HUVECs were exposed to 0,100μg/m L PM_2.5,1000nmol/L VD₃ + 100μg/m L PM_2.5 and 1000nmol/L VD₃ for 24 h,respectively.The effects of PM_2.5 on tube formation of HUVECs in vitro were detected by tube formation assay,and the cell mitochondrial membrane potential was detected by JC-1 staining.4.After HUVECs were exposed to 0,100μg/m L PM_2.5,1000nmol/L VD₃ + 100μg/m L PM_2.5 and 1000nmol/L VD₃ for different time（12h,24 h and 36 h）,DCFH-DA assay and Mito SOX? assay were used to assess the intracellular ROS and mitochondrial ROS level.5.HUVECs were incubated with 0,100μg/m L PM_2.5,1000nmol/L VD₃ + 100μg/m L PM_2.5 and 1000nmol/L VD₃ for 24 h,respectively.The expression of IL-1β,IL-18 and VEGF proteins were detected by ELISA assay.The levels of proteins and m RNA involved in NLRP3 inflammasome signaling pathway and VEGF were measured by western blot and RT-PCR.Results 1.COFs-derived PM_2.5 exposure could reduce HUVECs viability in dose-and time-dependent manners.2.The cell viability decreased gradually with the increase of COF-derived PM_2.5 concentration and exposure time.3.The levels of intracellular and mitochondrial ROS in COFs-derived PM_2.5 exposure group were significantly increased.However,when VD₃ was incubated with COFs-derived PM_2.5,the levels of intracellular and mitochondrial ROS could be effectively rescued.4.In COFs-derived PM_2.5 exposure group,the red fluorescence significantly reduced and the green fluorescence significantly increased.However,when VD₃ was incubated with COFs-derived PM_2.5,the red fluorescence effectively increased and the green fluorescence significantly decreased.5.Compared with the control group,the expressions of NLRP3、caspase-1、IL-18 and IL-1β m RNA and protein in COFs-derived PM_2.5 exposure group significantly increased,While treatment of PM_2.5 decreased the m RNA and protein expression of VEGF.However,co-incubation with VD₃ effectively reduced the m RNA and protein levels of NLRP3、caspase-1、IL-18 and IL-1β,effectively increased the m RNA and protein levels of VEGF.Conclusions This study demonstrated that VD₃ could rescue HUVECs damage induced by COFs-derived PM_2.5 and reveal some related mechanisms,suggesting that ROS/NLRP3/VEGF signaling pathway plays an important role.It provides the basis for VD₃ to interfere with adverse pregnancy outcome and widens the clinical application range of VD₃.