1,25（OH）₂D₃ Alleviates The Apoptosis Of HUVECs Induced By COFs-PM_2.5 Through MiR-29b-3p/SIRT1/ROS Pathway

ObjectiveAs the main ingredient of indoor air pollution,cooking oil fumes（COFs）are more vulnerable to pregnant women because of their longer indoor activity time and could lead to adverse birth outcomes.Appropriate levels of vitamin D are thought to be beneficial to maternal and infant health,but it is still unclear whether vitamin D supplementation can prevent adverse pregnancy outcomes and umbilical cord vascular injury induced by cooking oil fume derived PM_2.5(COFs-PM_2.5).In this study,the apoptosis model of human umbilical vein endothelial cells（HUVECs）induced by COFs-PM_2.5 and the intervention model of 1,25（OH）₂D₃ were constructed to observe the effects of 1,25（OH）₂D₃ intervention on the apoptosis level of HUVECs induced by COFs-PM_2.5.Meanwhile,the mechanism of miRNA was explored.Materials and methods1.COFs-PM_2.5 was collected and diluted to 100 mg/ml with DMSO.Calcitriol powder was dissolved in anhydrous ethanol and diluted to 1 mmol/L to prepare 1,25（OH）₂D₃ stock solution.2.HUVECs were incubated with different concentrations of COFs-PM_2.5（0,25,50,100,200,400μg/ml）or 1,25（OH）₂D₃（0,0.1,1,10,100,1000 n M）,then MTT assay was used to detect the changes of cell activity and finally to ensure the treatment concentrations of COFs-PM_2.5 and 1,25（OH）₂D₃ in subsequent trials.3.HUVECs were treated with different reagents(0,100μg/ml COFs-PM2.5,100μg/ml COFs-PM_2.5+1 n M 1,25（OH）₂D₃和single 1 n M 1,25（OH）₂D₃), respectively.After 12 h,the cell apoptosis level was detected with flow cytometry, the ROS level was detected with ROS detection assay kit,and the changes of the signal pathway related-factors were measured with Western blot and RT-PCR.4.The cells were transfected with Lipofectamine 2000 to overespress miR-29b-3p.The targeted relationship of miR-29b-3p and SIRT1 was confirmed with dual-luciferase reporter assay using HEK-293T cells.5.After transfected with miR-29b-3p mimics,HUVECs were treated with COFs-PM_2.5 and 1,25（OH）₂D₃.Western blot and q RT-PCR was used to detect the effect of miR-29b-3p overexpression on the expression of miR-29b-3p and signal pathway related factors.Flow cytometry and ROS detection assay kit was used to measure the effect of miR-29b-3p overexpression on the level of apoptosis and ROS,respectively.Results1.The results of MTT assay reported that the viability of HUVECs gradually decreased with the increased concentrations of COFs-PM_2.5.The treatment of 1,25（OH）₂D₃ could significantly increase the cell viability at low doses.Compared with single COFs-PM_2.5 exposure group,1,25（OH）₂D₃ co-incubation could also increase the cell viability in a dose-response manner at low doses.2.Compared with the control group,COFs-PM_2.5exposure significantly increased the level of miR-29b-3p and ROS,and decreased the protein expression of SIRT1.The co-incubation of 1,25（OH）₂D₃significantly inhibited the changes of miR-29b-3p/SIRT1/ROS signal pathway induced by COFs-PM_2.5.3.Compared with the control group,COFs-PM_2.5exposure significantly promoted the apoptosis of HUVECs and increased the expression of apoptosis-related factors both Bax and the ratio of cleaved-/caspase 3,and decreased the expression of bcl-2.The co-incubation of 1,25（OH）₂D₃ significantly reduced the apoptosis level and improved the expression of apoptosis-related proteins.4.According to the prediction of miRNA online database and the result of dual-luciferase reporter assay,SIRT1 was the potential target of miR-29b-3p.5.Compared with the transfected miR-NC group,the overexpression of miR-29b-3p down-regulated the expression of SIRT1 protein and up-regulated the ROS level,and then further promoted the apoptosis of HUVECs induced by COFs-PM_2.5.The overexpression of miR-29b-3p also reversed the improvement effects of 1,25（OH）₂D₃on the injury of COFs-PM_2.5.Conclusion1,25（OH）₂D₃could alleviate the apoptosis of HUVECs induced by COFs-PM_2.5 and miR-29b-3p/SIRT1/ROS signal pathway might be involved in the process.The overexpression of miR-29b-3p could aggravate the apoptosis level induced by COFs-PM_2.5 by targeting SIRT1 and reverse the anti-apoptosis effect of 1,25（OH）₂D₃.This study could provide biological evidence in vitro for vitamin D supplement rescuing the umbilical cord injury induced by COFs-PM_2.5 exposure.