Study On The Mechanisms Of Nicotine Affecting The Function Of Placental Trophoblast Cells And The Development Of Heart

Background and ObjectiveNicotine exposure during pregnancy does great harm to the growth and development of embryos,including the heart development,but the development depends on a stable placenta-heart axis.Abnormal placenta would lead to poor cardiac development outcomes,and the motility of trophoblast is indispensable for the placental development.Nicotinic acetylcholine receptors(n ACh Rs)may play a key role in these processes,but the effects of nicotine on n ACh Rs are not clear.This study attempts to explore the effect of nicotine on cardiac development and its mechanism from the perspective of placental function,and to reveal the effect of nicotine on the expression of n ACh Rs.MethodsNicotine was used to treat the human embryonic stem cell line H9,induced pluripotent stem cell(IPSC),human umbilical cord mesenchymal stem cell(UCMSC),human bone marrow mesenchymal stem cell(BMMSC),cardiac progenitor cell which induced by H9,trophoblast cell(HTR-8/SVneo)and pregnant C57BL/6J mice.The RNA level of related genes was detected by PCR,and the protein expression level was detected by Western blot.The n ACh Rs were localized by cellular immunofluorescence,and the protein phosphorylation levels were detected by protein phospho-array assays.And RNA sequencing was used to detect the changes of transcription level in trophoblast cells and mice fetal heart after nicotine treatment.The effects of nicotine on trophoblast cells were studied by transwell cell-invasion assay and cell-viability assay.ResultsDuring the differentiation of H9 into cardiomyocyte,the levels of Gata4,Mef2 c and c Tn I in nicotine group decreased.The detection of phospho-array assays showed that the phosphorylation level of CD4,ATP1A1,BLNK,Trk B,MKK4/SEK1,CDK5 and Ezrin increased while the phosphorylation level of PTEN,Tau,PKD1/PKCμ,IKK-α,HistoneH2 A.X decreased.There was stillbirth in the nicotine group of mice.And the RNA-sequencing of the fetal heart at the embryonic day 12.5(E12.5)showed that there were5563 differential genes,which mainly enriched in 8 pathways,including the pathway of oxidative phosphorylation,and 7 key genes were screened,including the Atg7,Keap1,Lnx,Ube2 a,skp1a,Trim63 and Nedd4 l.HTR-8/SVneo expressed α3,α7,α9 and β1 subunit of n ACh Rs.Nicotine down-regulated CXCL12 and inhibited trophoblast invasion.Methyllycaconitine,as an antagonist of the α7 homopolymer,blocked the inhibitory effect of nicotine.CXCL12 could rescue the nicotine-induced inhibitory effect on invasion of HTR-8/SVneo cells.The α2,α3,α4,α7,α9 and β1 subunits of n ACh Rs were detected in H9 and IPSC,but BMMSC expressed α4,α7,α9 and β1,while UCMSC expressed α2,α3,α4,α7,α9 and β1.After nicotine treatment,there was no significant change on α7 in these stem cells,and α2 was down-regulated in H9 and UCMSC,but up-regulated in IPSC,and β2 rised in BMMSC.ConclusionNicotine,interfering with the differentiation of cardiomyocytes and regulating the expression of some genes in cardiac development,can affect pathways or key genes common to heart and placenta,which means that placenta-heart axis is influenced.Nicotine can down-regulate the expression of CXCL12 through α7 receptor to inhibit trophoblast invasion,thus affecting placental function,which may be an intermediate process of nicotine affecting heart development.n ACh Rs express in stem cells differently and play an important role in growth and development.Most of the n ACh Rs are regulated by nicotine,while α7 is the most stable.