PM_2.5 Induces Cardiac Malformations Via PI3K/akt2/mTORC1 Signaling Pathway In Zebrafish Larvae

Background and Objective:Recent evidence suggests that maternal exposure to ambient fine particulate matter(PM2.5)is closely associated with congenital heart disease in offspring,but the molecular mechanisms remain unclear.We previouly found that the aryl hydrocarbon receptor(AHR)mediates PM2.5-induced apoptosis and Wnt pathway inhibition,leading to heart malformations.Due to the key roles of the PI3K/Akt/mTORC1 signaling pathway in regulating apoptosis and Wnt signaling pathway,we hypothesized that PM2.5 activates AHR and causes apoptosis and Wnt signaling inhibition via PI3K/Akt/mTORCl signal pathway,resulting in heart defects.This study aimed to investigate the role of PI3K/Akt/mTORC1 signaling pathway in PM2.5-induced cardiac defects in zebrafish embryos.Methods:PM2.5 was collected in Suzhou city and extractable organic matter(EOM)was purified by using Soxhlet extraction method.Zebrafish embryos were exposed to EOM from 2 h post fertilization(hpf),and small molecule inhibitors or activators were added based on experimental design,including BAY11-7082(BAY,NF-κB inhibitor,CAS,19542-67-7);SB203580(SB,p38 MAPK inhibitor,CAS 152121-47-6);MK2206(MK,akt1/2/3 inhibitor,CAS 1032350-13-2);LY294002(LY,PI3K inhibitor,CAS 154447-36-6);akt2-specific inhibitor CCT128930(CCT),AKT agonist SC79,mTOR inhibitor Rapamycin(RAPA)and reactive oxygen species(ROS)scavenger N-acetyl-L-cysteine(NAC).AHR was knocked down by microinjecting morpholino oligonucleotides(MO).Embryos at 72 hpf were observed under a microscope to assess cardiac malformation rate and survival rate.ROS levels and apoptosis in the heart area were detected by dichlorodihydrofluorescein diacetate(DCFH-DA)and acridine orange(AO)staining,respectively.Mitochondrial ROS and membrane potential levels in the heart of zebrafish embryos were detected by MitoSOXTM Red Mitochondrial Superoxide Indicator and MitoTrackerTMRed CMXRos,respectively.Hearts were isolated from zebrafish embryos under a dissecting microscopy.Total RNA was extractedfrom isolated hearts and qPCR was conducted to detect relevant mRNA expression levels.Immunofluorescence was used to detect the level of p-Akt,p-mTOR,β-catenin,Cleaved-Caspase3 and Cleaved-Caspase9.PTEN activity was detected by using a tissue PTEN activity quantification kit.Results:(1)Base on the effects of different inhibitors,we found that inhibition of PI3K/Akt signal pathway protected against EOM-induced cardiac defects in zebrafish emryos.Akt has three isoforms,aktl,akt2,and akt3,of which akt2 is critical for zebrafish survival,growth,and development.We further found that akt2 is involved in EOM-induced cardiac malformations by using genetic knockdown and specific pharmacological inhibitor CCT.EOM also enhanced akt phosphorylation in the heart of zebrafish embryos,which were counteracted by CCT supplementation,indicating that akt2 activation mediates EOMinduced heart defects.(2)Akt2 inhibitor CCT attenuated EOM-induced intracellular ROS overproduction in the heart of zebrafish embryos.We further found that EOM enhanced mtROS production and decreased MMP levels,which were counteracted by supplementation with CCT.Using MO mediated genetic knockdown,we confirmed that akt2 activation mediates EOMinduced mitochondria-mediated intrinsic apoptosis.In addition,EOM exposure decreased the levels of active β-catenin(non-phosphorylation),which were attenuated by addition of akt2 inhibitor CCT.EOM significantly downregulated the expression levels of Wnt signaling target genes axin2,nkx2.5 and sox9b,and addition of CCT attenuated the EOM-induced downregulation of these genes.The essential role of akt2 in EOM-decreased active β-catenin was confirmed by genetic knockdown.(3)We found that EOM significantly increased mTORC1 phosphorylation levels in the heart of zebrafish larvae,which were attenuated by supplementation with pharmaceutical inhibitor CCT or knockdown of akt2,supporting the fact that mTORC1 is activated by akt2 in the heart of zebrafish embryos exposed to EOM.We further found that EOM induced intracellular ROS overproduction and decreased PTEN activity,which were counteracted by the addition of either CH or NAC,suggesting that EOM-induced akt2 activation is mediated through aryl hydrocarbon receptor(AHR)/ROS-induced of PTEN inhibition.Conclusion:Our results demonstrate that EOM from PM2.5 activates PI3K/akt2/mTORC1 signaling via AHR/ROS-mediated PTEN downregulation,leading to mitochondria-mediated intrinsic apoptosis and heart malformations;PI3K/akt2/mTORC1 signaling activation also contributes to the EOM-induced heart defects by suppressing the canonical Wnt signaling pathway.Our findings suggest that the PI3K/akt2/mTORC1 signaling pathway can serve as a potential therapeutic target for the prevention and treatment of air pollution-related CHDs.