Effect And Mechanism Of PM2.5 Exposure In Disruption Of Cardiac Differentiation

Objective:To investigate the effect and mechanism of PM_2.5exposure on embryo cardiac differentiation.Methods:1.MTT assay to examine the cytoplasmic toxicity of PM_2.5The cells were exposed to PM_2.5at different concentrations of 0 mg/L、1mg/L、10 mg/L、100 mg/L,and the non-cytotoxic concentration was determined by MTT assay.2.The P19 mouse carcinoma stem cells were used as an in vitro cardiac differentiation model The P19 mouse carcinoma stem cells were cultured for 14 days to mimic different stages of embryonic heart development-including undifferentiated period,pan mesoderm and cardiac mesoderm period,cardiac progenitor period,and cardiac cell period.3.Measurement of the P19 cardiac differentiation interruption The undifferentiated P19 cells were exposed to PM_2.5at the non-cytotoxic concentration of 10 mg/L for 2 days.The morphology changes were recorded and the expression of cardiac specific gene was evaluated by PCR.Cardiac functional protein cTNT（cardiac troponin T）was examined by immunofluorescence and flow cytometry.4.Detection of DNA damage and apoptosis and analysis of AHR and WNT signaling pathway The undifferentiated P19 cells were exposed to PM_2.5at the non-cytotoxic concentration of 10 mg/L for 2 days.γ-H2 a.X was examined by immunofluorescence,and apoptosis was detected by flow cytometry with Annexin V FITC/PI Apoptosis and Cell Cycle Kit.AHR andβ-catenin distribution was examined by immunofluorescence.Results:1.Cytotoxicity of PM_2.5.There was no significant difference in cell viability when cells were exposed to PM_2.5below the concentration of 10 mg/L.High concentration of PM_2.5（100 mg/L）evidently affect the cell viability.10 mg/L PM_2.5was determined as the non-cytotoxic concentration.2.The P19 mouse carcinoma stem cell cardiac differentiation model was established successfully.Undifferentiated P19 cells showed clonal growth,embryoid bodies in suspension was tight and the edge was neat,cardiomyocytes limbed rapidly out of embryoid bodies growing adherently.3.Effect of PM_2.5exposure on cardiac differentiation.3.1 Alteration in cell morphology.The undifferentiated P19 cells showed alterations in clonal growth,embryoid bodies and cellular edges.3.2 Alteration in cardiac specific gene expression.PM_2.5.5 exposure decreased the expression of OCT4,ISL1 and MLC2 V genes.3.3 Exposure of PM_2.5.5 decreased the expression of cardiac functional protein cTNT（cardiac troponin T）.4.Alteration in cell biology.4.1 DNA damage,cell apoptosis,cell proliferation,and cell cycle distribution.PM_2.5exposure resulted in DNA damage,depressed cell proliferation and cell cycle arrest in S-phase.4.2 condition and distribution of AHR signaling and WNT signaling.PM_2.5exposure activated AHR signaling and inhibited WNT signaling.4.3 Role of AHR signaling and WNT signaling in PM_2.5effects on P19 cardiac differentiation.Cell-proliferation-inhibition and cell-cycle-arrest in S-period can be attenuated by AHR inhibitor CH2,but not so efficiently by WNT activator.Conclusion:1.PM_2.5exposure may disturb cardiac differentiation.2.Mechanisms underlying inhibited cardiac differentiation by PM_2.5exposure include DNA damage,cell cycle arrest,inhibition of cell proliferation and activated AHR signaling.