The Effects Of Wnt/β-catenin Signaling Pathway On Human Embryonic Lung Fibroblast Poisoned By Arsenite

Objective: Investigate the effects of Wnt/?-catenin signaling pathway on Human embryonic lung fibroblast(HELF) poisoned by arsenite(NaAsO2) to provide the basis for further elaboration of arsenic inducing lung injury. Methods: 1. Use the MTT to determine the LC50, exposure dose and inhibition rate. 2. Observe the cell microstructure changes by using Fluorescent Inverted Microscope and Transmission Electron Microscopy. 3. The activity of superoxide dismutase(SOD) and reducing glutathione peroxidase(GSH-PX) were detected by Hydroxylamine and Colorimetric Method, and the content of Malondialdehyde(MDA) was detected by TBA. 4. HELF cells treated by 30 umol/L NaAsO2 were transfected with ?-catenin siRNA. 5. Use7-ADD/Annexin V to detect apoptosis before and after transfected. 6. Expression of?-catenin, C-myc, Cyclin Dl, Axin and Wnt5 a mRNA were detected by real-time fluorescence quantitative(Real-Time Quantitative PCR), so as the ?-catenin and Wnt5 a mRNA after ?-catenin siRNA transfected. 7. Protein expression of ?-catenin, C-myc,Cyclin Dl, Axin and Wnt5 a were detected by western blot before and after transfected.Results: 1. The LC50 of HELF cells infected by NaAsO2 were 135.04 μmol/L(24 h), so NaAs O2 of 10.00, 20.00, 30.00 μmol/L were the experimental groups and NaAsO2 of 0.00μmol/L was the control group. 2. NaAsO2 induce HELF cell microstructure damages. 3.After 24, 48, 72 hours of poisoned with NaAs O2 at the three exposure doses, cell inhibition rates increased and positively correlated with NaAsO2 doses(r=0.93, P<0.05).4. SOD and GSH-PX in all experimental groups decreased(P<0.05), but MDA increased(P<0.05). 5. Cell early apoptosis rate increased(P<0.05). 6. Expression levels of?-catenin, C-myc, Cyclin D1 mRNA were decreased(P<0.05), but Axin and Wnt5 a mRNA were increased(P<0.05) after NaAs O2 exposuretion. 7. Protein expression of?-catenin, C-myc, Cyclin D1 were decreased(P<0.05), but Axin and Wnt5 a were increased(P<0.05) after NaAsO2 exposuretion. 8. The apoptosis rate of HELF treated by30 umol/L NaAs O2 transfected with ?-catenin siRNA was higher than no transfected(P<0.05). 9. There were no protein expression of ?-catenin, C-myc, CyclinD1, and Wnt5 a protein expression was increased(P<0.05), but Axin had no change(P>0.05)after transfected. 10. There was no ?-catenin mRNA expression, but Wnt5 a m RNA expression was increased(P<0.05) after transfected. Conclusion: 1. NaAs O2 induce microstructure damage of HELF cells. 2. NaAs O2 induce apoptosis, oxidative stress, and inhibit cell increased of HELF. 3. NaAs O2 induce Wnt negative regulation factors Axin and Wnt5 a expression increased, but ?-catenin and its target gene C-myc and CyclinD1 expression decreased on HELF cell, instructing that arsenic inhibitting Wnt/ ?-catenin signaling pathway is one of the molecular mechanism to induce cell proliferation suppression, apoptosis increases, and oxidative stress. 4. After ?-catenin siRNA transfection, apoptosis rate of HELF is higher than before, expression of ?-catenin,C-myc, CyclinD1 are inhibited, but the expression of Wnt5 a is increased. Further illustrate that arsenic inhibitting Wnt/ ?-catenin signaling pathway may be one of the important mechanisms of arsenic to HELF cells damage.