The Research Of CeO₂ Nanoparticles Effect On Alveolar Macrophages NR8383 And Alveolar Epithelial Cells RPAEpiC In Rat

Objective In this experiment,rat alveolar macrophages NR8383 and rat alveolar epithelial cells RPAEpi C were chosen as the research objects.To study the effect of nano cerium oxide on damage induced by hydrogen peroxide on alveolar macrophages cells and rat alveolar epithelial cells RPAEpi C,to investigate whether it can alleviate the damage caused by hydrogen peroxide.At the cellular level in vitro to investigated the biological effects and toxic effects of nano-cerium oxide on organisms.Method 20 nm nano cerium oxide of different final concentrations（5,10,20,50,100μg/m L）were applied to NR8383 cells and RPAEpi C cells for 24 hours,then 100μmol/L H₂O₂applied to cells for 2 h.Using CCK-8 method to detect the survival rate of NR8383 cells,the release of lactate dehydrogenase（LDH）in the culture supernatant,the contents of glutathione（GSH）and superoxide dismutase（SOD）were assayed by Enzyme-labeled method.Fluorescence microscopy was used to observe the changes of reactive oxygen species（ROS）.Western-blot was used to detect the expression levels of Caspase-3,GRP78 and Nrf-2 in NR8383 and RPAEpi C cells.Results（1）NR8383 cells:Compared with hydrogen peroxide group,the viability of NR8383 cells was higher in 5μg/m L nano cerium,while in 100μg/m L nano cerium oxide+hydrogen peroxide group,the survival rate of NR8383 cells was lower,the differences were statistically significant（P<0.05,P<0.01）;in the 50μg/m L nano cerium oxide+hydrogen peroxide group,the LDH release of NR8383 cells was significantly higher than that in control group（P<0.05）.With the increase of concentration of nano-cerium oxide,the amount of LDH released was fluctuation.The SOD and GSH in NR8383 cells fluctuated,and the level of ROS increased.Expression of Caspase-3,GRP78 and Nrf-2 proteins in NR8383 cells:After rat alveolar macrophages were treated with nano-cerium oxide for 24 h,compared with the control group,the expression of Caspase-3 protein was decreased under different concentrations and had a dose effect;The expression of GRP78 protein was not obvious at the concentration of 5,10,20 and 50μg/m L,the expression of GRP78 protein was significantly decreased at 100μg/m L（P<0.05）.At the concentration of 10μg/m L,the expression of Nrf-2 protein increased,at 100μg/m L,the expression of Nrf-2 decreased significantly（P<0.05）.After incubation with hydrogen peroxide,the expression of Caspase-3 was increased,at 5,10μg/m L,the expression of Caspase-3 was significantly decreased（P<0.05）,at 20,50 and 100μg/m L,the expression of protein was significantly decreased（P<0.01）.The expression of GRP78 protein was increased,the expression of Nrf-2 protein was increased at 5μg/m L,but there was no significant difference（P>0.05）.（2）RPAEpi C cells:Compared with hydrogen peroxide group,in 5,10μg/m L nano cerium oxide+hydrogen peroxide group,cell survival was higher,with the increase of cerium oxide concentration,the survival rate of cells decreased gradually,and the dose effect was not significant（P>0.05）.In the 50μg/m L nano cerium oxide+hydrogen peroxide group,LDH release rate was higher,the differences were statistically significant（P<0.05）.The content of SOD in the cells was higher than the hydrogen peroxide group.The intracellular GSH level fluctuated with the increase of the concentration of cerium oxide and the changes were not obvious.Hydrogen peroxide caused damage to cells,green fluorescence enhancement,in 50,100μg/m L nano cerium oxide+hydrogen peroxide group,the fluorescence intensity of cells increased.Expression of Casepase-3,GRP78 and Nrf-2 proteins in RPAEpi C cells:After rat alveolar Epithelial were treated with nano-cerium oxide for 24 h,compared with the control group,when the concentration of cerium oxide was 5,10,20μg/m L,the expression of Caspase-3decreased gradually,at 20μg/m L,there was a significant difference（P<0.05）;At each concentration,the expression of GRP78 protein increased gradually;the expression of Nrf-2 protein was fluctuated and it was not statistically significant（P>0.05）.After incubation with hydrogen peroxide:in 5,10μg/m L nano cerium oxide and hydrogen peroxide group,the expression of Caspase-3 protein in the cells was decreased and the concentration of 5μg/m L was statistically significant（P<0.05）;in 10,20,50,100μg/m L nano cerium oxide+hydrogen peroxide group,the expression of GRP78 protein in the cells increased gradually and the dose effect was not significant（P>0.05）;With the increase of the concentration of nano-cerium oxide,the expression of Nrf-2 protein in RPAEpi C cells fluctuated.Conclusions The low level of cerium oxide can alleviate the damage of NR8383 and alveolar epithelial cells RPAEpi C induced by hydrogen peroxide in rats,the mechanism may be that the cerium ions on the surface of cerium oxide crystal have two valence states changes of+3 and+4.With the change of the valence state of cerium on the crystal surface,it can resist the oxidation of ROS and reduce oxidative stress damage.The possible mechanism is that nano-cerium oxide activates the Nrf-2-mediated antioxidant stress response and endoplasmic reticulum stress,which affected the apoptotic signal pathway and inhibited cell apoptosis.High concentration did not show a protective effect,it may be the particles reunited in the solution presumably,particle size increases,the particle surface area decreases,the site which can produce valence change has a significant reduction in the crystal surface,the effect of antioxidant reduced.It may also be at high concentrations,nano cerium oxide particles are overload in cells,causing new damage to the cells,the damage is higher than the protective effect,showing no protective effect on cells.