Reduction Of Oxidative Damages Induced By Titanium Dioxide Nanoparticles Correlates With Induction Of The Nrf2 Pathway By GSPE Supplementation In Mice

Objective:Recent studies have found that exposure to TiO₂NPs is associated with cancer risk,and oxidative stress may be the mechanism of promoting cancer occurrence.Titanium dioxide nanoparticles（TiO₂NPs）was a new type of nano-material.It is widely used as additives in cosmetics,drugs,paints and even food.Grape seed proanthocyanidins extract（GSPE）is a natural compound with extensive pharmacological and biochemical properties,which has anti-inflammatory,anticancer and antioxidant properties.The purpose of this study was to detect the histopathology,tissue function,oxidative stress and DNA damage of heart,liver and kidney in mice induced by TiO₂ NPs nano-titanium dioxide.To evaluate the expression levels of Nrf2,NQO1,HO-1 and GCLC in the tissues of mice pretreated with GSPE,and to explore their potential antioxidant protection mechanism.Methods:In order to explore the main organ toxicology of TiO₂NPs after gastrointestinal exposure and to find the potential improvement effect of GSPE intervention as well as the underlying molecular mechanisms,we established a mouse model of TiO₂ NPs intragastric administration and GSPE intervention.Mice were divided into four groups,means the control group,TiO₂ NPs treatment group（2 g/kg body weight）,the TiO₂ NPs treatment+low-dose GSPE intervention group（2 g/kg body weight,TiO₂ NPs+167mg/kg）,the TiO₂ NPs treatment+high-dose GSPE intervention group（2 g/kg body weight TiO₂ NPs+500 mg/kg body weight GSPE）.After one week of treatment,the organ coefficients and pathological changes of heart,liver and kidney were evaluated;the changes of heart function were evaluated by the activities of serum LDH,CK and CKMB;the changes of liver function were evaluated by the activities of serum ALT,AST and TBIL;and the changes of kidney function were evaluated by serum BUN and Cr content;SOD,GSH-Px activities and ROS,MDA contents were used to evaluate the oxidative stress level of these tissues;the DNA damage of tissues was evaluated by the Comet assay.The protein expression of nuclear factor E2 related factor 2（Nrf2）and its downstream molecules quinone oxidoreductase 1（NQO1）,heme oxygenase 1（HO-1）and glutamate cysteine ligase catalytic subunit（GCLC）were detected by Western blot.Results:1.TiO₂ NPs characterization.Transmission electron microscope（TEM）and scanning electron microscope（SEM）showed that TiO₂ NPs was spherical.TiO₂ NPs is well dispersed in PBS.2.Organ coefficient.There was no significant difference in the organ coefficients of heart,liver and kidney among each group.3.Histopathological changes.After TiO₂ NPs exposure,the arrangement of cardiomyocytes was disordered,the hepatic sinusoid dilated to varying degrees,and the renal tubules dilated and the lumen was filled with protein fluid.A small amount of inflammatory cell infiltration was observed in the myocardium of TiO₂+GSPE-167mice,and some hepatocytes were disarranged in liver tissue,but the degree of injury was slighter than that in TiO₂ NPs exposure group.Renal tubular dilatation was found in kidney,but no obvious pathological changes were found in glomeruli.In TiO₂+GSPE-500 group,there was no abnormality in myocardial tissue,only mild disorder of hepatocyte arrangement in liver tissue,and only a small amount of inflammatory cell infiltration in kidney.4.Serological index.Cardiac injury index:compared with the control group,the serum LDH,CK,CKMB of TiO₂ NPs exposed group increased 1.8,16.83,2.68 times respectively.However,the three markers decreased significantly after the intervention of GSPE,which was significantly lower than that of mice exposed to TiO₂ NPs（P<0.05）.Liver injury index:compared with the control group,the levels of serum ALT and AST in the TiO₂ NPs exposure group were2.95 times and 1.37 times higher than those in the control group.After adding GSPE intervention,the level of ALT decreased by about 50%（P<0.05）.Kidney damage index:compared with the control group,the serum BUN and Cr of the TiO₂ NPs exposed group increased by 2.4 fold and 3.0 fold respectively.After adding GSPE intervention,the level of Cr decreased by about 20%,and the difference was statistically significant（P<0.05）.5.DNA damage results and oxidative damage.After TiO₂ NPs exposure,DNA damage occurred in heart,liver and kidney of mice.However,in TiO₂+GSPE-500 group,the degree of DNA damage in each tissue of mice was significantly less than that of TiO₂ NPs exposure group,and the difference was statistically significant（P<0.05）.Compared with the control group,ROS in heart,liver and kidney tissues of mice exposed to TiO₂ NPs increased by 3.42,3.66 and 2.71times,respectively.After adding 500 mg/kg GSPE pretreatment,the ROS of heart,liver and kidney decreased by 27%,64%and 47%respectively,and the difference was statistically significant（P<0.05）.Conclusion:1.TiO₂ NPs exposure can induce heart,liver and kidney pathological changes in mice by oxidative stress.2.GSPE can effectively prevent oxidative damage of heart,liver and kidney induced by TiO₂ NPs in mice.3.GSPE can activate Nrf2 and its downstream genes NQO1,HO-1 and GCLC to play an antioxidant role.