the attention to the worldwide epidemic of obesity is heightened as it is the fifth leading risk factor for global deaths . mortality rate from all causes in obese population is at least 20% higher compared to normal - weight . incidence of obesity strongly correlates with a wide range of the related diseases , such as cardiovascular diseases ( except congestive heart failure ) , type 2 diabetes , all types of cancers ( except esophageal cancer in females ) , asthma , gallbladder disease , osteoarthritis and chronic back pain . in obese individuals lipid metabolism one of the main sites of potential fat deposition is the liver that is a prime cause of the development of nonalcoholic fatty liver disease ( nafld ) . nafld is the most common cause of liver dysfunction in the western world because of its close association with obesity , insulin resistance and dyslipidaemia . nafld is defined as the accumulation of lipids within the hepatocytes exceeding 5% of liver weight in the absence of excessive ethanol intake ( conventionally defined as an intake of ethanol 20 g / day ) and without other causes of liver diseases . the mechanisms involved in the development of fatty liver and in the progression of the disease are unclear , but these may be related to a metabolic profile in the context of a genetic predisposition . insulin resistance , oxidative stress , cytokines and obesity are identified as the major risk factors involved in nafld / nash pathogenesis . these factors can promote intra - hepatic fat accumulation and lipotoxicity , development of an inflammatory status , oxidative stress , apoptosis and fibrogenesis that determine the progression of the disease . recently , antioxidant effects of cerium dioxide nanoparticles ( nceo2 ) , a type of engineered nanomaterials , have been reported . nceo2 possess catalytic activity , which arises from the presence of two valence states ( ce3 + and ce4 + ) . due to the oxygen vacancies present in these nanoparticles , they are able to react with the surrounding reactive oxygen species , thus introducing nceo2 as a potential in vivo mimetic for endogenous anti - oxidants like superoxide dismutase . however , there are conflicting results about the efficiency of nceo2 anti - oxidant activity . one of the pathogenic mechanisms of the nafld is the accumulation of reactive oxygen species , which in turn aggravate the disease progress . thus , we aimed at investigating the novel cerium dioxide nanoparticles ( nceo2 ) , which have shown promising antioxidant auto - regenerative ability and low toxicity   the aim of this study was to investigate the influence of nceo2 on lipid peroxidation , and antioxidant enzymes activity in rats with experimental induced nafld . in order to develop the experimental nafld in rats the model of monosodium glutamate ( msg)-induced obesity was used [ 1315 ] . white male wistar rats ( n=30 ) were used in the study and divided into 3 groups : control , msg and msg+ nceo2 groups . msg and msg+nceo2 groups were injected with msg ( 4 mg / g , 8 l / g volume ) at 2nd-10th day of life subcutaneously . at the age of 1 month , rats of group ii were administered water in a volume of 2.9 ml / kg orally , msg+nceo2 group was treated with 1 mm solution of nceo2 ( 1 mg / kg orally ) . the treatments were given intermittently in two - week courses alternated with two - week breaks for 3 months . during the experiment , rats aged between one and four months were fed with standard laboratory chow and tap water ad libitum . the specimen were examined under a xs-4130 micromed microscope . to assess morphological changes in the liver we used nas ( nafld activity score ) , which included histological features and has been defined as unweighted sum of scores for steatosis ( 03 ) , lobular inflammation ( 03 ) and ballooning ( 02 ) . according to nas scores 5 are considered as non - alcoholic steatohepatitis ( nash ) , while those with a nas <3 are considered as not nash . the content of diene conjugates ( dc ) , tba - active products and schiff bases were determined by standard biochemical methods . for evaluation of diene conjugates   and schiff bases 5 ml of mixture of heptane and isopropyl alcohol ( 1:1 ) was added to the sample containing 100 g of protein , homogenized ( 10 min ) and centrifugated ( 1000 g , 15 min ) . ethanol ( 1.5 ml ) was added to an upper heptane phase ( 0.3 ml ) and this mixture was subjected to spectrophotometric analysis at 233 nm for the detection of diene conjugates . schiff bases were detected in heptane phase with fluorophotometer rf-510 , shimadzu ( japan ) at l=360 nm ( excitation ) and l=420 nm ( emission ) and expressed as absorbance units ( a.u.)/mg of protein . the tba - active products were detected by formation of the colored complex with tba . briefly , sample with 0.5 mg of protein in tris was centrifuged with 0.2 ml of 17% trichloroacetic acid . 0.25 ml of 0.8% tba was added to 0.5 ml of supernatant and the obtained solution was heated in a boiling water bath for 10 min . the concentration of tba - active products was presented as nmol / mg of protein . antioxidant systems activity was assessed by detection of the activity of superoxide dismutase ( sod ) and catalase . sod activity was determined by chevari s method   based on the ability of sod to compete with nitroblue tetrazolium for superoxide anions , which are formed during the interaction of nadh with phenazine methosulfate . in the presence of sod percentage of superoxide anions recovery by nitroblue tetrazolium h2o2 forms a colored complex with molybdenum salts . the sample containing 100 g of protein after 10 min the reaction was stopped by adding of 1 ml of 4% ammonium molybdate . all data in this study were expressed as means  standard error ( mse ) or % . continuous variables with parametric distribution were analyzed using analysis of variance ( anova ) and if the differences were significant , a post - hoc tukeys test was performed . for data with non - parametric distribution kruskall - wallis and post - hoc dunn s test were conducted for multiple comparisons . for comparisons of categorical variables we conducted 2 test . the difference between groups was defined to be statistically significant when a p - value was less than 0.05 . in order to develop the experimental nafld in rats the model of monosodium glutamate ( msg)-induced obesity was used [ 1315 ] . white male wistar rats ( n=30 ) were used in the study and divided into 3 groups : control , msg and msg+ nceo2 groups . msg and msg+nceo2 groups were injected with msg ( 4 mg / g , 8 l / g volume ) at 2nd-10th day of life subcutaneously . at the age of 1 month , rats of group ii were administered water in a volume of 2.9 ml / kg orally , msg+nceo2 group was treated with 1 mm solution of nceo2 ( 1 mg / kg orally ) . the treatments were given intermittently in two - week courses alternated with two - week breaks for 3 months . during the experiment , rats aged between one and four months were fed with standard laboratory chow and tap water ad libitum . the specimen were examined under a xs-4130 micromed microscope . to assess morphological changes in the liver we used nas ( nafld activity score ) , which included histological features and has been defined as unweighted sum of scores for steatosis ( 03 ) , lobular inflammation ( 03 ) and ballooning ( 02 ) . according to nas scores 5 are considered as non - alcoholic steatohepatitis ( nash ) , while those with a nas <3 are considered as not nash . the content of diene conjugates ( dc ) , tba - active products and schiff bases were determined by standard biochemical methods . for evaluation of diene conjugates   and schiff bases 5 ml of mixture of heptane and isopropyl alcohol ( 1:1 ) was added to the sample containing 100 g of protein , homogenized ( 10 min ) and centrifugated ( 1000 g , 15 min ) . ethanol ( 1.5 ml ) was added to an upper heptane phase ( 0.3 ml ) and this mixture was subjected to spectrophotometric analysis at 233 nm for the detection of diene conjugates . schiff bases were detected in heptane phase with fluorophotometer rf-510 , shimadzu ( japan ) at l=360 nm ( excitation ) and l=420 nm ( emission ) and expressed as absorbance units ( a.u.)/mg of protein . the tba - active products were detected by formation of the colored complex with tba . briefly , sample with 0.5 mg of protein in tris was centrifuged with 0.2 ml of 17% trichloroacetic acid . 0.25 ml of 0.8% tba was added to 0.5 ml of supernatant and the obtained solution was heated in a boiling water bath for 10 min . the concentration of tba - active products was presented as nmol / mg of protein . antioxidant systems activity was assessed by detection of the activity of superoxide dismutase ( sod ) and catalase . sod activity was determined by chevari s method   based on the ability of sod to compete with nitroblue tetrazolium for superoxide anions , which are formed during the interaction of nadh with phenazine methosulfate . in the presence of sod percentage of superoxide anions recovery by nitroblue tetrazolium h2o2 forms a colored complex with molybdenum salts . the sample containing 100 g of protein after 10 min the reaction was stopped by adding of 1 ml of 4% ammonium molybdate . continuous variables with parametric distribution were analyzed using analysis of variance ( anova ) and if the differences were significant , a post - hoc tukeys test was performed . for data with non - parametric distribution kruskall - wallis and post - hoc dunn s test were conducted for multiple comparisons . for comparisons of categorical variables we conducted 2 test . the difference between groups was defined to be statistically significant when a p - value was less than 0.05 . neonatal subcutaneous injection of msg led to the development of visceral obesity and nafld as consequence of metabolic disorder in 4-month rats . there was evidence of steatosis , lobular inflammation and ballooning degeneration in liver tissue in 4-month treated with msg neonatally . in contrast , there was significantly lower total score ( 1.30.26 vs. 3.60.34 , p<0.001 ) , degree of steatosis ( 1.10.18 vs. 2.10.18 , p<0.001 ) , manifestation of lobular inflammation ( 0.20.13 vs. 1.20.2 , p<0.001 ) and ballooning degeneration ( 0.00.0 vs. 0.30.15 , p=0.034 ) due to nas in the nceo2 group as compared to msg - group ( table i , figures 1 , 2 ) . nash was confirmed only in 30% of the msg - group of rats ( p=0.036 ) . biochemical analysis showed the increase of lipid peroxidation in the liver tissue under conditions of nafld development . dc in msg - group was higher by 62.2% ( p<0.05 ) , tba - products  by 92.3% ( p<0.05 ) and schiff bases  by 72.3% ( p<0.05 ) compared to control values ( figure 3 ) . short - term periodic oral administration of nceo2 significantly decreased the lipid peroxidation in liver tissue , namely reduced the dc content by 27% ( p<0.05 ) , tba - products  by 43% ( p<0.05 ) and schiff bases  by 21% ( p<0.05 ) ( figure 4 ) . worsening of pro / antioxidant balance in the rat liver in case of nafld is also caused by alteration of antioxidant enzyme activity . sod activity in obese rats was decreased by 46.1% ( p<0.05 ) , while catalase activity was increased by 90.0% ( p<0.05 ) compared to control ( figure 4 ) . treatment with nceo2 led to the restoration of sod activity to the control values and decrease of excessive catalase activity by 22.1% ( p<0.05 ) compared to the msg - group ( figure 4 ) . thus , the studied nanoparticles improved the impaired pro / antioxidant state in the liver under conditions of msg - induced nafld . oxidative stress is one of the main fundamental phenomena during the development of obesity and nafld . nceo2 mimic sod and/or catalase activity and have shown promise as a therapeutic application due to their antioxidant auto - regenerative ability and low toxicity . the electronic structure of cnps at the nanoscale underlines their antioxidant activity . both large surface - to - volume ratio with the reduction in particle size   and the ability to reversibly switch between ce+and ce + present on the surface   result in the formation of oxygen defects in the crystal lattice that act as  reactive sites  or  hot spots  for reactive oxygen species ( ros ) scavenging . physiochemical properties ( size , shape , agglomeration status in liquid and coating ) of nceo2 also could influence their biological response and catalytic activity . several nanocomposites of cnps which are used for coating biocompatible polymers ( polyethylene glycol , dextran , polyacrylic acid , citric and oleic acid ) have been described . in our study , we used near shaped , citrate coated nceo2 with particle size of 25 nm . sodium citrate in nanocomposites serves not only as a stabilizer , which determines the compositional stability of nceo2 in water and biologically relevant media , but also plays an important role in the pharmacokinetics of nceo2 in the cells . citrate as a component of krebs cycle provides nceo2 to the mitochondria , where ros form under the pathological conditions , leading to oxidative stress . in recent years , there has been a substantial interest in nceo2 as a therapeutic agent , and many examples can be found where these nanoparticles have been tested for the treatment of several pathologies , where an imbalance of the redox state occurs . in the present study , we first investigated the influence of nceo2 on liver lipid peroxidation and antioxidant enzymes activity in rats with experimental nafld . several previous studies clearly demonstrated the biodistribution of nceo2 mainly in the spleen and liver , also with trace amounts found in the lungs and kidneys , and virtually none in the heart or brain . despite the strong accumulation in the spleen and liver for a period of at least 30 days , histological analysis does not reveal any alteration of the organ cytology and show the typical morphology of the tissues without difference between treated and control rats . only in one study granulomatous formations were found after 30 days in the liver of rats exposed to a single dose of nceo2 . however , this hepatic damage was probably due to the high dose of administered nceo2 ( 85 mg / kg ) , which resulted in an impaired nanoparticle clearance . reported that weekly administration of nceo2 for 2 or 5 weeks with 0.5 mg / kg to mice with induced liver toxicity ( by ccl4 ) showed similar findings to mice treated with n - acetyl cystine ( nac ) , a common therapeutic to reduce oxidative stress . these data are in agreement with our findings , where we demonstrate that attenuation of pathological lipid peroxidation after administration of nceo2 reduces damaging influence of ros on the liver tissue in msg - induced animal model of nafld . there is an interesting study reported by rocca et al , in which nceo2 were tested both in vitro and in vivo as novel anti - obesity pharmaceutical formulation . they mentioned that nceo2 interfere with the adipogenic pathway by reducing the mrna transcription of genes involved in adipogenesis , and by hindering the triglycerides accumulation in 3t3-l1 pre - adipocytes . intraperitonal administration of 0.5 mg / kg nceo2 to wistar rats , did not have toxic effects , but caused significant reduction of weight gain and decrease of plasma levels of insulin , leptin , glucose and triglycerides as compared to control group . demonstrated that the beneficial antioxidant properties are inherent only to nanoparticles of ceo2 and can be strengthened by additional administration of sodium selenite . the improvement of antioxidant enzymes activity and decrease of cholesterol , triglyceride and low density lipoprotein levels have been demonstrated in streptozotocin - induced diabetic rats after 2 week intraperitoneal injection of nceo2 with sodium selenite alone or in combination , but the metal form of ceo2 showed no significant improvement . the administration of nceo2 under conditions of oxidative stress can reduce / prevent its pathological effects . due to the ability of ce3 + conversion into ce4 such attenuation of pathological lipid peroxidation reduces the pathological influence of ros on the liver tissue , which is confirmed by significant improvement of the main nafld histological features and restoration of antioxidant enzymes activity . in summary , nceo2 may have the potential to be used as a treatment for nafld , but it should be tested using other animal models of obesity .
backgroundone of the pathogenic mechanisms of the nonalcoholic fatty liver disease ( nafld ) is the accumulation of reactive oxygen species , which in turn aggravates the disease progress . 
 we have investigated novel cerium dioxide nanoparticles ( nceo2 ) due to their promising antioxidant auto - regenerative ability and low toxicity.methods30 white male wistar rats were divided into 3 groups : control , monosodium glutamate ( msg)-induced obesity and msg treated with nceo2 ( msg+nceo2 ) groups . 
 newborn rats of control group were injected with saline ( control ) . 
 msg and msg+nceo2 groups were injected with msg ( 4 mg / g concentration , 8 l / g volume ) between the 2nd and the 10th days of life subcutaneously [ 13 ] . at the age of 1 month , rats of group ii were administered water 2.9 ml / kg orally , msg+nceo2 group received 1 mm solution of nceo2 1 mg / kg orally . 
 4-months rats were sacrificed and the liver was harvested for histological and biochemical analysis . to assess the morphological changes in the liver we used nas ( nafld activity score ) . 
 the content of lipid peroxidation products and enzymatic activity of superoxide dismutase ( sod ) and catalase in the liver were studied by standard biochemical methods [ refs].resultsin 4-month rats we found significantly lower total score ( 1.30.26 vs 3.60.34 , p<0.001 ) , degree of steatosis ( 1.10.18 vs 2.10.18 , p<0.001 ) , manifestation of lobular inflammation ( 0.20.13 vs 1.20.2 , p<0.001 ) and ballooning degeneration ( 0.00.0 vs 0.30.15 , p=0.034 ) due to nas in the nceo2 group compared to the msg - group . 
 nceo2 significantly decreased lipid peroxidation in the liver tissue , namely it reduced the conjugated dienes content by 27% ( p<0.05 ) , tba - products  by 43% ( p<0.05 ) and schiff bases  by 21% ( p<0.05).conclusionsdue to its antioxidant properties nceo2 significantly reduces the incidence of nash and improves the main nafld histological features .