Protective effects of cerium oxide nanoparticles in non-alcoholic fatty liver disease (NAFLD) and carbon tetrachloride-induced liver damage in rats: Study on intestine and liver

Background and aims Nanoparticles could represent a therapeutic approach for the treatment of various diseases. It has been reported that cerium oxide nanoparticles (CeO2 NPs) have potential useful effects. Therefore, we aimed to examine the protective effects of the CeO2 NPs in two models of liver injury, non-alcoholic fatty liver disease (NAFLD) and carbon tetrachloride (CCl4)-induced liver fibrosis, in rats. Methods In this experimental study, male rats were randomly divided into different experimental groups including: Experiment 1; group1: healthy rats received normal saline, 2: CCl4 group, 3: CCl4 + nanoparticle. Experiment 2; group1: healthy rats received chow diet, 2: NAFLD group, 3: NAFLD + nanoparticle. The oxidative stress markers were determined in the liver and intestine. Tumor necrosis factor-α (TNF-α) levels were measured by ELISA. Histopathological changes of liver and intestine were evaluated by light microspore. Results Total antioxidant capacity (TAC) and glutathione (GSH) levels significantly decreased, while malondialdehyde (MDA) and total oxidant status (TOS) were significantly increased in the liver, and intestine of the NAFLD and CCl4 group compared with control rats. However, the use of nanoparticles significantly normalized these markers. The levels of the TNF-α were significantly reduced in the nanoparticle group as compared with NAFLD model and CCl4-treated rats. CeO2 NPs also normalized the liver and intestinal histological changes. Conclusions Our finding revealed that CeO2 NPs has potential protective effects by increasing antioxidant activity, and reducing inflammation.


Introduction
Liver is known as the main organ that is involved in the metabolism of macromolecules, excretion and detoxification of circulating agents, synthesis of proteins, and bile acids. Experimental animal models are vital to know the mechanisms responsible for liver diseases. Among the animal models of liver cirrhosis and fibrosis, the most generally used is the carbon tetrachloride (CCl 4 ) and high-fat diet (HFD), which closely resembles the histological and hemodynamic features of human disease [1]. HFD is known as a main risk factor for the prevalence of various disorders such as non-alcoholic fatty liver disease (NAFLD), obesity, dyslipidemia, cardiovascular disease (CVD), and diabetes. NAFLD is known as the main form of chronic liver disorders throughout the world [2], considering approximately 24% of the worldwide population with the highest estimates reaching in the Middle East (32%) and in South America (31%) [3]. The pathogenesis of NAFLD is defined in terms of the "two hits", including lipid accumulation (first hit) and increased oxidative stress, inflammation, mitochondrial dysfunction and lipid peroxidation (second hit) which are mainly are responsible for the onset NAFLD to develop non-alcoholic steatohepatitis (NASH) and liver cirrhosis [4]. On the other hand, hepatotoxins, such as carbon tetrachloride (CCl 4 ) which is recognized by variable grade of hepatocyte degeneration and cell death [5,6]. CCl 4 is a chemical pollutant that has numerous adverse effects on the kidney, liver, blood and heart by elevating lipid peroxidation and generating free radicals [7]. CCl 4, as a prominent toxin among the other hepatotoxins, is commonly used to induce experimental animal models that mimic human hepatotoxicity. In the liver, the cytochrome P 450 enzymes catalyzed CCl 4 into trichloromethyl radical (CCl 3 • ), which quickly reacts with oxygen to form trichloromethyl peroxy radical (CCl 3 OO • ), the extremely reactive derivative. Both of these radicals by covalent binding to the cell proteins induce lipid peroxidation and oxidative stress (OS), consequently can lead to liver damage [5,7]. The CCl3 • and CCl3OO • mediated lipid peroxidation and is known as a major mechanisms of liver damage induced by carbon tetrachloride [5]. Moreover, CCl 4 can increase inflammatory markers in the body [8]. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α) worsen pathological progression, and lead to liver fibrosis that complicates the liver treatment [9]. Oxidative stress itself has been confirmed to mediate various cellular responses causing diverse outcomes such as cell growth and apoptosis [10]. Oxidative stress is a consequence of the imbalance between cellular antioxidant capacity and reactive oxygen species (ROS) generation [11]. These free radicals are involved in the etiology of various disorder conditions such as neurodegenerative disorders, cancers, cardiovascular diseases, and aging [12]. Therefore targeting lessening of inflammation and oxidative stress are a beneficial strategy to combat liver injury [13]. Unfortunately, therapeutic strategies designed to relieve liver injury have progressed at a slow pace, perhaps because of the adverse effects of chemical medicines [14,15]. Hence, patients often resort to natural products or new agents as an alternative therapy for their illnesses [16][17][18][19].
The nanoparticles administration has been documented as a potential therapeutic because of a better cellular uptake and distribution than other chemical medicines. The cerium oxide nanoparticles (CeO 2 NPs) are one of the main favourable nanoparticles for anti-inflammatory and antioxidant applications [20]. Cerium has two oxidation states, including Ce +3 and Ce +4 . The beneficial effect is attributed to its capability to mimic superoxide dismutase (SOD), acting as effective ROS and reactive nitrogen species (RNS) scavengers (Ce +3 to Ce +4 ) and mimic catalase activity (conversion of H 2 O 2 into oxygen and water) and peroxidase activity (reducing H 2 O 2 into hydroxyl radicals). CeO 2 NPs related to antioxidant activity renders the nanoparticles a precious agent for treatment of oxidative-related disorders [20,21]. Hence, we hypothesize that CeO 2 NPs can potentially reduce liver injury, inflammation, and oxidative stress in experimental HFD (NAFLD model) and CCl 4 -induced liver fibrosis. CCl 4 can lead to liver fibrosis, but no insulin resistance, nor obesity, and it is no NAFLD model by itself, which is why we use two different liver animal models. In this experiment, we evaluated the effects of CeO 2 NPs on liver and intestine by assessing the antioxidant activity, chemical factors and histological changes. The aim of the study was to reveal whether CeO 2 NPs can prevent oxidative stress, and inflammation in the liver and intestine in NAFLD rats models and CCl 4 -induced liver injury.

Material and methods
All chemicals agents had analytical grade and were purchased from Sigma-Aldrich (Poole, UK). CeO 2 NPs were obtained from NanoSany (NanoSany Corporation, Mashhad, Iran).

Animal handling and treatment
Male Wistar rats weighing 170-200 g, and aged 7-week were housed in the animal hosue under standard conditions (60-70% humidity, 25 ± 2 • C and 12 h light/dark cycle). The rats were fed on a standard diet and water. The animals were maintained for 7 days prior to the beginning of the experiments. After adaptation, animals randomly divided into different groups as bellow: Experiment 1; group1: healthy rats received normal saline, 2: healthy + nanoparticle 3: CCl 4 group, 4: CCl 4 group + nanoparticle. Nanoparticle (NanoSany Corporation, Mashhad, Iran. Fig. 1.) was administered for two weeks (0.1 mg/kg, i.v. twice a week for 2 weeks) [22,23], and 2 h after the last administration, liver injury was induced by CCl 4 (1 ml/kg of 50% CCl 4 , mixture in olive oil, i.p.) [24]. After 24 h, all rats were euthanized (by diethyl ether) and blood samples were collected from the heart. Experiment 2; group 1: healthy rats, 2: healthy rats + nanoparticle, 3: NAFLD group) 60 kcal% fat), 4: NAFLD group + nanoparticle. Nanoparticle was administered for 4 weeks (0.1 mg/kg, i.v. twice a week). After 24 h, all rats were euthanized (by diethyl ether) and blood samples were collected from the heart.
To achieve serum, the blood sample was allowed to clot and then centrifuged at 3000 g for 10 min. Serum was used for the detection of biochemical tests. Then the animals were sacrificed by cervical dislocation and the liver, and intestine from each animal was removed, and washed in ice-cold saline. Small portion of the liver (left lobe), and intestine (same part for each animal) immersed in liquid N 2 , and stored at − 80 • C for antioxidant tests [25]. All steps of this experiment were done in accordance with the Hamadan Medical University ethics committee (Ethic code: IR.UMSHA.REC.1398.520).

Biochemical factors
The serum levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, total protein, albumin, fasting blood sugar, total cholesterol and triglyceride concentrations were measured by colorimetric methods using automated chemical analyzer (Cobas Integra 400 Plus, China).

Protein estimation
About 0.1 g of tissue was homogenized in an 800 μL phosphatebuffered saline (PBS) buffer. The homogenates then were centrifuged to get supernatant, which was used for antioxidant tests.
Protein concentrations were measured by Bradford reagent. Bovine serum albumin was used as the standard [24].

Lipid peroxidation
The concentration of malondialdehyde (MDA), a lipid peroxidation marker, in tissues homogenate was determined by assay of thiobarbituric acid reactive substances (TBARS) formation. The absorbance of the TBARS-MDA complex was read by a spectrophotometer at 532 nm. Results were presented as nmol of MDA/mg protein [26,27].

Total antioxidant activity (TAC)
TAC levels were determined by ferric reducing antioxidant power (FRAP) method. Homogenate samples reduce ferric ions (Fe 3+ ) to ferrous (Fe 2+ ) in the presence of tripyridyl-s-triazine (TPTZ). The absorbance of the blue Fe 2+ -TPTZ complex was measured by a spectrophotometer at 593 nm. The amounts of TAC were expressed as nmol/ mg protein [28].

Total oxidative status (TOS)
The total oxidative status (TOS) of the sample was determined by the oxidation of ferrous iron to ferric in the samples. The ferric measurement was performed by xylenol orange. Light intensity was measured by a spectrophotometer at 560 nm [26,27].

Glutathione levels
The level of glutathione (GSH) was determined according to the manufacture instruction (Zellbio, Germany). The amount of GSH was expressed as nmol/mg protein.

Tumor Necrosis Factor-α (TNF-α) levels
The TNF-α levels were measured in the serum by ELISA kit according to manufacture instruction (BioLegend, UK). The results were expressed as pg/mg protein.

Histopathological analyses
For morphological evaluation, the liver and intestine samples from different groups were taken and fixed in 10% formalin. Then, fixed samples were embedded in paraffin and cut into 5 μm thick sections.
Samples were then stained with haematoxylin and eosin (H & E), and observed by optical microscope. The severity of lesions was classified according to the previous published paper [29].

Statistical analysis
The data were analysed using SPSS 20 software and presented as mean ± standard error of mean (Mean ± SEM). The results were analysed by ANOVA followed by Tukey as post-hoc test. A p value less that 0.05 was assumed significant.

Body weight
Tables 1 and 2 revealed the body weight of animal groups. The body weight significantly reduced in CCl 4 -treated rats and increased in NAFLD group. We did not find any significant change in body weight between the hepatotoxic and CeO 2 NPs group, while NPs reduce body weight in the NAFLD group.

Blood chemical markers
Tables 1 and 2 exhibit the biochemical factors in different treated animals. The hepatotoxic rats displayed markedly (p < 0.05) higher serum activity of ALP, AST and ALT than the normal group. Pretreatment with nanoparticles significantly normalized ALP, ALT and AST levels in hepatotoxic rats. High levels of liver enzymes induced by HFD and CCl 4 were alleviated markedly in nanoparticles treatment rats. Furthermore, exposure to CCl 4 significantly reduced (p < 0.05) total protein, and increased total and direct bilirubin levels. CeO 2 NPs effectively ameliorated these markers as compared with the NAFLD and CCl 4 group.
Changes in the glucose levels were not significant. Table 1 also reveals a significant (p < 0.05) increase in cholesterol and triglyceride levels in the NAFLD group compared to the control rats. Nanoparticle administration significantly reduced these markers.

Oxidative stress marker
GSH levels markedly reduced in the liver and intestine of NAFLD group CCl 4 -treated animals compared with the control rats. Administration of CeO 2 NP was increased GSH concentration in these tissues in the hepatotoxic rats. We found a significant rise in TOS in the liver, and intestine of the hepatotoxic and NAFLD group compared with the healthy rats. Furthermore, HFD and CCl 4 decreased TAC concentration and increased MDA levels in the liver and intestine of animals.
Treatment with CeO 2 NPs significantly increased TAC concentrations and reduced MDA levels as compared with the NAFLD and CCl 4 group (p < 0.01), which showed the mitigation of oxidative stress in these organs (Figs. 2-5).

TNF-α level
The TNF-α level of rats in NAFLD and CCl 4 groups were significantly higher as compared to control rats (Fig. 6). However, the TNF-α level in the NAFLD and CCl 4 group which were treated with CeO 2 NPs was obviously (p < 0.05) lower than the untreated group.

Histological alteration
The histological finding supported the liver function test and oxidative stress. Liver samples from healthy group revealed normal lobular structure and cells with a well preserved cytoplasm, and welldefined nucleus without any irregular histological alterations in hepatocytes and lobular architecture (Fig. 7). Liver of NAFLD and CCl 4 -treated rats showed wide liver injuries revealed by moderate necrosis around the central vein, cholangiocyte hyperplasi, hepatic fibrosis, vacuolization and hepatocellular hydropic degeneration. Nonetheless, administration of CeO 2 NPs significantly alleviated the pathological damages.
The histological results revealed mucosal injury, mild edema, disruption, mononuclear cell infiltration, shortening and loosening of intestinal villi, accompanied by spotty hemorrhage, change in the crypt structure, and necrosis in the intestine of NAFLD and CCl4-treated rats. But these alterations were not present in the nanoparticle treated animals (Fig. 8).

Discussion
In this study we evaluate the hepatoprotective effects of CeO 2 NPs in the animal models. Previous studies also documented the beneficial effects of CeO 2 NPs against hepatic oxidative damage caused by the acetaminophen and pyrrolizidine alkaloid monocrotaline [30]. Considering the useful properties of CeO 2 NPs, we further examined whether this nanoparticle may also restore liver functions in NAFLD rats models and CCl 4 -treated rats. The common dose used in the previous experiment was 0.1 mg/kg [22,23]. In this effective dose, the CeO 2 NPs decrease oxidative stress, alleviate liver steatosis, and showed anti-inflammatory properties. Therefore, in this study we administered CeO 2 NPs at the dose of 0.1 mg/kg.
It is well known that HFD substantially alters intestinal physiology and structure. Moreover, HFD promotes intestinal inflammation, oxidative stress and altered barrier integrity [31]. CCl 4 also can induce oxidative stress in the intestine and increase the production of proinflammatory cytokine and inflammatory cell infiltration in the intestine [32].
In this experiment, the ability of CeO 2 NPs to protect against HFD and CCl 4 -induced liver injury, oxidative stress and inflammation were examined. Nanoparticles absorption may decrease due to agglomeration/aggregation of the particles in the intestine. Therefore, intravenous administration can be distributed to various organs [33]. In this study, CeO 2 nanoparticle was administered by intravenous route. CCl 4 is a lipophilic agent and is extremely toxic to the hepatocyte [7]. CCl 4 is metabolized in the liver to generate potentially reactive free radicals and ROS. It is also identified that its oxy metabolite could cause liver toxicity by the depletion of liver GSH. Furthermore, HFD can induce ROS production, accompanied by increased nitric oxide and TNF-α secretion, which promotes chronic inflammation and tissue damage [1]. Our results propose that CeO 2 NPs can have potential antioxidant properties (by increasing GSH and TAC as well as reducing TOS and MDA).
Liver necrosis causes raises of ALP, AST and ALT levels and an elevated severity of histological hepatic injuries in the animals. In agreement with previous studies [14,15], our result showed a noticeable raise in the ALT, AST, and ALP levels in the hepatotoxic group. CeO 2 NPs normalized the serum enzymes (ALT, AST and ALP) and led a subsequent recovery towards normalization as compared to the healthy rats, indicating the liver protective effects of CeO 2 NPs. The noticeable raise in the liver enzyme activity is a sign of the liver injury in the experimental animals [13,34]. Liver is the main organ involved in the blood protein synthesis, particularly albumin. In this experiment, circulating albumin Results are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with Hepatotoxic group. ### P < 0.01 compared with control. Results are expressed as means ± SEM of six rats/group TNF-α: tumor necrosis factor-α, NPs: nanoparticles. b. TNF levels in different treated groups.
Results are presented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared with NAFLD group. ### P < 0.01 compared with control. Results are expressed as means ± SEM of six rats/group TNF-α: tumor necrosis factor-α, NPs: nanoparticles. concentration was used to determine liver synthetic ability. The assessment of this protein concentration indicates that CeO 2 NPs can prevent the reduction of protein likely through neutralizing ROS by scavenger compounds or stabilizing endoplasmic reticulum [35]. Toxic chemicals such as CCl 4 are oxidized by cytochrome P450 with the following release of liver tissue damaging RNS or ROS resulting in the leakage of liver enzymes into blood. Production of trichloromethyl free radicals (active metabolite of CCl 4 ) lead to liver necrosis, malondialdehyde (MDA) production and extracellular matrix destruction. Trichloromethyl radical in the presence of oxygen is converted to trichloromethyl peroxyl radical. These free radicals can covalently bind to protein and membrane lipids to produce MDA, leading to damage to the cells. MDA formation is one of the main reasons of CCl 4 induced liver and intestine injury. The reduced MDA levels in the liver of the treatedhepatotoxic groups (CCl 4 and NAFLD), propose the antioxidant and hepatoprotective properties of CeO 2 NPs [36].
In the present study, marked restorations of glutathione levels and TAC in nanoparticle group were observed when compared with the NAFLD group and CCl 4 treated groups [37]. TAC is a main defense system against hydroperoxide, ROS, and environmental toxicity. Similarly, glutathione is the first line of defense against oxidative stress [13]. GSH, a main cellular antioxidant, has been recognized as a vital factor needed for liver detoxifications. The depletion of glutathione levels in the liver and intestine may be because of augmented glutathione use in the elimination of trichloromethyl peroxyl radical. Glutathione has a key role in detoxifying the toxic metabolites and liver damage begins when glutathione levels are significantly reduced. Glutathione has long been considered as a key factor in detoxification of the toxic metabolites of CCl 4 . CCl 4 and high consumption high fat diets, induce a permanent state of inflammatory cytokines. Inflammation is usually related with liver fibrosis. In this respect, TNF-α is a main factor that induces the inflammatory pathways involved in liver injury [9]. Administration of CeO 2 NPs significantly reduced TNF-α concentration in different organs as compared to hepatotoxic groups. TNF-α activates the different pathways after liver damage, consequently induces hepatocyte apoptosis, hepatocyte proliferation, and liver inflammation [38].
Liver has a vital role in the metabolism of macronutrients. Administration of CCl 4 and HFD lead an obvious rise in the total cholesterol (TC) and triglyceride (TG) levels. The disturbance in the phospholipids metabolism and decrease in protein synthesis may result in dyslipidemia. Pretreatment with CeO 2 NPs modulated blood lipid profiles. It has been reported that CCl 4 induces the transfer of acetate into hepatocytes and causes rise in cholesterol synthesis. CCl 4 also elevate the triglyceride synthesis from acetate and increases lipid esterification. The TG accumulation in the liver may happen because of the suppression of lipase activity and secretion of very-low-density lipoprotein (VLDL) [35]. On the basis of our results, a better lipid clearance rate in nanoparticle treated rats was observed.
The hepatoprotective influence of CeO 2 NPs was further approved by histopathological analyses. High fat diet and CCl 4 induced liver damages, including necrosis, steatosis, foam cell formation, degeneration of biliary and vacuolization. Nevertheless, in the CeO 2 NPs treated rats, less degeneration was observed, indicating that this nanoparticle can prevent liver injury or cause the restoration of damaged liver parenchyma [35]. CeO 2 NPs also normalized histopathological change in the intestine. Our results showed that CeO 2 NPs normalized intestine and liver function by reducing oxidative stress and inflammation. Therefore, this nanoparticle treatment may be consider as a potential agent for liver diseases.

Availability of data and material
Data are available upon reasonable request.

Declaration of competing interest
The authors report no conflict of interest.