A short-term rodent model for non-alcoholic steatohepatitis induced by a high-fat diet and carbon tetrachloride

Abstract Several models of mice-fed high-fat diets have been used to trigger non-alcoholic steatohepatitis and some chemical substances, such as carbon tetrachloride. The present study aimed to evaluate the joint action of a high-fat diet and CCl4 in developing a short-term non-alcoholic steatohepatitis model. C57BL6/J mice were divided into two groups: standard diet-fed (SD), the high-fat diet-fed (HFD) and HFD + fructose-fed and carbon tetrachloride (HFD+CCl4). The animals fed with HFD+CCl4 presented increased lipid deposition compared with both SD and HFD mice. Plasma cholesterol was increased in animals from the HFD+CCl4 group compared with the SD and HFD groups, without significant differences between the SD and HFD groups. Plasma triglycerides showed no significant difference between the groups. The HFD+CCl4 animals had increased collagen deposition in the liver compared with both SD and HFD groups. Hydroxyproline was also increased in the HFD+CCl4 group. Liver enzymes, alanine aminotransferase and aspartate aminotransferase, were increased in the HFD+CCl4 group, compared with SD and HFD groups. Also, CCl4 was able to trigger an inflammatory process in the liver of HFD-fed animals by promoting an increase of ∼2 times in macrophage activity, ∼6 times in F4/80 gene expression, and pro-inflammatory cytokines (IL-1b and TNFa), in addition to an increase in inflammatory pathway protein phosphorylation (IKKbp). HFD e HFD+CCl4 animals increased glucose intolerance compared with SD mice, associated with reduced insulin-stimulated AKT activity in the liver. Therefore, our study has shown that short-term HFD feeding associated with fructose and CCl4 can trigger non-alcoholic steatohepatitis and cause damage to glucose metabolism.


Introduction
Non-alcoholic fatty liver disease (NAFLD) begins with the liver's accumulation of triacylglycerides (TAG).It is defined as lipid droplets in the cytoplasm of more than 5% of hepatocytes [1], and it is usually associated with hepatic insulin resistance [2,3].This disease develops when the rate of hepatic TAG synthesis, as a result of increased uptake of fatty acids and their esterification to TAG, as well as de novo lipogenesis from carbohydrate and protein metabolism, exceeds the rate of TAG catabolism via fatty acid oxidation or TAG secretion in the form of very low-density lipoproteins (VLDL) [1].NAFLD represents a broad spectrum of histological abnormalities ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), which may progress to cirrhosis or hepatocellular carcinoma [4].Long-term fat feeding stimulates the development of fibrosis, a feature highlighted in the NASH [5][6][7].
In addition to fat feeding, chemical substances induce the fibrotic process such as carbon tetrachloride (CCl 4 ) [8].CCl 4 is used to study liver fibrosis and cirrhosis in rodents.CCl4 biotransformation depends on CYP2E1 to form the trichloromethyl radical, which is involved in the process of lipid peroxidation [9,10], contributing to necrosis, Kupffer cell activation and inflammatory response [11].This inflammatory process contributes to the production of several cytokines, which promote the activation of liver stellate cells and, consequently, fibrosis [12].
Studies have shown that a diet rich in fat and enriched with cholesterol (western diet), fructose (23.1 g/L) and glucose (18.9 g/L) in drinking water and treatment with CCl 4 at the lowest dose (0.2 μl/g) once a week for 12 and 24 weeks, triggered the progression from NASH to extensive fibrosis and cancer in C57BL/6J model [13].
Studies have used various animal models of diets and chemicals to propose models of NASH.However, these models usually go through a long-term diet feeding of approximately 12 weeks with lower doses of CCL 4 [14].Therefore, the present study aimed to characterize and propose a new model of NASH, with a short-term developing time (4 weeks), in C57BL/6 mice fed with a high-fat diet supplemented with fructose in the drinking water and treated with CCl 4 .

Animals
Male C57BL/6 mice (20-25 g) were kept in a temperature-controlled room at 22 + − 2 • C with ad libitum access to food and water, submitted to a 12-h light-dark cycle (light from 6 a.m. to 6 p.m.).These animals were fed with a standard diet (SD) (Nuvilab ® -CR-1), consisting of 19% protein, 56% carbohydrate, 3.5% lipids, 5% cellulose and 4.5% vitamins and minerals, providing 3.2 kcal/g of feed; a high-fat diet (HFD) (Research diet -D12079B), consisting of 17% proteins, 43% carbohydrates, and 40% lipids, providing 4.7 kcal/g of feed; and the other group was fed a HFD and supplemented with 10% fructose in the drinking water, and received an intraperitoneal injection of carbon tetrachloride (CCl 4 ) (dose: 0.5 μl/g of animal weight) three times a week, the CCl 4 was diluted in olive oil [8,15].HFD started when the animals were approximately 8 weeks old.The experiments were carried out after 4 weeks of the onset of HFD.Therefore, we had three groups of experimental animals: animals fed with a standard diet (SD), animals fed only with a high-fat diet (HFD), animals fed with HFD + fructose (10%) and treated with CCl 4 (HFD+CCl 4 ).Ten animals were used per group.The animals were sacrificed under an anesthetic overdose (isoflurane).All animal experiments took place at Ribeirao Preto Medical School at the University of Sao Paulo and all experimental procedures were performed following the animal care principles of the 'Guidelines for the ethical use of animals in applied etiologies studies' [16] and previously approved by the FMRP/USP Ethics Committee on Animal Use (No. 1082/2022).

Body composition
The awake animals were submitted to body composition using the minispec LF50 (Bruker's, Massachusetts, U.S.A.).Where total body mass, fat mass, lean mass and net weight were analyzed.

Glucose tolerance test
After 6 h of food restriction, mice were injected intraperitoneally (i.p.) with glucose (1 mg/kg body weight-10% dextrose).Blood samples for measuring glucose were taken by tail bleeding at 0, 15, 30, 45, 60, 90, and 120 min after injection, as previously described [17].The area under the curve (AUC) was calculated using the statistical software GraphPad Prism 9.0 in order to use it for statistical analysis.Glucose levels were measured in duplicate.

Lipid and hepatic enzymes measurement
After 6 h of food restriction, the animals were euthanized, and the tissues were removed for lipid content analysis.Tissue TAGs were extracted using the method of Bligh and Dyer [18] and measured using a TAG reagent.The blood of the animals was collected and centrifuged (12000 rpm, 2 min) for analysis of liver enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), plasmatic triglycerides, and cholesterol.The tests were performed using commercial kits (Bioclin, Brazil).The samples were measured in duplicate.

Lipid peroxidation measurement
Lipid peroxidation was determined by measuring thiobarbituric acid reactive substances (TBARS).Aliquots of 200 μl of samples (blood and tissues) were added to a 400 μl mixture composed of by equal parts of 15% trichloroacetic acid (TCA), 0.25 N HCl and 0.375% TBA, plus 2.5 mM butylated hydroxytoluene (BHT) and 40 μl of 8.1% sodium dodecyl sulfate (SDS), being heated for 30 min at 95 • C in an oven.The mixture pH was adjusted to 0.9 with concentrated HCl.
BHT was used to prevent lipid peroxidation during heating.After cooling to room temperature and adding 4 ml of butanol, the material was centrifuged at 800 × g for 15 min at + − 4 • C and the supernatant absorbance was measured at 532 nm.The molar extinction coefficient used was 1.54 × 10 5 M −1 cm −1 and the TBARS result was expressed in nmol Eq MDA/ml for plasma and tissue samples [19].

Hydroxyproline measurement
A liver fragment (80-100 mg) was homogenized in 1 mL hydrolyzed solution.The solution was incubated in a water bath at 95 • C for 20 min.After, the pH of the solution was adjusted, pH between 6.0 and 6.8, as described in the commercial kit.Then, the volume was completed with distilled water to a final volume of 10 ml and mixed.After, 3-4 ml of this solution was taken and placed in another tube, and 20-30 mg of acticarbon was added, mixed, and centrifuged at 3500 rpm for 10 min.After this procedure, take 200 μl and pipette into a 96-well plate to carry out the assay protocol described in the commercial kit.The kit used was the Hydroxyproline (Hyp) colorimetric assay kit (Alkali Hydrolysis method) (Elabscience, U.S.A.).The reading was performed at 550 nm on the Accuris SmartReader UV-Vis -Microplate reader.The samples were measured in duplicate.

Morphological analyzes
Oil Red O (ORO) staining Liver fragments were embedded in a mould with tissue-tek (Thermo Scientific) and frozen in liquid nitrogen (N2).Twelve μm (three sections per slide) were made from different tissue parts.A cryostat (Microm H560) was used to perform the cuts.The slides were stained with ORO (5 min), then washed in running water (30 min).Images (10 of each animal) were obtained using a Nikon Eclipse Ti-U microscope at the 20× objective, with a Nikon DS-Ri1 digital camera and NISElements BR 3.1 software.The quantification of fat accumulation in the tissue was performed using Image J.

Sirius red staining
The slides were submitted to Sirius Red staining to identify collagen fibers.Sections were stained with Picrosirius (1 h) and washed in running water (3 min).The red color represented collagen deposition.Ten fields of histological sections (10 images) were analyzed using a 20× objective, taken from three different sections.Collagen was quantified using ImageJ.

Immunohistochemistry (IHC)
The liver was fixed in a 10% formaldehyde solution for 8 h for immunohistochemistry studies.After dehydrating the samples, the tissue samples were embedded in paraffin at 60 • C. Five-micron thick tissue sections were cut transversely on a microtome (Zeiss, Jena, Germany).The tissue was deparaffinized and then hydrated in phosphate buffer (0.2 M) for 10 min.The sections were washed with PBS, incubated with 3% hydrogen peroxide to inhibit endogenous peroxidation for 30 min and then treated with 10% goat serum for 30 min; these procedures were performed at 22 • C in a humidified chamber.Next, all the sections were then incubated overnight at 4 • C with the primary rabbit antibody F4-80 (Cell Signaling, U.S.A.) diluted in triton X-100 (0.3%) (1:200).The sections were then incubated with the Biotinylated goat anti-polyvalent (Abcam, U.S.A.) for 10 min.Then the sections were washed with PBS and applied Streptavidin peroxidase (Abcam, U.S.A.) was for 10 min and washed with PBS.After, the 3,3-diaminobenzidine (DAB) was utilized as the chromogen, yielding the overall brown colour.Negative controls were conducted for each antibody by omitting the primary antibody.At least two samples from each animal were independently analyzed.Images were acquired with a Nikon DS-R1 digital camera connected to a Nikon Eclipse Ti-U microscope.The immunostained sections were quantified by using the ImageJ software.At least 5 areas per slide were selected and photographed blinded.The expression of F4/80 was calculated as follows: the total area of the liver (specimen) was outlined, and the area occupied by cells expressing F4/80 was quantified using the image analyzer within the reference area.A color for F4/80 was pre-defined and applied to the selected area.The result was expressed as a percentage of the positive area concerning the total area.

Protein expression
Animals were deeply anesthetized with isoflurane (5%), and after the loss of corneal reflexes, the abdominal wall was opened, and the liver was removed.After that, pieces of the liver (∼50 mg) were homogenized in RIPA buffer with protease and phosphatase inhibitor cocktail (TermoFisher, U.S.A.).The tissue extracts were centrifuged at 15000 g, at 4 • C, for 20 min.The protein content of the supernatants was measured by the Bradford method.Aliquots of the

Gene expression
Total RNA from the liver was extracted with a Total RNA purification kit (Cellco, Brazil) and reverse transcribed into cDNA (High-Capacity cDNA kit, Applied Biosystems, Waltham, MA, USA).Gene expression was evaluated by RT-PCR using Rotor-Gene Q (Qiagen, Hilden, Germany) and SYBR Green as the fluorescent dye (Platinum ® SYBR ® Green qPCR Supermix UDG, Invitrogen, Waltham, MA, U.S.A.).The gene expression analysis was carried out using a method previously described [20,21].The samples were measured in duplicate.The primers used are described in Table 1.

Statistical analysis
The results were analyzed using the GraphPad Prism version 9.0 ® program (GraphPad Software, La Jolla, CA, U.S.A.).The minimum sample size per group for each parameter analyzed was defined by an n sufficient to analyze the distribution of samples through the 'D Agostino and Pearson omnibus normality test' recommended by the GraphPad Prism version 9.0 ® program.All samples were evaluated for normal distribution and subjected to either a one-way ANOVA followed by the post-hoc Bonferroni test (Bonferroni Multiple Comparison Test) (P<0.05).The results were expressed as mean + − standard error of the mean (mean + − SEM).

Effect of CCl 4 on the accumulation of lipids in animals fed a high-fat diet
After feeding a high-fat diet and injecting CCl 4 for 4 weeks, the animals were euthanized, and the liver and plasma were collected for analysis.According to the morphological parameters, the animals fed with HFD+CCl 4 presented hepatic steatosis, as can be seen in the image below (Figure 1A), and in the quantification of fat droplets in these images, with an increase in this lipid deposition of ∼13%, when compared with animals fed a standard diet (0.2%) and HFD (1.2%) group.The HFD and SD groups had not significant difference (Figure 1B).Tissue triglyceride measurement was also increased in the HFD+CCl 4 group (12.5 + − 0.8 mg/g of tissue weight) when compared to the SD group (9.5 + − 0.5 mg/g of tissue weight) and HFD (11.2 + − 0.6 mg/g of tissue weight), without significant difference between the SD and HFD groups (Figure 1C).Plasma triglycerides showed no significant difference between groups (SD: 78.4 + − 4.2, HFD: 82.0 + − 0.4, HFD+CCl 4 : 84.0 + − 3.3 mg/dL) (Figure 1D).Plasma cholesterol was increased in animals from the HFD+CCl 4 group (163.3 + − 20.5 mg/dL) when compared with the SD group (61.1 + − 3.9 mg/dL) and HFD (95.0 + − 5.5 mg/dL), without significant difference between the SD and HFD groups (Figure 1E).Tissue cholesterol measurement was increased in 57% in both HFD and HFD+CCl 4 groups, when compared with SD (Figure 1F).The increase of lipids in the tissue can promote lipid peroxidation, so we evaluated lipid peroxidation markers such as malondialdehyde (MDA).However, we observed no significant difference in MDA in either plasma or tissue between the groups (Figure 1G,H).We also evaluated the gene expression of enzymes related to de novo lipogenesis, such as Acetyl-CoA carboxylase (ACC) and Fatty acid synthase (FASN), and observed an increase by 9-and 5-fold, respectively, in the HFD+CCL 4 group compared with the SD and HFD groups (Figure 1I,J).Thus, the high-fat diet with fructose and CCl 4 promotes hepatic steatosis.

Effect of CCl 4 in the hepatic inflammation in animals fed a high-fat diet
CCl 4 showed that it triggers an inflammatory process in the liver of animals fed a high-fat diet by promoting an increase of approximately ∼2 times in macrophage activity, as can be seen in the image below and its quantification (Figure 2A,B), in addition to the increase of 6 times in the gene expression of the macrophage marker F4/80, and pro-inflammatory cytokines, 9 times for IL-1b and 5 times for TNFa (Figure 2C-E).In addition to the 190% increase in inflammatory pathway protein phosphorylation (IKKbp) (Figure 2F), these data were compared with the SD and HFD groups.JNK and NFkB activity did not differ significantly between groups.Thus, treatment with CCl 4 has been shown to stimulate the inflammatory process in the liver.

Effect of CCl 4 on collagen deposition in animals fed a high-fat diet
Parameters related to liver fibrosis were analyzed, and it was seen that the animals fed with HFD and treated with CCl 4 had an increase of 4-fold in the liver collagen deposition compared with the SD and HFD groups.These results demonstrated an increase in collagen deposition in the HFD+CCl 4 group, as seen in the figure below and the quantification of collagen through the images (Figures 3A,B).In addition, hydroxyproline, an amino acid that is in the composition of the collagen, was measured, and there was also an increase of ∼130% in this parameter in the HFD+CCl 4 group when compared with the SD and HFD groups, which did not have a significant difference between them (Figure 3C).Liver enzymes were analyzed, alanine aminotransferase (ALT) and aspartate aminotransferase U/L, respectively), both had no significant difference between HFD and HFD+CCl 4 (Figure 3D,E).In addition, the expression of some genes that encode proteins related to collagen production, such as TGF-b and collagen 1a (col1a), was demonstrated.Col1a expression was increased 5 times in the HFD+CCl 4 group compared with the SD and HFD groups, which did not show a significant difference between them.At the same time, the gene expression of TGF-b showed no significant difference between groups (Figure 3F,G).These results show that the high-fat diet with fructose and CCl 4 stimulates steatohepatitis's progression since liver collagen deposition was increased.

Effect of CCl 4 in the body mass and glucose metabolism in animals fed a high-fat diet
The total body mass of the animals increased with the high-fat diet (25.6 + − 0.6 g) and in the HFD animals treated with CC l4 (25.9 + − 0.5 g) when compared with the SD (23.1 + − 0.7 g) (Figure 4A), and there was no significant difference between the HFD and HFD+CCl 4 groups.Similar results were obtained concerning fat mass (SD: 3.1 + − 0.1 g; HFD: 4.4 + − 0.3 g; HFD+CCl 4 : 3.9 + − 0.1 g).No significant difference was observed in the lean mass of the animals between the groups (Figures 4B,C).After 4 weeks of a high-fat diet and treatment with CCl 4 , the animals were submitted to a glucose tolerance test (GTT).It was seen that both animal groups fed with HFD and HFD treated with CCl 4 were glucose intolerant, maintaining increased values during the entire glycemic curve, with values for the area under the curve of 355.5 + − 26.3 mg/dL-min and 344.7 + − 17.6 mg/dL-min, respectively, when compared with the control SD (236.2 + − 13.7 mg/dL-min) (Figure 4D).Fasting glucose increased in both the HFD (172.6 + 11.7 mg/dL) and HFD+CCL 4 (184.9+ 7.8 mg/dL) groups when compared to the SD (138.0 + 5.8 mg/dL) (Figure 4E).Furthermore, a reduction in the activity of AKT, a protein of the insulin signaling pathway, was observed in both HFD (46%) and HFD+CCl 4 (41%) groups compared with the SD group (Figure 4F).Thus, the high-fat diet promotes glucose intolerance and impaired insulin signaling, results that are maintained with treatment with CCl 4 .

Discussion
We investigated the effect of CCl4 toxin and a high-fat diet enriched with fructose on steatohepatitis and glucose metabolism parameters.We observed that CCl 4 , coupled with a high-fat diet and fructose, stimulates an increase in hepatic triglycerides and plasmatic cholesterol, as well as collagen deposition in the liver and hepatic transaminases in the plasma.In addition, we saw an increase in macrophage activity, proteins and cytokines pro-inflammatory.Moreover, the high-fat diet was able to promote glucose intolerance and decrease the protein activity of the insulin pathway.
CCl 4 applied orally can lead to increased liver weight, lipids accumulation, liver enzyme activity, inflammation, damage and cell death, which leads to the development of fibrosis, cirrhosis and hepatocellular carcinoma if this oral exposure is long-term [22,23].The isolated application of CCl 4 (dose: 0.5-0.7 ml/g body weight), without the presence of a high-fat diet, twice a week and for 6 weeks, or three times a week for four weeks, demonstrated the deposition of collagen in the liver, which was already characterized as fibrosis.However, this work did not characterize lipid deposition [23].
Animal model fed only with a high-fat diet promotes hepatic steatosis after 12 weeks of diet [24].Our results have shown that a high-fat and fructose-enriched diet for only 4 weeks, associated with intraperitoneal injections of CCl 4 three times a week during the same period as the diet, promotes an increase in the deposition of lipids in the liver.Fructose has been associated with non-alcoholic fatty liver disease, and this has been confirmed in different studies [25][26][27].It is not fructose itself that causes the accumulation of triglycerides.The increase of fructose promotes the activation of de novo lipogenesis and the blocks of the fatty acid oxidation [28].In our study, we demonstrated an increase in enzymes related to de novo lipogenesis, such as ACC and FASN, in the group that received fructose, which was also the group in which CCl 4 was administered.
In addition, we showed an increase in collagen deposition in the liver, both by quantification of collagen by images and by measuring hydroxyproline in the liver, an amino acid that makes up collagen [29], associated with an increase in gene expression 1 alpha collagen in the tissue.These data corroborate results previously published [23].Other studies have described that HFD with CCl 4 administration has promoted inflammation and apoptosis, leading to fibrosis in mice.In one of these studies, the animals were fed for 12 weeks with HFD, and in the last 4 weeks, they were treated with CCl4 twice a week (dose: 0.05-0.1 ml/kg) [14].In another study, the animals were fed a Western diet, a high-fat diet enriched with cholesterol, for 12 and 24 weeks, with simultaneous administration of CCl 4 once a week at doses of 0.32 mg/g.Also, they presented steatosis and fibrosis [13].Concerning our study, we reduced the time on a high-fat diet and increased the dose (0.5 ml/g) and the number of applications per week of CCl 4 (three times a week) in the animals, and we obtained similar results.
Non-alcoholic fatty liver disease (NAFLD) is classified into two subtypes, non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), the progressive form of the disease.NASH is characterized by steatosis, lobular inflammation and ballooning of hepatocytes, with or without fibrosis [30].A previously described mechanism that leads to NAFLD is related to hepatic energy imbalance, where the increased energy intake by an elevated consumption of carbohydrates and lipids exceeds the oxidation of this energy in CO2 or exports it as very-low-density lipoproteins (VLDLs).This imbalance promotes the accumulation of lipids as TAG in the liver [31][32][33][34].The accumulation of lipids in the liver is toxic, generating what is known as lipotoxicity, as well as CCl 4 itself, which is a hepatotoxin.This lipotoxicity disrupts normal cellular processes, promoting cell damage and death, leading to inflammatory processes and the replacement of functional cells by fibroblasts and extracellular matrix [35].
Our model presented hepatic steatosis and fibrosis, as previously described, and an inflammatory condition, which we demonstrated through of increase in the macrophage activity by the F4/80 marker, by the increase in the gene expression of F4/80 and pro-cytokines (IL-1B and TNF-α), in addition to increased activity of the IKKB protein, proteins of the inflammatory pathway.From this set of results, our model represents a model of steatohepatitis.In our laboratory, another NASH model was established using ApoE knockout mice and fed with different diets, HFD and western diet, both groups showed similar results to those described in this study, such as imbalance in glucose metabolism, hepatic steatosis and inflammation, as well as increased collagen deposition in the liver when compared with the group fed the standard diet [6].
Type 2 diabetes is considered a risk factor for the progression of NASH, increasing the progression of fibrosis and subsequently may lead to cirrhosis, promoting increased mortality from liver diseases.Like obesity and dyslipidemia, however, diabetes is still a significant risk factor [36].Given this strong relationship between Type 2 diabetes and NASH, we evaluated some parameters related to glucose metabolism, and we saw that the high-fat diet promoted an increase in the body mass of the animals and fat mass.In addition, we observed glucose intolerance, fasting glucose increased and damage to the insulin signaling pathway, which may indicate insulin resistance.A high-fat diet promotes insulin resistance in adipose tissue, which leads to hyperglycemia and lipotoxicity due to excess of lipolysis, as insulin plays a crucial role in glucose uptake and inhibition of lipolysis in adipose tissue [3,37,38].In addition to the adipose tissue, insulin acts on the liver, inhibiting gluconeogenesis and glycogenolysis.Once there is a change in glucose metabolism, such as insulin resistance triggered by HFD, whether, in patients or an animal model, the liver will produce more glucose, which leads to increased plasma glucose [35,37].

Figure 3 .
Figure 3. Carbon tetrachloride promotes increase of collagen in the liver of animals fed a high-fat diet Representative images of collagen deposition in the liver.(A) Histological sections stained with Sirius Red.(B) Collagen quantification.(C) Hydroxyproline measurement in liver tissue.(D) Alanine aminotransferase (ALT) measurement in plasma.(E) Plasma aspartate aminotransferase (AST) measurement.(F,G).Genes expression that encode proteins that participate in collagen production.The black bar in the images represents a scale of 100 μm and objective 20×.Columns and vertical bars represent mean and e.p.m., respectively.One-way ANOVA followed by Bonferroni post-test, *P<0.05;**P<0.01;****P<0.0001(compared with SD), # P<0.05; #### P<0.0001 (compared with HFD) (Collagen quantification by sirius red, n=5; hydroxyproline in tissue, n=4; Plasma levels of ALT and AST, n=5; Gene expression, n=3-5).