Investigation of Non-alcoholic steatohepatitis development in newly designed short-term insulin resistance-fatty liver rat models. From chronic to acute

doi:10.21203/rs.3.rs-3810049/v1

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Investigation of Non-alcoholic steatohepatitis development in newly designed short-term insulin resistance-fatty liver rat models. From chronic to acute

https://doi.org/10.21203/rs.3.rs-3810049/v1

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Background

The progression of insulin resistance (IR) and diabetes into non-alcoholic steatohepatitis (NASH) is a chronic process. The pathogenesis of diabetes progression into NASH is very complex and not fully understood. The lack of reproducible IR-NASH animal models that mimic human pathogenesis is the main roadblock. Therefore, we designed this study to investigate the development of IR-NASH within one month using new combinations of diets, fructose, and a small dose of streptozotocin (MFD or HFD/HFrD/STZ). And compare them with the known IR-NASH model using HFD/STZ.

Methods

Thirty-two male Wistar rats were randomized into four different groups and fed by either a different combination of diets or a chow diet for one month. After two weeks, a single dose of STZ was injected in all groups except the healthy group to develop diabetes. Body weights, animal mortality, eating, and drinking behaviors were recorded during the study. Glucose intolerance, hepatotoxicity indices, and fat accumulation were investigated. Also, we examined the hepatic histopathological alternations in rats.

Results

Animals’ body weights, eating, and drinking behaviors were extremely affected in high fructose diet (HFrD)-feed groups. Also, all groups showed a significant IR and glucose intolerance. However, the clear progressive NASH with aggressive hepatic steatosis, inflammation, and fibrosis was visualized in the investigated liver sections’ of HFrD-feed groups only. At the same time, the HFD/STZ group showed progressive non-alcoholic fatty liver disease (NAFLD) only.

Conclusion

MFD or HFD/ HFrD/ STZ models were successful short-term NASH models that can be used for pharmacological screening studies and dose selection. While HFD/STZ is a suitable model for chronic pharmacological investigations.

non-alcoholic steatohepatitis

insulin resistance

high fat diet

high fructose diet

liver fibrosis

Modern life besides the consequences of coronavirus (COVID-19) disease lockdown showed high consumption of junk food and beverages riching in different obesogenic factors such as high fats and sugars (Nolan et al., 2011; Singh et al., 2021). Lack of physical activity and staying home for a long time together with the presence of genetic factors are some of the causes of obesity and fat accumulation (Nolan et al., 2011; Dabelea, and Pettitt, 2001). Total and saturated fats enriched in the western diet play a fundamental role in type 2 diabetes (T2D) development (van Dam et al., 2002).

Diabetes is a metabolic syndrome producing intracellular metabolic changes in most human tissues including the liver. While IR and accumulated fats are strongly associated with NAFLD (Bugianesi, 2010). Although NAFLD is a mirror image of systemic insulin impairment, it is considered as one of the major risk factors of diabetes (Adams, 2009; Bugianesi, 2010). NAFLD represents a wide range of diseases starting with simple steatosis to NASH, progressing into cirrhosis and hepatocellular carcinoma. Triglyceride (TG) accumulation, inflammation, oxidative stress (OS), and mitochondrial dysfunction are the main factors of NAFLD pathogenesis (Takaki et al., 2013).

Different animal models are designed to study diabetes and its complications and therefore, investigate new treatments (Islam, and du Loots, 2009). Some of them are genetic models and others are using a diet with or without diabetes-inducing agents such as STZ or alloxan (Islam, and du Loots, 2009; Lozano et al., 2016).

Feeding rats, a high-fat diet alone for two months is easy to induce metabolic disorder without hyperglycemia induction (Auberval et al., 2014). Also, IR induction through the combination of a 60% HFD and a small dose of STZ is considered the most promising model for IR and T2D (Chao et al., 2018). Besides, Mohammed et al., 2020 have investigated HFD/STZ-IR model in NASH initiation and progression and showed positive results. Although the histopathological examination revealed the presence of fibrous connective tissue and microvascular steatosis, it showed a few macrovascular oil droplets and mild NASH. However, some epidemiological studies correlated the amount of sugar ingestion and diabetes; showing the strong impact of fat and sugar such as fructose combination in metabolic syndrome than either fat or sugar alone (Basu et al., 2013). García-Berume et al. 2019 showed the severe liver injury presented in profound hepatic steatosis and inflammation in rats’ liver feed by HFD and 25% fructose diet for six weeks. But they did not investigate the presence of fibrosis and NASH in liver sections. Only Barrièr et al., 2017 have studied the effect of a high-fat/high-fructose (HFD/HFrD) diet and low-dose STZ in the development of liver fibrosis and NASH as one of the complications of diabetes. Despite the promising results of this model, it consumed too long time and exhaust a high number of diets and drugs.

For this reason, we designed this study to investigate the effect of combining different percentages of HFD with 60% fructose together with single to double doses of STZ as an IR-diabetes model in NASH initiation and development. And compare the degree of liver injury and NASH of the two novel models with the previously published stable HFD/STZ model of IR-NASH.

2.1 Drugs and Chemicals

The following materials were purchased from Sigma Chemical co. (St. Louis, Mo, USA): STZ, citric acid monohydrate (C₆H₈O₇•H₂O), trisodium citrate dihydrate (C₆H₅O₇Na₃•2H₂O), fructose, and D-glucose. The other chemicals used were of the highest grade commercially available.

2.2 In-vivo study

2.2.1 Animals

The study was established according to the ethical guidelines of the animal house of Drum Tower Hospital, Nanjing Medical University, Nanjing, China. Eight weeks male Wistar albino rats (190–225 g) were obtained from the animal house of Drum Tower Hospital, Nanjing, China. Rats were acclimated in an air-conditioned atmosphere of 25°C with alternatively 12-hour light and dark cycles. Before experimentation, animals were accommodated for one week and kept on a standard diet pellet (Research Diet INC, New Brunswick, NJ 08901 USA) included not less than 20% protein, 5% fiber, 3.5% fat, 6.5% ash, and a vitamin mixture beside water ad libitum.

2.2.2 Experimental design

After the accommodation, thirty-two animals were randomized into four distinct groups’ eight animals in each and treated for one month as follows (Fig. 1):

Group 1 was served as control and received a normal diet and 0.25/kg, Interperitoneally (i.p) 0.1 M sodium citrate buffer (PH: 4.4) on the same day of ST Z injection.

Group 2 was served as a high-fat diet /STZ (HFD/STZ) model and given HFD 60% fats (Research Diet INC, New Brunswick, NJ 08901 USA) for one month and double doses of STZ 35mg/kg, i.p. (prepared in 0.1M sodium citrate buffer PH 4.4) on day 15 and 22. (Srinivasan et al., 2005; Chao et al., 2018).

Groups 3 was served as middle fat diet/ high fructose diet/ STZ (MFD/HFrD/STZ) model and given MFD 45% fats (Research Diet INC, New Brunswick, NJ 08901 USA), HFrD of 60% dissolved in distilled water for one month and double doses of STZ 35mg/kg, i.p. (prepared in 0.1M sodium citrate buffer PH 4.4) on days 15 and 22.

Groups 4 were served as HFD/HFrD/STZ model and given HFD 60% fats, HFrD of 60% dissolved in distilled water for one month, and double doses of STZ 35mg/kg, i.p. (prepared in 0.1M sodium citrate buffer PH 4.4) on day 15 and 22.

The amount of ingested diet and drink were measured twice weekly. Also, animals’ body weights and FBG were measured twice a week using ACCU-Check (Roche) glucometer.

3.3 Oral glucose tolerance test (OGTT), collection of animals’ blood and tissue samples

After four weeks from starting the experiment, rats were fastened for 12 hrs., and OGTT was performed using 20% of glucose solution orally given to all groups and blood glucose was determined (0, 30, 60, 90, 120 min) using ACCU Chick Glucometer (Roche). The day following OGTT, rats were anesthetized using isoflurane inhalation and blood samples were collected from the retro-orbital plexus and allowed to clot. The serum was separated by centrifugation at 4500 rpm for 10 min and used for the assessment of liver function tests. Then, rats were sacrificed, and specimens from the three major lobes of each liver were fixed in 10% formalin for histopathological examination.

3.4 Histopathological examination using Hematoxylin and eosin (H&E) and Masson's trichrome stains

After liver dissection, liver specimens were fixed in 10% formalin for one day and paraffin blocks were prepared. Then, sledge microtome was used for liver sectioning at 4 µm thickness followed by staining with either H&E or Masson’s trichrome stains which were previously described (Lillie and Ashburn, 1943; Bancroft and Gamble, 2008). Finally, all sections were visualized under the light microscope, histopathological alternations were identified, and fibrous connective tissue was quantified using Image J imaging software (Image J, 1.46a, NIH, USA) and expressed as area percentage.

3.5 Biochemical investigation of liver injury and fats accumulation

Liver injury parameters such as serum levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were calorimetrically determined using commercially available kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instruction. Also, fat accumulation was determined through the estimated serum levels of total cholesterol (TC) and TG using readymade kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Besides, the liver index was calculated according to the following formula (liver weight/body weight).

2.6 Statistical Analysis

The statistical significance was detected by one-way analysis of variance (ANOVA) followed by Tukey-Kramer post-hoc test. For initial statistical analysis, Instat software package (version 3) was used, and significance was considered when p < 0.05. whereas Graph pad prism® software package, version 7 (GraphPad Software, CA, USA) was used for the final statistical analysis and charts establishment. All results were expressed as mean ± standard errors mean (S.E.M).

3.1 Mortality rate, animals’ body weight, chow, and drink consumption of different NASH rat models

According to the results in Table 1, there was no mortality in the first two weeks of diet in all NASH models. But after STZ injection, animals’ mortality started in the groups provided by HFrD particularly in HFD/HFrD/STZ group. One rat died after three days of the 1st STZ injection in the HFD/HFrD/STZ group. While three animals died in MFD/HFrD/STZ group after the 2nd dose of STZ exceeded the total number of animals that died in the HFD/HFrD/STZ group. On the other hand, no animal death was recorded in both normal control and HFD/STZ groups during the whole experiment.

Following the chart in Fig. 2A, we can see the significant increase of animals’ body weight in the HFD/STZ group during the first two weeks of the experiment by (25, 22.6%, respectively) when compared to the normal control group. However, feeding animals with either MFD or HFD besides the HFrD for two weeks did not significantly increase the animals’ body weight or even decrease after the 1st STZ injection in comparison to the normal control animals. Although the continuous decrease of HFD/STZ animals’ body weight after the 1st dose of STZ, is still significantly higher than normal control animals by 9% and returns to the normal weight after the 2nd dose of STZ. The dramatic decrease in animals’ body weight after the 2nd dose of STZ in both MFD and HFD feed groups besides the HFrD by (31, 24.5, respectively) when compared with the control animals support the high mortality rate in these groups. Comparing the change in animals’ body weight between different animal models showed a significant increase in HFD/STZ body weights than both MFD/HFrD/STZ and HFD/HFrD/STZ groups during the whole experiment. Despite the absence of significant difference in animals’ body weights of both MFD/HFrD/STZ and HFD/HFrD/STZ groups during the first three weeks of the experiment, a 9% significant weight reduction in MFD/HFrD/STZ group was observed after the 2nd dose of STZ in comparison with HFD/HFrD/STZ group (Fig. 2A).

In HFD/STZ group, the amount of diet chewed was significantly decreased after the 1st and 2nd STZ injections (18.5, 20%, respectively) in comparison to the control group (Fig. 2B). But the amount of water consumption was significantly higher than control in the first three weeks of the experiment and normalized after the 2nd STZ injection (Fig. 2C). Feeding animals with HFrD had a toxic effect in the amount of either MFD or HFD chewed which significantly decreased after the first and second weeks of the experiment by (35.5, 51.5%, respectively) in comparison with the control group. A huge reduction was observed in the amount of chewed diet in both MFD/HFrD/STZ and HFD/HFrD/STZ groups after the 1st and 2nd doses of STZ by (66.3, 66.5%, respectively) in comparison with the control group (Fig. 2B). But there was no significant difference between the two groups in eating behavior during the whole experiment although a significant difference in the amount of fructose consumption was found. Animals in MFD/HFrD/STZ drank (26%) fructose less than normal control while animals in HFD/HFrD/STZ drank (12%) fructose higher than the control during the 1st week. In the 2nd week, fructose consumption increased by (24%) in MFD/HFrD/STZ and normalized in HFD/HFrD/STZ animals in comparison with the normal control. After the 1st dose of STZ, fructose consumption significantly decreased by 23% in both groups of MFD/HFrD/STZ and HFD/HFrD/STZ in comparison with the normal control. And the dramatic decrease continued after the 2nd dose of STZ in comparison with the normal control without any significant difference between the two groups (Fig. 2C).

3.2 OGTT and fasting blood glucose (FBG) in different NASH rat models

In Fig. 3A, the highly significant baseline (0 minutes) of FBG in the HFD/STZ group increased (4 folds) after 30 min of the administrated oral glucose in OGTT in comparison with the control group confirming the IR. Also, the FBG was (2.5 folds) significantly higher than the control group (Fig. 3B). On the other hand, HFrD-fed groups besides the MFD or HFD didn’t show any significance in (0 minutes) of FBG in OGTT from the control or even from each other although the significant difference between them and HFD/STZ group. But after 30 min of oral glucose administration, FBG of MFD/HFrD/STZ increased (4.5 folds) exceeding the (3 folds) increases of the HFD/ HFrD/STZ group when compared to the normal control group (Fig. 3A). Also, the measured FBG levels in Fig. 2B of MFD/HFrD/STZ and HFD/ HFrD/STZ groups were (2 folds) significantly higher than the normal control with no significant difference between them and were significantly lower than HFD/STZ group.

4.3 Biochemical investigation of liver injury and fats deposition of different NASH rat models

Insulin resistance (IR) and T2D are leading causes of liver injury together with excessive fat deposition progress into NASH. Although HFD/STZ group didn’t show a highly significant liver index from the control, it still showed significant hepatomegaly which was significantly lower than HFD/HFrD/STZ group (Fig. 4A). But the investigated rat livers’ from MFD/HFrD/STZ and HFD/ HFrD/STZ groups showed highly significant hepatomegaly by (1.4 folds) in comparison to the control group. Measured serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of HFD/STZ group showed (2.5, and 2 folds, respectively) increase when compared to the normal control showing chronic liver injury (Fig. 4B and C, respectively). While the ALT levels of MFD/HFrD/STZ and HFD/ HFrD/STZ groups were (4 and 6 folds, respectively), higher than the control group indicating excessive liver injury. Also, the AST levels were significant by (2 and 2.5folds, respectively) in comparison to the normal control group (Fig. 4B and C, respectively).

Although the duplicated serums TC level in HFD/STZ animals, wasn’t significantly higher than the control animals. At the same time, serum TG were (3 folds) higher than the control level (Fig. 4D and E, respectively). While the measured lipid profile of MFD/HFrD/STZ and HFD/ HFrD/STZ groups showed a (2 and 5 folds, respectively) increase in TC and (4 and 6 folds, respectively) increase in TG levels in comparison to the control group (Fig. 4D and E, respectively).

3.4. Histopathological alternations and fibrosis identification of different NASH rat models

The normal control animals showed normal liver sections with intact hepatic architecture with normal hepatocytes after H&E routine stain. While the H&E-stained liver sections of HFD/STZ animals showed inflammatory infiltrates, excessive microvascular steatosis, and fibrous connective tissue confirming a successful NASH model Table 2 (Fig. 5). Providing animals with HFrD resulted in an excessive liver injury with progressive NASH showed in H &E-stained liver sections of both MFD/HFrD/STZ and HFD/HFrD/STZ groups (Table 2 and Fig. 5). For fibrosis detection, liver sections of different groups stained with Masson's trichrome stain were imaged then quantified using Image J software. The healthy sections of the control group showed minimal blue stains with no fibrosis. HFD/STZ sections showed significant fibrosis (7 folds) higher than normal control sections. Both MFD/HFrD/STZ and HFD/HFrD/STZ groups showed an excessive blue stain of the progressive fibrosis with defined NASH. The quantified fibrous connective tissue of MFD/HFrD/STZ sections was ten times higher than normal. While in HFD/HFrD/STZ sections it was sixteen folds higher than healthy animals (Fig. 6).

This study is a pharmacological screening of different new NASH rat models in compression with the previously studied model. We investigated a combination of HFrD with either MFD or HFD together with multiple low doses of STZ inducing either IR or type 2diabetes and its progression into NASH. Using HFD with multiple low doses of STZ was previously studied and proved as a most stable IR and T2D animal model-human analogue (Chao et al., 2018). Also, the pathogenesis in which HFD/STZ progresses from diabetes into NASH was previously studied and confirmed (Mohammed et al., 2020; Oligschlaeger and Shiri-Sverdlov, 2020).

Researchers showed the importance of fructose to induce NASH and accelerate its progression, thus they used different diet combinations including high fructose with or without accelerating hepatotoxic or diabetes-inducing agents to induce steatosis (Oligschlaeger and Shiri-Sverdlov, 2020). Only one published study by Barrière et al., 2018, studied hepatic steatosis and fibrosis as a complication of T2D using 25% HFD, 46.5% HFrD for 56weeks, and a small dose of STZ (25 mg/kg) injected after two weeks of diet and repeated three times during the study. Although the promising effect of this model, it takes too long and a high amount of diet. Therefore, we designed this study for one month with a new combination of different percentages of fat diets with 60% HFrD beside single to double doses of STZ as a diabetes-inducing agent to accelerate NASH induction and progression.

According to Mohammed et al, 2020, feeding animals with an HFD diet for a month resulted in significant obesity and compensated the toxic effect of STZ injections without any animal death and slow NASH progression. Fructose added to HFD showed a higher percentage of hepatocytes damage, steatosis, and inflammation reflected in animals’ body weight, eating, and drinking behaviors (García-Berumen, et al., 2019). In HFrD with either MFD or HFD groups, animals didn’t show obesity before STZ injections confirmed by the previous study of Lee et al., 2015. After STZ injections, the toxicity augmented and resulted in an extreme reduction in animals’ body weight and animal death. From the results, the number of dead animals in the MFD/HFrD/STZ group at the end of the experiment exceeded the number in HFD/HFrD/STZ group explaining the role of the protective effect of the high percentage of fats in HFD. Observing animals’ eating and drinking behavior in HFD/STZ group was increasing normally before STZ and highly affected after STZ (Mohammed et al., 2020). While HFrD fed groups eating behaviors were identical during the whole experiment and extremely lower than both normal control and HFD/STZ groups showing the high toxicity of HFrD. But the amount of fructose drunk was significantly higher in HFD/HFrD/STZ group than MFD/HFrD/STZ group before STZ injection and became the same after STZ injection. This is because of the endotoxemic effect of fructose which is considered a macronutrient that deteriorates the intestinal barrier through hepatic de novo lipogenesis (DNL) stimulation, and fat deposition (Vos and Lavine 2013; Softic et al., 2016; Todoric et al., 2020).

In OGTT, HFD/STZ animals showed a high FBG baseline with a typical IR-OGTT pattern confirming a successful IR model (Mohammed et al., 2020). While both MFD/HFrD/STZ and HFD/HFrD/STZ groups showed T2D-OGTT pattern with lesser FBG baseline when compared with HFD/STZ group. It was surprising to see a higher response of MFD/HFrD/STZ than both HFD/HFrD/STZ and HFD/STZ groups after 30 and 60 mins of OGTT. In addition, the measured FBG levels of both groups were significantly higher than the normal control group showing a prediabetic phase with hyperinsulinemia, modest hyperglycemia, and glucose intolerance (Barrière et al., 2018).

To investigate the extent of liver injury, liver indexes were calculated, and different liver indices were measured. Due to the short duration of the experiment, HFD/STZ animals showed mild hepatomegaly with a highly significant increase in aminotransferases indicating liver injury and either non or a low TC and TG fat accumulation. On the other side, HFrD feed animals showed severe hepatomegaly, a highly significant increase in aminotransferases, and high significance in TC and TG fat accumulation indicating progressive liver injury with significant hepatic steatosis. These results are supported by Lozan et al., 2016 findings that showed significant hypertriglyceridemia and hypercholesterolemia in animals fed with a western diet together with 25% fructose solution for two months. The mechanism in which fructose increases fat accumulation is referred to as its carbohydrate nature. Excessive carbohydrates supply causes excessive Adenosine triphosphate (ATP) exhaustion due to the excessive carbohydrates’ phosphorylation leading to phosphate depletion, Adenosine Monophosphate (AMP) accumulation, and excessive TG synthesis (Lanaspa et al., 2012; Satapati et al., 2012; García-Berumen, et al., 2019). This situation is augmented when fructose is combined with a high or med fat diet unlike the administration of HFD alone.

Confirming NASH occurrence and presence of liver fibrosis, both routine histopathological investigation and Masson’s trichrome were performed. The H&E stained HFD/STZ liver sections showed 40% hepatic steatosis, inflammation, and presence of fibrous tissue confirmed by the quantified highly significant blue staining of Masson’s trichrome confirming mild NASH development. According to Mohammed et al, 2020; using 60% of HFD besides STZ for six weeks results in microvascular steatosis without a significant presence of macrovascular steatosis. While the study of García-Berumen, et al., 2019, showed a mixture of hepatic steatosis (40% of microvascular steatosis and 60% of macrovascular steatosis) in H&E liver sections of animals treated with the prepared HFD containing 10% lard.

On the other hand, investigated MFD/HFrD/STZ and HFD/HFrD/STZ H&E liver sections showed an excessive macrovascular steatosis together with microvascular steatosis and inflammation indicating severe liver injury and the excessive blue stains of Masson’s confirm the severe NASH. These findings are in the same line as the previously published study of García-Berumen, et al., 2019.

Comparing HFrD fed sections with HFD/STZ, liver sections of HFrD groups showed a clear progressive NASH with defined fibrous connective tissue while HFD/STZ group showed a NAFLD with mild NASH. These results were completely agreed with Lozan et al., 2016 findings which showed the superiority of HFrD/HFD to HFD alone in different axes during different periods of time. HFrD/HFD resulted in hepatic macrophages accumulation, triglyceride increase, and excessive glycogen secretion in comparison with the HFD alone. The mechanism in which NAFLD initiates and progresses into NASH stands in two arms. Hepatic TG accumulation is considered the first arm and oxidative stress followed by aggressive inflammation is the second arm (Day and James, 1998; Lozan et al., 2016). Thus, the development of NASH as a result of metabolic consequences of insulin resistance doesn’t depend on the hyperinsulinemia only but it depends on the changed adipocytokines output in fat tissue creating a pro-inflammatory and pro-fibrotic state (Lozan et al., 2016). Moreover, released tumor necrosis factor (TNF), an inflammatory cytokine, increases lipid secretion and is correlated with sterol regulatory element binding protein (SREBF1), ACACA, and fatty acid synthase (FASN) mRNAs secretions triggering NASH initiation and progression (Todoric et al., 2020).

In conclusion, we infer the combination of HFrD together with either MFD/STZ or HFD/STZ are diabetes models that progress into highly successful NASH models in a short time. But at the same time using a high dose of fructose with a low dose of STZ showed high hepatotoxicity and resulted in animal mortality. Also, HFD/HFrD/STZ NASH model is much better and safer than MFD/HFrD/STZ NASH model. Therefore, the standard HFD/STZ NASH model is suitable for chronic pharmacological investigations while HFrD-NASH models are much more suitable for pharmacological screening and short duration studies.

Insulin resistance: IR

Non-alcoholic steatohepatitis: NASH

Med-fat diet/ High-fat diet/ Streptozotocin: MFD/HFrD/STZ

High-fructose diet: HFrD

High-fat diet: HFD

Non-alcoholic fatty liver disease: NAFLD

Coronavirus disease: COVID-19

Type 2 diabetes: T2D

Triglyceride: TG

Oxidative stress: OS

Interperitoneally: i.p

Oral Glucose tolerance test: OGTT

Hematoxylin & eosin: H&E

Serum alanine aminotransferase: ALT

Aspartate aminotransferase: AST

Total cholesterol: TC

One-way analysis of variance: ANOVA

Standard errors mean :S.E.M

Fasting blood glucose: FBG

De novo lipogenesis: DNL

Adenosine triphosphate: ATP

Adenosine monophosphate: AMP

Tumor necrosis factor: TNF

Sterol regulatory element binding protein: SREBF

Fatty acid synthase: FASN

Messenger RNA: mRNA

Ethical Approval

The study was established according to the ethical guidelines of the animal house of Drum Tower Hospital, Nanjing Medical University, Nanjing, China.

Funding

This declaration is “not applicable”.

Availability of data and materials

All the data is included in the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

ED and BS conceived and designed research. ED conducted experiments and analyzed data. ED and BS wrote the manuscript.

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Table 1

Mortality rate of different NASH rat models
Groups/Mortality Rate	After the 1st week	After the 2nd week	After the 3rd week (1st dose of STZ)	After the 4th week (2nd dose of STZ)
Control	No death	No death	No death	No death
HFD/STZ	No death	No death	No death	No death
MFD/HFrD/STZ	No death	No death	No death	3 animals died
HFD/HFrD/STZ	No death	No death	1 animal died	1 animal died

Non-alcoholic steatohepatitis: NASH; High-fat diet/ Streptozotocin: HFD/STZ; Med-fat diet/ High-fat diet/ Streptozotocin: MFD/HFrD/STZ; high-fat diet/ High-fat diet/ Streptozotocin: HFD/HFrD/STZ.

Table 2

The degree of fatty changes and hepatic steatosis in different NASH rat models
Steatosis	Control	HFD/STZ	MFD/HFrD/STZ	HFD/HFrD/STZ
Fatty changes in the periphery of lobules	0	2	2	2
Microvesicular steatosis	0%	40%	15%	10%
Macrovesicular steatosis	0%	0%	30%	50%

Non-alcoholic steatohepatitis: NASH; High-fat diet/ Streptozotocin: HFD/STZ; Med-fat diet/ High-fat diet/ Streptozotocin: MFD/HFrD/STZ; high-fat diet/ High-fat diet/ Streptozotocin: HFD/HFrD/STZ.

No competing interests reported.

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Investigation of Non-alcoholic steatohepatitis development in newly designed short-term insulin resistance-fatty liver rat models. From chronic to acute

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1 Drugs and Chemicals

2.2 In-vivo study

2.2.1 Animals

2.2.2 Experimental design

3.3 Oral glucose tolerance test (OGTT), collection of animals’ blood and tissue samples

3.4 Histopathological examination using Hematoxylin and eosin (H&E) and Masson's trichrome stains

3.5 Biochemical investigation of liver injury and fats accumulation

2.6 Statistical Analysis

3. Results

3.1 Mortality rate, animals’ body weight, chow, and drink consumption of different NASH rat models

3.2 OGTT and fasting blood glucose (FBG) in different NASH rat models

4.3 Biochemical investigation of liver injury and fats deposition of different NASH rat models

3.4. Histopathological alternations and fibrosis identification of different NASH rat models

4. Discussion

List Of Abbreviations

Declarations

References

Tables

Additional Declarations

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