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Histological Analysis of Hepatic Steatosis, Inflammation, and Fibrosis in Ascorbic Acid-Treated Ovariectomized Mice
Biomed Sci Letters 2022;28:101-108
Published online June 30, 2022;  https://doi.org/10.15616/BSL.2022.28.2.101
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Mijeong Lee* , Suyeon Jeon* , Jungu Lee* , Dongju Lee* and Michung Yoon†,* *

Department of Biomedical Engineering, Mokwon University, Daejeon 35349, Korea
Correspondence to: Michung Yoon. Department of Biomedical Engineering, Mokwon University, Daejeon 35349, Korea.
Tel: +82-42-829-7581, Fax: +82-42-829-7590, e-mail: yoon60@mokwon.ac.kr
*Graduate student, **Professor.
Received April 26, 2022; Revised May 24, 2022; Accepted June 2, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
High-fat diet (HFD)-fed ovariectomized (OVX) female mice were used as an animal model of obese postmenopausal women. We investigated the effects of ascorbic acid on the histological changes induced in the liver. Plasma alanine aminotransferase levels and liver weights were higher in mice fed an HFD for 18 weeks than in mice fed a low-fat diet, effects that were inhibited by ascorbic acid. Similarly, mice fed an ascorbic acid-supplemented HFD had less hepatic lipid accumulation than did mice fed an HFD alone. Moreover, administration of ascorbic acid reduced inflammatory cells, including mast cells and CD68-positive cells, and inflammatory foci in the liver and inhibited hepatocyte ballooning. Hepatic collagen levels were lower in ascorbic acid-treated versus non-treated mice. These results suggest that ascorbic acid inhibits hepatic steatosis, inflammation, and fibrosis in obese OVX mice. Thus, ascorbic acid intake may be useful for postmenopausal women with nonalcoholic fatty liver disease.
Keywords : Ascorbic acid, Ovariectomized mouse, Liver, Steatosis, Inflammation, Fibrosis
INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is the most common type of chronic liver disease that includes hepatocellular steatosis, steatohepatitis, fibrosis, and cirrhosis (Clark, 2006; Erickson, 2009). The rapid increase in the incidence of NAFLD is in line with the worldwide increase in obesity and type 2 diabetes (Khashab et al., 2008). The NAFLD prevalence is estimated to be approximately 25% of the adult population worldwide and 80% of obese people (Powell et al., 2021).

A major feature of NAFLD is liver steatosis, which is histologically defined when more than 5% of hepatocytes have intracellular triglycerides (Kleiner et al., 2005). Hepatic steatosis may lead to hepatocyte injury and initiate the activation of hepatic inflammatory cells such as macrophages (Koyama and Brenner, 2017). Hepatocyte injury and hepatic inflammation can also cause liver fibrosis by stimulating hepatic stellate cells and secreting extracellular matrix including collagen (Cai et al., 2020).

Ascorbic acid, also known as vitamin C, inhibits diet-induced obesity and adiposity (Campion et al., 2006; García-Díaz et al., 2007; Garcia-Diaz et al., 2014). Ascorbic acid administration also resulted in weight loss in guinea pigs and reduces the number of adipocytes in rats (Senen et al., 2002; Jun et al., 2010); likewise, lower levels of circulating ascorbic acid increase body mass index and waist-to-hip ratio, ascorbic acid supplementation accelerates weight loss and reduces abdominal obesity in obese populations (Canoy et al., 2005; Johnston et al., 2007; Bahadoran et al., 2012). These results suggest that ascorbic acid can regulate obesity-related metabolic diseases such as NAFLD.

Female mice are more resistant to obesity than male mice, whereas ovariectomy removes the protection against weight gain in female mice (Hong et al., 2009). Furthermore, ovariectomized (OVX; surgical removal of both ovaries) female mice experience body weight gain and body fat levels similar to male mice (Hong et al., 2009). OVX mice have been used as animal models of postmenopausal women to study the metabolic syndrome and changes in metabolic parameters like weight gain (Nishio et al., 2019). In addition, postmenopausal women have a higher prevalence of NAFLD than men and premenopausal women (Ascha et al., 2010). Accordingly, we investigated the effects of ascorbic acid on histological changes in the livers of high-fat diet (HFD)-fed obese OVX female mice.

MATERIALS AND METHODS

Animal studies

For all experiments, seven-week-old female C57BL/6J wild-type mice (n=5/group) were obtained from Central Lab Animal (Seoul, Korea) and housed at Mokwon University with a standard 12-hr light/dark cycle. Prior to the administration of special diets, mice were fed standard rodent chow and water ad libitum. Mice were OVX, divided into three groups, and fed for 18 weeks with a low-fat diet (LFD, 13 kcal% fat, Research Diets, New Brunswick, NJ, USA), an HFD (45 kcal% fat, Research Diets), or an HFD supplemented with 5% (w/w) ascorbic acid (Sigma-Aldrich, Saint Louis, MO, USA) (HFD-AA). Plasma alanine aminotransferase (ALT) levels were measured using a blood chemical analyzer (Cobas 8000, c502, Grenzach-Wyhlen, Roche, Germany). All animal experiments were approved by the Institutional Animal Care and Use Committees of Mokwon University (permit number: NVRQS AEC-19), and followed National Research Council Guidelines.

Histological analysis

Liver tissues were fixed in 10% phosphate-buffered formalin for 1 day and subsequently embedded in paraffin. Paraffin-embedded liver sections (5 μm) were cut and stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) & eosin (Thermo, Runcorn, UK), toluidine blue (Sigma-Aldrich), and Masson's trichrome (Sigma-Aldrich) for microscopic examination. For Oil Red O staining, liver tissues were embedded in a frozen section compound (Leica, Wetzlar, Germany). Cryosections (7 μm) were stained with 0.5% Oil Red O (Sigma-Aldrich) and counterstained with Mayer's hematoxylin (Dako, Carpinteria, CA, USA). The stained sections were examined using ImageJ software (http://imajej.nih.gov/ij/).

Immunohistochemistry

Immunohistochemical staining was performed using a Mouse on MouseTM basic kit (BMK-6100, Vector laboratories, Burlingame, CA, USA) and a VectastainTM Elite ABC kit (PK-6100, Vector laboratories). Liver sections were incubated with an anti-CD68 primary antibody (1:200 dilution, ab955, Abcam, Canbrude, UK) and a biotinylated anti-mouse IgG secondary antibody (Vector laboratories). Stained sections were visualized using ImmPACTTM DAB (SK-4105, Vector laboratories) and then counterstained with Mayer's hematoxylin. The stained sections were investigated using ImageJ software.

Statistical analysis

All values were presented as mean ± standard deviation (SD). Statistical analysis was performed by the analysis of variance followed by Tukey's tests using SigmaPlot 13 (SPSS, Chicago, IL, USA). Statistical significance was set as P < 0.05.

RESULTS

Regulation of plasma ALT levels and liver weights by ascorbic acid

Eighteen weeks of HFD feeding increased plasma ALT levels by 230% compared with LFD feeding (P<0.05), whereas ALT levels were decreased by 64% in mice fed the HFD + ascorbic acid (HFD-AA mice) compared with mice with the HFD alone (HFD mice) (P<0.05; Fig. 1A). Liver weights of HFD mice were elevated by 90% compared with those of LFD-fed mice (LFD mice) (P<0.05; Fig. 1B). However, ascorbic acid treatment reduced liver weights by 43% in HFD mice compared with HFD mice that did not receive the ascorbic acid treatment (P<0.05). Representative liver photographs are shown in Fig. 1C.

Fig. 1. Effects of ascorbic acid on plasma ALT levels, liver morphology, and liver weight in obese OVX female mice.
Mice (n=5/group) were fed an LFD, an HFD, or an HFD-AA for 18 weeks. (A) Plasma ALT levels, (B) liver gross morphology, and (C) liver weight. All values are expressed as mean ± SD. #P<0.05 compared with LFD. *P<0.05 compared with HFD.

Inhibition of hepatic steatosis by ascorbic acid

Hematoxylin & eosin-stained liver sections revealed that lipid accumulation increased by 101% in HFD mice compared with LFD mice (P<0.05; Fig. 2A and B), whereas lipid droplets decreased by 61% in HFD-AA mice compared with HFD mice (P<0.05). Similarly, Oil Red O staining showed that hepatic triglyceride droplets increased by 521% in HFD mice compared with LFD mice (P<0.05; Fig. 2C and D). However, ascorbic acid treatment decreased liver triglyceride levels by 79% (P<0.05).

Fig. 2. Effects of ascorbic acid on hepatic steatosis in obese OVX female mice.
Mice (n=5/group) were fed an LFD, an HFD, or an HFD-AA for 18 weeks. (A) Hematoxylin & eosin-stained liver sections (original magnification ×100). (B) Relative lipid droplet area. (C) Oil-Red O-stained liver sections (original magnification ×400). (D) Relative Oil Red O-stained area. All values are expressed as mean ± SD. #P<0.05 compared with LFD. *P<0.05 compared with HFD.

Modulation of hepatic inflammation by ascorbic acid

Mast cells stained with toluidine blue increased by 118% in HFD mice compared with LFD mice (P<0.05; Fig. 3A and B). In contrast, mast cells decreased by 57% in HFD-AA mice compared with HFD mice (P<0.05). To confirm the effects of ascorbic acid on hepatic inflammation, liver sections were stained with an antibody against CD68, which is highly expressed in macrophages. CD68-positive cells increased by 56% in HFD mice compared to those in LFD mice (P<0.05; Fig. 3C and D). In contrast, ascorbic acid treatment decreased CD68-positive cells in HFD mice by 24% (P<0.05).

Fig. 3. Effects of ascorbic acid on mast cells and CD68-positive cells in livers of obese OVX female mice.
Mice (n=5/group) were fed an LFD, an HFD, or an HFD-AA for 18 weeks. (A) Liver sections were stained with toluidine blue. Dark arrows indicate mast cells (original magnification ×400). (B) Relative mast cell number. (C) CD68-positive macrophages are shown in brown color (original magnification ×100). (D) Relative CD68-positive area. All values are expressed as mean ± SD. #P<0.05 compared with LFD. *P<0.05 compared with HFD.

Hematoxylin & eosin-stained liver sections showed that hepatocellular ballooning increased by 200% in HFD mice compared with that in LFD mice (P<0.05; Fig. 4A and B). In contrast, ballooned hepatocytes decreased by 66% in HFD-AA mice compared with HFD mice (P<0.05). Lobular inflammatory foci increased by 270% in HFD mice compared with LFD mice (P<0.05; Fig. 4C and D). In contrast, ascorbic acid treatment decreased inflammatory foci in HFD mice by 63% (P<0.05).

Fig. 4. Effects of ascorbic acid on hepatocyte ballooning and inflammatory focus in obese OVX female mice.
Mice (n=5/group) were fed an LFD, an HFD, an HFD-AA for 18 weeks. (A) Hematoxylin & eosin-stained liver sections. Dark arrows indicate ballooned hepatocytes (original magnification ×400). (B) Hepatocyte ballooning score. (C) Hematoxylin & eosin-stained liver sections. Dark arrows indicate inflammatory foci (original magnification ×200). (D) Lobular inflammation score. All values are expressed as mean ± SD. #P<0.05 compared with LFD. *P<0.05 compared with HFD.

Regulation of hepatic fibrosis by ascorbic acid

Masson's trichrome was used to quantify collagen levels. Hepatic collagen levels increased by 74% in HFD mice compared with those in LFD mice (P<0.05; Fig. 5A and B). In contrast, ascorbic acid treatment decreased collagen levels in the livers of HFD mice by 42% (P<0.05).

Fig. 5. Effects of ascorbic acid on hepatic fibrosis in livers of obese OVX female mice.
Mice (n=5/group) were fed an LFD, an HFD, or an HFD-AA for 18 weeks. (A) Masson's trichrome-stained liver sections (original magnification ×100). (B) Relative collagen positive area. All values are expressed as mean ± SD. #P<0.05 compared with LFD. *P<0.05 compared with HFD.
DISCUSSION

NAFLD significantly correlates with metabolic disorders like dyslipidemia, hypertension, insulin resistance, type 2 diabetes, and obesity (Onyekwere et al., 2015; Neuschwander -Tetri, 2017). NAFLD comprises a spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), represented by lobular inflammation and ballooning. Across the spectrum of this disease, fibrosis can progress to cirrhosis; fibrosis progression is generally more common and rapid in NASH than in simple fatty liver disease (Sarwar et al., 2018). Postmenopausal women have a greater risk of NAFLD than premenopausal women due to lower estrogen levels in the former; postmenopausal women also have an increased incidence of liver fibrosis and/or hepatocellular carcinoma (Ascha et al., 2010; Quinn et al., 2018). We thus studied the effects of ascorbic acid on hepatic histological changes in HFD-fed obese OVX mice, an animal model of obese postmenopausal women.

HFD feeding induces NAFLD through the accumulation of lipids in the liver (Van Herck et al., 2017; Song et al., 2018). In the present study, hematoxylin & eosin- and Oil Red O-stained liver sections indicated significantly increased liver lipid accumulation after 18 weeks of HFD feeding in female OVX mice compared with LFD mice. These results are in accordance with the increases in liver weights caused by the HFD in OVX mice, suggesting that increases in liver weights may be due to the rise in liver lipid droplets. In contrast, ascorbic acid treatment decreased hepatic triglyceride droplets in obese OVX mice. These results indicate that ascorbic acid may alleviate hepatic steatosis in HFD-fed obese female OVX mice. Our findings are consistent with the results that ascorbic acid improves hepatic steatosis in obese male C57BL/6J mice and N-marry rats (Rezazadeh and Yazdanparast, 2014; Lee et al., 2019).

To examine the effect of ascorbic acid on liver inflammation, liver sections were stained with hematoxylin & eosin and toluidine blue and immunostained using an antibody to CD68 (Jeftic et al., 2015; Tai et al., 2016). Toluidine blue staining showed that HFD feeding elevated the number of mast cells in the livers, an effect reduced by ascorbic acid treatment. Ascorbic acid treatment also reduced the area of CD68-positive cells. Hepatocyte ballooning and inflammatory foci are histologically important indicators in the diagnosis of NASH (Caldwell et al., 2010; Wang et al., 2020). Hematoxylin & eosin-stained liver sections showed that hepatocyte ballooning and inflammatory foci were higher in HFD mice than in LFD mice, effects decreased by ascorbic acid administration. Accordingly, ascorbic acid reduces hepatocyte ballooning, inflammatory foci, mast cell numbers, and CD68-positive cell numbers, suggesting the role of ascorbic acid in the regulation of liver inflammation in HFD-fed OVX mice.

In approximately 25% of patients, NASH progresses to liver fibrosis (Schwabe et al., 2020). A remarkable feature of liver fibrosis is the increased accumulation of extracellular matrix proteins, such as collagens, fibronectin, and laminins (Wu et al., 2016). HFD feeding for 24 weeks causes a small amount of collagen to accumulate in the liver (Matsumoto et al., 2013). In the present study, hepatic collagen levels were determined by Masson's trichrome staining (Gao et al., 2016; Asokan et al., 2020). HFD feeding for 8 weeks slightly increased Masson's trichrome-stained collagen levels in the livers of OVX mice. On the other hand, hepatic collagen levels were reduced by ascorbic acid treatment in HFD mice, which were similar to those observed in LFD mice. Thus, ascorbic acid may reduce liver fibrosis in obese OVX mice.

Liver injury causes the release of the hepatocyte enzyme ALT into the circulation, leading to elevated plasma ALT levels (Smith et al., 2020). The abnormal increase of ALT is considered a marker of liver injury and necrosis (Smith et al., 2020). HFD mice had greatly higher levels of plasma ALT, an effect decreased with ascorbic acid treatment. It is likely that ascorbic acid may reduce liver damage in obese OVX mice.

In conclusion, the present study demonstrates that ascorbic acid effectively suppresses liver steatosis, inflammation, and mild fibrosis in obese female mice without functioning ovaries. In addition, ascorbic acid also normalizes liver weights and plasma ALT levels. Therefore, ascorbic acid may be an effective treatment for NAFLD in obese postmenopausal women.

ACKNOWLEDGEMENT

None.

CONFLICT OF INTEREST

The author declares no conflict of interest.

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