Potent Efficacy of 3-Amino-4-hydroxy Benzoic Acid, a Small Molecule Having Anti-fibrotic Activity, in a Mouse Model of Non-alcoholic Steatohepatitis

Abstract

Non-alcoholic steatohepatitis (NASH), which is on the rise due to the increasing obese population and changing lifestyles, causes fibrosis over time and carries the risk of progression to cirrhosis and hepatocellular carcinoma. However, there are no approved effective treatments for NASH. Recent studies suggest that increased lipid metabolism and reduced nitric oxide content are responsible for NASH; 3-amino-4-hydroxy benzoic acid (AHBA) was identified as an inhibitor for the phosphatase activity of soluble epoxy hydrolase, which in turn inhibits lipid metabolism and endothelial nitric oxide synthase activity. The aim of this study was to assess the efficacy of AHBA in a mouse model of NASH. NASH was induced in mice by streptozotocin administration and a high-fat diet loading. The efficacy of AHBA was determined by measuring liver function using serum and liver samples and conducting a morphological assessment. AHBA considerably attenuated the increase in the liver weight and alkaline phosphatase content, which occurred due to the progression of NASH. Hepatocellular steatosis, inflammatory cell infiltration, and hepatocellular ballooning of hepatocytes remained unaltered. In contrast, AHBA treatment significantly ameliorated the fibrotic alterations within liver tissue that were induced by the onset of NASH. These results demonstrate the potential of AHBA as a therapeutic pharmaceutical compound that can treat NASH.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is pathologically characterized by hepatic lipidosis and has been associated with metabolic syndromes, including diabetes and dyslipidemia. NAFLD encompasses a wide spectrum of liver diseases ranging from simple lipid accumulation in the liver to non-alcoholic steatohepatitis (NASH), which is characterized by large-drop fatty degeneration of liver cells, inflammatory cell infiltration, balloon-like degeneration of hepatocytes, and hepatocyte damage.¹⁾ The global prevalence of NAFLD is estimated to hover around 25%.²⁾ The incidence of NAFLD and NASH is increasing along with the epidemics of obesity, diabetes, and metabolic syndrome. NASH causes fibrosis and increases the risk of progression to cirrhosis and hepatocellular carcinoma. Fibrosis represents a pivotal focal point within the realm of NASH since numerous studies have demonstrated fibrosis to be a principal determinant of mortality in NASH.^3,4) The preferred therapeutic approach for NAFLD and NASH involves pharmaceutical interventions targeting underlying causative maladies, namely: diabetes or dyslipidemia. However, as NASH advances, hepatocytes are replaced by fibrotic scar tissues, which attenuate the efficacy of the therapeutic targets addressing the fundamental causative factors. Consequently, NASH treatment primarily revolves around fibrosis amelioration. Thus, new therapeutic agents are urgently needed to combat the disease.

The hypothesis of multiple parallel hits has been postulated to elucidate the etiology and pathogenesis of NAFLD/NASH development.⁵⁾ This concept posits that diverse factors engaged in lipid accumulation, inflammation, and fibrosis development within the hepatic milieu act concurrently on the liver, thus culminating in the development of NASH. Recent accounts have highlighted that the hepatoprotective effect of nitric oxide is nullified in livers with NASH,^6,7) which potentially facilitates the development and progression of NASH. Furthermore, in addition to vascular endothelium, endothelial nitric oxide synthase (eNOS) finds expression in hepatic hepatocytes, and there are accounts suggesting that the diminished activity of eNOS might contribute to the pathogenesis of NASH.^8–10)

Lately, 3-amino-4-hydroxy benzoic acid (AHBA) has emerged as an inhibitor of the phosphatase activity (N-phos) within the N-terminal domain of soluble epoxyhydrolase (sEH)¹¹⁾; sEH, a bifunctional enzyme, exhibits both C-terminal epoxide hydrolase activity (C-EH) and N-terminal domain phosphatase activity (N-phos).^12,13) Although the hydrolase function of C-terminal domain has been comprehensively studied,^14,15) N-phos remains relatively unexplored. Our research group has focused on inhibiting sEH via Stachybotrys microspora triprenyl phenols (compounds derived from fungi), but mainly on the C-terminal domain.^16–20) Meanwhile, isoprenoid pyrophosphate, a key player in cholesterol synthesis and blood pressure regulation, has been suggested as a substrate for N-phos.²¹⁾ Regulation of N-phos is also believed to influence eNOS activation and increase nitric oxide production.²²⁾ Recent investigations, which have employed a novel strain of rats selectively deficient in N-phos activity, indicate that N-phos activity contributes to the increase in lipid metabolism and body weight.²³⁾ Consequently, N-phos inhibitors hold promise as prospective therapeutic agents for NASH, given their role in fat metabolism and eNOS activation.

However, the effects of AHBA on NASH have not been investigated. The aim of the present study was to investigate the effects of AHBA in a mouse model wherein NASH was induced via streptozotocin (STZ) administration and a high-fat diet (HFD).

MATERIALS AND METHODS

Materials

STZ, rosuvastatin, isoflurane, and 10% formalin neutral buffer solution (pH 7.4) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). AHBA was purchased from BLD Pharmatech Ltd. (Shanghai, China).

Animals

All animal experimentation procedures were meticulously conducted in strict adherence to the protocols recommended by the Institutional Animal Care and Use Committees of Showa University, and also complied with the Standards Governing the Care and Oversight of Experimental Animals within the jurisdiction of Japan (Approval No. 25015). Pathogen-free 12-d pregnant C57BL/6J mice (CLEA Japan, Inc., Tokyo, Japan) were used. The mice, entrusted to our scientific endeavor, were accommodated within a climate-controlled animal facility, maintained at a temperature of 23 ± 2 °C, with a relative humidity spanning 50 ± 20%. This environment observed a structured 12-h light–dark cycle, with the illuminative phase commencing at 08 : 00 h and concluding at 20:00 h. Study mice were generously granted unrestricted access to a conventional diet and plentiful supply of water throughout the course of the study.

NASH Model Mice

Animals were randomly divided into control group (n = 8) and NASH group (n = 24). NASH model mice were induced, as previously described, with some modifications.²⁴⁾ NASH was induced in male mice by a single subcutaneous injection of 200 µg STZ at 2 d after birth and feeding with HFD32 (CLEA Japan, Inc.) ad libitum after 4 weeks of age. The control group received a single subcutaneous injection of the same volume of normal saline 2 d after birth and was fed the normal diet during the entire term. The NASH model mice were then randomly divided into NASH (n = 8), AHBA treatment (n = 8), and rosuvastatin treatment (n = 8) groups at 4 weeks of age. The efficacy of AHBA (30 mg/kg) was compared with rosuvastatin (30 mg/kg) and a vehicle-treated NASH group (n = 8). Because rosuvastatin was effective for the NASH model in previous reports, rosuvastatin was selected in this study as a control reagent.^25,26) AHBA (30 mg/kg), rosuvastatin (30 mg/kg), and the vehicle were administered by intraperitoneal injection every other day for 16 weeks from 8 weeks of age.

Sample Collection

At the age of 24 weeks, the mice were weighed and subjected to anesthetic induction employing 5% isoflurane, with the maintenance of anesthesia at a steady 2% isoflurane concentration. Blood specimens were meticulously harvested from the abdominal aorta. Serum was separated via centrifugation at 800 × g for 15 min at 25 °C and stored at −20 °C until needed for analysis. The liver was excised, followed by a quantification of its weight. The liver weight (LW) was assessed relative to the body mass, expressed in milligrams per gram of the murine body weight. Then, the liver was used for morphological assessment.

Biochemical Analysis

Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), triglyceride (TG), and total cholesterol (TC) were measured by Fuji DRI-CHEM NX500V (FUJIFILM Corporation, Tokyo, Japan) as described in the manufacturer’s instruction.

Histological Analysis

Liver tissues fixed in 10% neutral buffered formalin were embedded in paraffin and stained with hematoxylin–eosin (H&E). Hepatic fibrosis was confirmed using Sirius Red/Fast Green staining. Histopathological examination was conducted using an optical microscope (BHS, Olympus Corporation, Tokyo, Japan) equipped with a digital camera (OPLYMPUS digital camera E−330, Olympus Corporation). NAFLD activity scores (NAS) were assessed for steatosis, intralobular inflammation, and balloon-like degeneration of hepatocytes, as shown by Kleiner et al.²⁷⁾ and Brunt et al.²⁸⁾ Hepatocellular carcinoma (HCC) was also detected by H&E staining. Briefly, hepatocellular steatosis (grade 0: no fat; grade 1: steatosis occupying <33% of the hepatic parenchyma; grade 2: 34–66%; grade 3: more than 66%); inflammatory cell infiltration (grade 0: none; grade 1: 1–2 foci per 200× field; grade 2: 3–4 foci per 200× field; grade 3: more than 4 foci per 200× field); hepatocellular ballooning (grade 0: none; grade 1: few balloon cells; grade 2: many balloon cells); HCC (grade 0: none; grade 1: 1 foci per 100× field; grade 2: 1 foci per 40× field; grade 3: 2 foci per 40× field; grade 4: more than 2 foci per 40× field). For quantitative analysis of Sirius Red/Fast Green-positive areas, bright field images of stained sections were captured at 6.25- and 20-fold magnification; positive areas in three fields were measured using ImageJ software (National Institute of Health, Bethesda, MD, U.S.A.). The results were the means of three different fields of each section.

Statistical Analysis

All values are shown as means ± standard error of the mean (S.E.M.). Data were analyzed using one-way ANOVA followed by Bonferroni test. A value of p < 0.05 was considered statistically significant.

RESULTS

Assessment of Liver Function and Liver Weight

Changes in body weight (BW) and LW in NASH model mice were assessed to evaluate the effect of AHBA as shown in Fig. 1. BW, in the NASH group (24.9 ± 1.0 g), was significantly lower than in the control group (28.0 ± 0.5 g) (p < 0.01). BW of NASH mice treated with AHBA ranged from 24.4 ± 0.6 g, and did not show any significant improvement. The value of BW in NASH mice treated with rosuvastatin was 25.1 ± 0.8 g, and also did not show any significant improvement. The values of LW in the NASH group (158.7 ± 16.6 mg/g) were significantly higher than in the control group (41.5 ± 0.9 mg/g) (p < 0.01). On the contrary, the values of LW were 125.4 ± 4.4 mg/g in AHBA-treated NASH group. The differences between the NASH and AHBA treated groups were statistically significant at p < 0.01. The value of LW in rosuvastatin-treated NASH group was 139.7 ± 8.7 mg/g and did not show any significant improvement.

Fig. 1. Effects of AHBA on BW and LW in a Mice Model Where NASH Is Induced via STZ Administration and an HFD Loading

Each column represents the mean ± S.E.M. of eight animals. p < 0.05 indicates statistical significance and was calculated using the one-way ANOVA followed by Bonferroni test.

Assessment of Liver Function

Changes in AST, ALT, and ALP values in NASH model mice were assessed to evaluate the effect of AHBA on the liver function as shown in Fig. 2. The values of AST, ALT, and ALP in the NASH group (252.1 ± 44.8 U/L, 191.1 ± 38.5 U/L, and 1078.0 ± 209.6 U/L) were significantly higher than in control mice (44.4 ± 6.1 U/L, 34.0 ± 3.4 U/L, and 267.4 ± 11.8 U/L) (p < 0.01). AHBA treatment did not improve AST and ALT values (200.4 ± 27.7 U/L and 135.0 ± 28.6 U/L), but decreased ALP value (716.4 ± 103.0 U/L) (p < 0.05). The treatment with rosuvastatin also did not improve AST and ALT values (184.3 ± 15.4 U/L and 117.5 ± 17.5 U/L), but decreased ALP value (513.0.4 ± 54.9 U/L) (p < 0.01).

Fig. 2. Effects of AHBA on AST, ALT, and APL in a Mice Model Where NASH Is Induced via STZ Administration and an HFD Loading

Each column represents the mean ± S.E.M. of eight animals. p < 0.05 indicates statistical significance and was calculated using the one-way ANOVA followed by Bonferroni test.

Assessment of Lipid Metabolism

The changes in TG and TC values in NASH model mice were assessed to evaluate the effect of AHBA on lipid metabolism as shown in Fig. 3. TG and TC values in the NASH group (445.5 ± 142.2 and 236.1 ± 15.1 mg/dL) were significantly higher than in the control group (93.6 ± 19.9 and 69.4 ± 2.2 mg/dL) (p < 0.01). The treatment of AHBA did not improve TG value (357.7 ± 85.0 mg/dL), but decreased TC value (189.0 ± 20.8 mg/dL) (p < 0.05). Rosuvastatin also did not improve TG value (314.7 ± 136.8 mg/dL), but decreased TC value (164.3 ± 17.8 mg/dL) (p < 0.01).

Fig. 3. Effects of AHBA on TG and TC in a Mice Model Where NASH Is Inducted via STZ Administration and an HFD Loading

Each column represents the mean ± S.E.M. of eight animals. p < 0.05 indicates statistical significance and was calculated using the one-way ANOVA followed by Bonferroni test.

Morphological Assessment and Liver Injury Evaluation

H&E staining was used to assess changes in steatosis, intralobular inflammation, balloon-like degeneration, NAS, and HCC in NASH model mice in effect to AHBA as shown in Fig. 4. The values of steatosis, intralobular inflammation, NAS, and HCC in the NASH group (1.4 ± 0.2, 0.6 ± 0.3, 2.1 ± 0.5, and 1.9 ± 0.8) were significantly higher than in the control (0.0 ± 0.0, 0.0 ± 0.0, 0.0 ± 0.0, and 0.0 ± 0.0) (steatosis and NAS, p < 0.01; intralobular inflammation and HCC, p < 0.05). The values of balloon-like degeneration in the NASH group (0.1 ± 0.1) did not significantly increase compared to those in the control group (0.0 ± 0.0). The values of HCC in the AHBA-treated NASH group (0.0 ± 0.0) were significantly lower than in the NASH group (p < 0.01). The values of steatosis, intralobular inflammation, balloon-like degeneration, and NAS in the AHBA-treated NASH group (1.5 ± 0.2, 0.3 ± 0.2, 0.0 ± 0.0, and 1.8 ± 0.3) did not significantly improve compared to those in the NASH group. The values of steatosis and NAS in the rosuvastatin-treated NASH group (2.7 ± 0.2 and 4.0 ± 0.3) were significantly higher than those in the NASH group (p < 0.01).

Fig. 4. Histological Changes in Liver Tissues in a Mice Model Where NASH Is Induced via STZ Administration and an HFD Loading

Arrows indicate the sites of steatosis (a), intralobular inflammation (b), balloon-like degeneration (c), and HCC (d). Each column represents the mean ± S.E.M. of eight animals. p < 0.05 indicates statistical significance and was calculated using the one-way ANOVA followed by Bonferroni test.

The values of intralobular inflammation, balloon-like degeneration, and HCC in the rosuvastatin-treated NASH group (0.9 ± 0.3, 0.4 ± 0.2, and 1.6 ± 0.8) did not significantly improve compared to those in the NASH group.

Morphological Assessment and Liver Fibrosis Evaluation

Sirius Red/Fast Green staining was used to assess changes in fibrosis of liver tissue in NASH model mice subjected to AHBA as shown in Fig. 5. The values of fibrosis in the NASH group (1.0 ± 0.2%) were significantly higher than in the control group (0.5 ± 0.1%) (p < 0.01). The values of fibrosis in the AHBA-treated NASH group (0.5 ± 0.1%) and rosuvastatin-treated NASH group (0.6 ± 0.1%) were significantly lower than in the NASH group (p < 0.01).

Fig. 5. Anti-fibrotic Effect of AHBA in a Mice Model Where NASH Is Induced via STZ Administration and an HFD Loading

Each column represents the mean ± S.E.M. of eight animals. p < 0.05 indicates statistical significance and was calculated using the one-way ANOVA followed by Bonferroni test.

DISCUSSION

In this study, we evaluated the effects of AHBA on the liver damage and fibrosis caused by NASH. We observed that AHBA decreased the ALP content and LW (Figs. 1, 2). AHBA did not reduce the AST and ALT levels, which are regarded as the principal indicators of hepatic function. Moreover, AHBA did not decrease the TG levels; however, it did decrease the TC level (Fig. 3). It has been reported that sEH knockout downregulated 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase expression and reduced cholesterol levels.²⁹⁾ AHBA treatment lowered serum cholesterol levels possibly through downregulation of HMG-CoA reductase expression due to N-phos inhibition. Furthermore, AHBA demonstrated a notable antifibrotic effect and restrained hepatocellular carcinoma within the hepatic milieu of the mouse model of NASH, which resembles to clinical conditions in pathological progression (Figs. 4, 5). Steatohepatitis induced by STZ administration and HFD loading is widely used to generate experimental animal models of NASH.²⁴⁾ Such NASH models are characterized by inflammatory cell infiltration in the liver and fibrosis of liver tissue, as observed in human NASH. eNOS activity, which is one of the factors involved in liver fibrosis, reduces in fibrotic livers.³⁰⁾ It has been experimentally ascertained that AHBA effectively impedes the phosphatase activity of sEH within mice.¹¹⁾ Notably, the abrogation of sEH phosphatase activity has been unequivocally associated with direct and indirect facilitation of eNOS activation.²²⁾ The enzymatic activity of eNOS, which is notably diminished in fibrotic hepatic tissues, stands as a major contributory factor underpinning the intricate process of hepatic fibrogenesis. However, the current study is the first to report that AHBA does not completely improve liver function in NASH model mice; however, it can partially improve lipid metabolism and effectively protect against fibrosis. In the current investigation, AHBA was administered every other day to mice aged eight weeks until they were 24 weeks old; these mice were inculcated with NASH through STZ administration and an HFD. This temporal window for intervention was meticulously selected based on the established scientific observations that indicated the manifestation of elevated liver enzymes (such as AST and ALT) and pro-inflammatory cytokines, which were exemplified by tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6); these results are observed around the eighth week,²⁴⁾ thereby confirming the emergence of NASH. Meanwhile, hepatic fibrosis, a hallmark manifestation, becomes evident at the twenty-fourth week of this model. Therefore, the administration of AHBA continued until the twenty-fourth week, thus aligning with the manifestation of liver tissue fibrosis. Furthermore, administrating AHBA every other day suppressed histological fibrosis in liver during the onset of NASH to fibrosis. It is desirable to evaluate fibrosis from multiple angles by measuring the expression of multiple fibrosis-related genes such as acta2, col1a1, tgfb, and timp1. However, these fibrosis-related genes of the 24-week-old model mice peaked out. Therefore, the anti-fibrotic effect of AHBA could not be evaluated by measuring fibrosis-related genes.

Generally, it takes approximately seven years for NASH to advance toward hepatic fibrosis by one stage, while an even more accelerated progression is observed in older patients and those afflicted with diabetes or dyslipidemia.³¹⁾ As fibrosis advances, hepatocytes are partially replaced by fibrotic scar tissues, thus significantly compromising the therapeutic targeting of the underlying ailment.³²⁾ From a clinical perspective, during both NASH and NAFLD, hepatic fibrosis has a profound and statistically significant correlation with overall mortality and mortality specifically related to liver complications.³⁾ Moreover, NAS and other histological assessments exhibit notably weak correlations.^33–35) In light of its antifibrotic properties, AHBA may emerge as a viable therapeutic approach for managing NASH-related fibrosis.

The progression of fibrosis mechanisms entails the activation of M2 macrophages and the inordinate accumulation of extracellular matrix (ECM).^36–38) These processes are incited by augmented inflammatory cues, including TNF-α and IL-1β emanating from immune cells.^39–41) Consequently, fibrosis is postulated as the ultimate pathological sequel of numerous chronic inflammatory maladies. The regulation of chronic inflammation is thus posited as one of the modalities for governing fibrosis.

N-phos of sEH is known to catalyze the conversion of lysophosphatidic acid (LPA) into monoacylglycerol (MAG) within hepatocytes.²³⁾ Recent evidence has illustrated the capability of AHBA to impede the restitution of LPA to MAG by inhibiting N-phos activity.⁴²⁾ This inhibition results in heightened stimulation of the LPA receptor, which in turn is postulated to incite an anti-inflammatory response by reducing phospholipid metabolic turnover, activating the AKT pathway, and enhancing thromboxane A₂ (TXA₂) receptor stimulation in macrophages. Conceivably, AHBA showcases anti-fibrotic properties through its anti-inflammatory mechanisms. However, the inflammatory cytokines of the 24-week-old model mice peaked out, and anti-inflammatory effects of AHBA could not be assessed.

Compared to the NASH group, the rosuvastatin group also showed fibrosis inhibition; however, further deterioration was observed in NAS; this finding is consistent with clinical reports.⁴³⁾ Meanwhile, NAS has been used to classify the severity of NASH, although it has been shown not to correlate with mortality. AHBA and rosuvastatin are consistent in showing an anti-fibrotic effect. However, the rosuvastatin-treated group showed an exacerbation of hepatic lipidosis. There are reports suggesting that statins inhibit phospholipid synthesis in HepG2 cells and cause a defective in very low density lipoprotein (VLDL) secretion.⁴⁴⁾ In this study, it is thought that rosuvastatin administration developed histological steatosis through the above mechanism. On the other hand, AHBA did not exacerbate hepatic lipidosis during administration. Thereby, it is suggested that AHBA results in a lower NAS than that observed in the rosuvastatin-treated group.

Several therapeutic agents have been developed for NASH, and compounds such as obeticholic acid and selonsertib have shown fibrosis-inhibiting effects in animal studies and some clinical trials.^45,46) Obeticholic acid possesses the capacity to regulate glycolipid metabolism and enhance insulin sensitivity through the modulation of bile acid metabolism.⁴⁷⁾ Selonsertib acts as an instigator of the p38/c-Jun N-terminal kinase (JNK) pathway and governs the process of hepatocyte apoptosis.⁴⁸⁾ Nonetheless, neither compound was effective in mitigating liver fibrosis during phase II or phase III trials, which ultimately resulted in the cessation of these clinical investigations. AHBA operates through a distinct mechanism when compared to the above-mentioned compounds and exerts a triple effect : modulating cholesterol levels, suppression dephosphorylation of LPA, and activation of eNOS. Consequently, there exists a promising prospect that AHBA could markedly curtail liver fibrosis among individuals with NASH.

Our data revealed that AHBA, while not affecting the complete restoration of liver function, does facilitate the partial amelioration of lipid metabolism. Furthermore, based on its antifibrotic properties, AHBA curtails the transition from NASH to hepatocellular carcinoma. The main mechanism underlying the etiology of NASH is lipid accumulation, and potential aetiologies include reduced eNOS activation. AHBA may be a candidate for a new class of NASH drugs that modulate cholesterol levels and activate eNOS. In patients with NASH for whom the inhibition of lipid accumulation alone proves insufficient, utilizing AHBA in their treatment holds significant promise in forestalling the progression from NASH to cirrhosis and HCC. In the next phase, fibrosis-related gene expression will be evaluated comprehensively to analyze the AHBA attenuation of hepatic fibrosis in NASH from multiple angles. Subsequent studies should explore the anti-inflammatory attributes of AHBA and the modulation of eNOS activity by AHBA in NASH model mice, conducting a comprehensive exploration into the operational mechanisms of these models. Moreover, optimal dosage and administration regimens may require further refinement.

CONCLUSION

In summary, based on the data acquired in this study, it can be postulated that AHBA harbors a therapeutic potential for treating NASH because it had a pronounced antifibrotic influence within the hepatic milieu of NASH-afflicted mice. Future inquiries are warranted to elucidate the intricate mechanisms underpinning the impact of AHBA on NASH.

Acknowledgments

This study received financial support through a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science KAKENHI (Grant Numbers 22K20897 (T.Y.) and 22K08318 (K.S.)).

Conflict of Interest

The authors declare no conflict of interest.

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