The Influence of Emodin Succinyl Ethyl Ester on Non-alcoholic Steatohepatitis Induced by a Diet High in Fructose, Cholesterol, and Fat in Mice

Abstract

Nonalcoholic steatohepatitis (NASH) is a subtype of nonalcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis and evidence of hepatocyte injury (ballooning) and inflammation, with or without liver fibrosis. In this study, after 12 weeks of induction, the mice were treated with emodin succinyl ethyl ester (ESEE) for four weeks at doses of 10/30/90 mg/kg/d. The blood analysis of experimental endpoints showed that ESEE exhibited significant therapeutic effects on the progression of disorders of glycolipid metabolism and the induced liver injury in the model animals. Histopathological diagnosis of the liver and total triglyceride measurements revealed that ESEE had a significant therapeutic effect on the histopathological features of nonalcoholic fatty liver disease/hepatitis, such as cellular steatosis and activation of intrahepatic inflammation. Additionally, ESEE was able to improve hepatocyte fat deposition, steatosis, and the course of intrahepatic inflammatory activity. Furthermore, it showed some inhibitory effect on liver fibrosis in the model animals. In summary, this study confirms the therapeutic effects of ESEE on the NAFLD/NASH model in C57BL/6J mice induced by a high-fat, high cholesterol, and fructose diet. These effects were observed through improvements in liver function, inhibition of fibrosis, and inflammatory responses. Changes in blood glucose levels, blood lipid metabolism, liver histopathological staining, liver fibrosis staining, and related pathological scores further supported the therapeutic effects of ESEE. Therefore, this study has important implications for the exploration of novel drugs for nonalcoholic fatty liver disease.

INTRODUCTION

The metabolic syndrome is closely linked to nonalcoholic fatty liver disease (NAFLD), a common liver condition. It is primarily characterized by the accumulation of fat in liver tissue, resulting from an imbalanced uptake and breakdown of liver fat.¹⁾ NAFLD can be caused by various factors, including obesity, insulin resistance, hypertension, and dyslipidemia. The incidence of NAFLD has been steadily increasing due to the development of our socioeconomic system, changing lifestyle habits, and dietary patterns. It is estimated that approximately 25% of people worldwide have NAFLD, with around 20% progressing to nonalcoholic steatohepatitis (NASH). NASH, a severe form of NAFLD, is characterized by excessive fat buildup in the liver. This leads to stress and damage to hepatocytes, resulting in inflammation, fibrosis, and potentially progressing to cirrhosis, liver failure, cancer, and even death.^2,3) Finding a cure or specific treatment for NASH has posed a significant challenge for the pharmaceutical industry, and the existing treatments are not curative or adequately tailored for this condition.^4,5) Hence, there is an urgent need for innovative and effective interventions or alternative therapies to effectively manage NASH.

Emodin is the primary active component of rhubarb and possesses significant pharmacological effects and clinical efficacy.⁶⁾ It exhibits various therapeutic effects, such as anticancer and antifibrotic effects, and is widely used in the treatment of encephalitis, diabetic cataract, and organ fibrosis.^7–10) However, its application is greatly limited due to its inherent instability in vivo and short duration of action. To overcome this limitation, emodin has been structurally modified, resulting in the synthesis of a novel anthraquinone compound known as emodin succinyl ethyl ester (ESEE). Several studies have demonstrated that ESEE possesses a highly potent lipid-lowering effect¹¹⁾ and provides protection to the vascular endothelium and myocardium,^12,13) thereby alleviating the inflammatory response and promoting wound healing in diabetes.¹⁴⁾ ESEE also exhibits potent cardioprotective and antifibrotic activities, effectively inhibiting the expression of NLRP3 inflammasome and suppressing proinflammatory cytokines.¹²⁾ Recent research has shown that ESEE can alleviate metabolic syndrome and nonalcoholic fatty liver disease induced by a high-fat diet by activating AMP activated protein kinase (AMPK). Based on the extensive body of previous studies, we have investigated the effects of ESEE on NASH.

NAFLD is characterized by the presence of not only steatosis, but also hepatic inflammation and fibrosis.¹⁵⁾ Thus, in this study, we utilized a mouse model of Nash induced by a high-fat, high cholesterol, high fructose diet. The objective was to assess the inhibitory effects of ESEE therapeutic intervention on the progression of disorders related to body glycolipid metabolism and the pathological advancement of NASH resulting from it. The findings demonstrated the potential of ESEE as a promising drug for the treatment of NASH caused by metabolic disorders.

MATERIALS AND METHODS

Chemicals and Reagents

ESEE (purity > 98%) was synthesized and provided by Kanion Pharmaceutical Co., Ltd. The Alanine aminotransferase (ALT/GPT) test kit, aspartate aminotransferase (AST/GOT) test kit, alkaline phosphatase (ALP/AKP) assay kit (microplate method), total cholesterol (TC/TCH) assay kit, triglycerides (TG) assay kit, high density lipoprotein cholesterol (HDL-C) assay kit, low-density lipoprotein cholesterol (LDL-C) assay kit, and Total bilirubin (T-Bil) kits were all purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Animal Models and Treatment

C57BL/6J male mice, aged 6–8 weeks, were acquired from Beijing Sibeifu Biotechnology Co., Ltd. (Production License Number: ZYXK-Beijing 2019-0010, and Permit Number: SYXK-Jiangsu 2018-0026). Compliance with relevant laws and regulations for the use and management of laboratory animals, as well as our institution's Laboratory Animal Use and Management Committee (IACUC) regulations of Kanion (Record Number: 2023022401), was ensured throughout the animal testing process. Following a week of adaptation, the mice were subjected to a high-fat, high-cholesterol, and high-fructose diet (named Western diet, 40 kcal% Fat, 2% Cholesterol, 20% Fructose) to induce a model of non-alcoholic fatty liver disease/hepatitis. The experimental cycle was divided into two phases. The first phase was the model induction phase, where experimental animals were randomly divided into two groups based on their body weight: the normal control group (n = 10) and the model induction group (n = 40). The model diet induction spanned 12 weeks, during which the mice were weighed weekly. At the end of the 12-week period, mice were sacrificed via the inner canthal region, serum was separated, and alanine transaminase (ALT) levels in the serum were measured. The second phase focused on ESEE treatment, where animals in the model induction group were further divided into four groups based on endpoint body weight and serum ALT levels from the first stage. These groups included the model group and low-dose (10 mg/kg), middle-dose (30 mg/kg), and high-dose (90 mg/kg) ESEE treatment groups. ESEE treatment began on the day of grouping in the second stage, with once daily oral gavage administration for four weeks. The blank control group received the corresponding dose of the drug solvent. After completion of the experiment, the animals were euthanized, and both the liver and serum were immediately subjected to pathological examination and biochemical analysis.

RNA Isolation and Real-Time PCR

Total RNA was extracted from liver using the Novozan kit (Nanjing, China). Reverse transcription was performed after determining the RNA concentration following the instructions of the TaKaRa reverse transcription Kit. The reaction conditions were 35 °C for 15 min, 85 °C for 5 s, and 4 °C for 15 min. For amplification, a mixture of 2 µL of cDNA, 10 µL of SYBR Green, 1 µL of upstream primer, 1 µL of downstream primer, and 6 µL of ribonuclease (RNase)-free water was prepared in a 20 µL system. The reaction conditions were as follows: 95 °C for 30 min, 95 °C for 5 min, 60 °C for 30 s, and 60 °C for 1 min after 40 cycles. After PCR, the target gene expression was calculated using the 2-ΔΔCT method. Sequences of the forward and reverse primers are listed below: TNFα (F:5′-AAGCCTGTAGCCCACGTCGTA-3′, R:5′-GGCACCACTAGTTGGTTGTCTTG-3), interleukin (IL)-1β (F:5′-TCCAGGATGAGGACATGAGCAC-3′, R:5′-GAACGTCACACACC-AGCAGGTTA-3), Collagen 1 (Col1a1) (F:5′-GCACGAGTCACACCGGAACT-3′, R:5′-AAGGGAGCCACATCGATGAT-3).

Western Blot Analyses

The expression of related proteins was detected using Western blot analysis. A small amount of liver tissue was weighed and pre-cooled Ripa buffer (containing protease inhibitors) was added. The mixture was homogenized and then centrifuged at 12000 rpm for 15 min at 4 °C. After protein quantification, 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) electrophoresis was performed. The protein was then transferred to a polyvinylidene difluoride (PVDF) membrane at 90 V for 2 h and blocked for 1 h. The primary antibody was incubated overnight at 4 °C, followed by incubation with the secondary antibody at room temperature for 1 h. Enhanced chemiluminescence (ECL) was used for signal detection, and the gray value was analyzed using ImageJ software. The primary antibodies used and β-actin were from Abcam (Cambridge, U.K.), anti-NLRP3, cleaved caspase-1, and caspase-1 from CST (Danvers, MA, U.S.A.).

Serum Biochemical Analysis

After being fed a high-fat, high cholesterol, high fructose diet for 12 weeks, all animals were subjected to a 12-h fast and blood samples were collected from the inner canthus. Serum was obtained through centrifugation. ALT levels were determined using a kit from Nanjing Jiancheng in China. Following a 4-week period of drug treatment, mice were sacrificed via the abdominal cardinal vein. Before blood collection, all experimental animals were fasted for 12 h and 1.0 mL of blood was collected. Serum was isolated to assess levels of ALT, aspartate transaminase (AST), ALP, TG, TC, HDL-C, and LDL-C using kits from Nanjing Jiancheng in China.

Haematoxylin–Eosin (H&E), Sirius Red and Oil Red O (ORO) Staining

Mice were dissected at the end of the dosing cycle. Whole livers were collected and weighed. Liver tissues were fixed in 10% formaldehyde for 48 h. Subsequently, the tissues were embedded in paraffin and cut into 3 µM-thick sections. These sections were subjected to hematoxylin–eosin staining (H&E) examination. Liver samples from experimental animals were then scored for histopathological diagnosis. The relevant pathological scoring criteria of the guide for the diagnosis and treatment of nonalcoholic fatty liver disease, developed by the hepatology branch of the Chinese Medical Association, were used as a reference. The scoring focused on steatosis, lobular inflammation, and hepatocellular swelling. Afterwards, the individual scores for each parameter were summed to produce the NAFLD score (NAS).¹⁶⁾ To assess the extent of fibrosis in liver samples, it was necessary to complete several steps. Firstly, the sections were deparaffinized in xylene, followed by the hydration process using graded ethanol. Subsequently, they were stained using Sirius red. After staining, the sections were dehydrated, made transparent, mounted, and then captured at various magnifications using a microscope equipped with ImageJ software. By analyzing the Sirius red staining, the sections were examined under a light microscope at 100× magnification to determine the level of fibrosis present in each liver sample. ORO staining was used to assess hepatic lipid accumulation.

Statistical Analysis

Results (mean ± standard error of the mean (S.E.M.)) were analyzed using one-way ANOVA followed by post hoc Tukey’s test for pairwise comparisons (GraphPad Prism 7; GraphPad Software, La Jolla, CA, U.S.A.). A p-value of less than 0.05 was considered statistically significant.

RESULTS

Effects of ESEE on Body Weight Growth and Liver Coefficients in Model Animals

During the induction period, the body weight of the control animals on a normal diet exhibited normal growth. On the other hand, the body weight of the model-induced animals started to increase rapidly after being fed a high-fat, high-cholesterol and high fructose diet. This increase in body weight was significantly higher compared to the growth observed in the normal control animals during the same period. Additionally, the body weight of the model group showed a significant and gradual increase over time. When monitoring the body weight of the animals during the ESEE treatment period and at the test endpoint (Figs. 1A, B), it was observed that both the ESEE (30 mg/kg) and ESEE (90 mg/kg) treated groups experienced a decrease in body weight during the dosing cycle, in comparison to the model control group during the same period. Notably, the body weight of the ESEE (90 mg/kg) treated group decreased significantly. These findings indicate that ESEE demonstrated a certain degree of inhibitory effect on the body weight growth of the model animals under the specified treatment regimen. The calculation of the liver index of the animals in each experimental group (Fig. 1C) revealed that the liver index of the model animals was significantly higher compared to the normal control group when continuously fed a high-fat, high-cholesterol and high fructose diet. However, it was observed that ESEE had a certain degree of inhibitory effect on the elevated liver index response of the model animals, as per the treatment regimen employed in the experiment's anatomical endpoints. Moreover, the animals in each treatment dose group exhibited a decrease in liver body indices, with the high-dose treatment group with ESEE displaying the most noticeable ameliorative effect.

Fig. 1. Effects of Treatments upon Body Weight and Liver Weight

(A) Effects of treatment period on body weight. (B, C) Whole-body weight and liver index were obtained for each animal at euthanasia. (^### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the model group).

Effects of ESEE on Lipid Metabolism and Liver Injury in Model Animals

Experimental endpoints, serum samples were collected from animals in each experimental group to analyze lipid and functional indexes. The analysis primarily focused on two aspects: glycolipid metabolism (TG, glutamic acid (Glu), TC, LDL-C, and HDL-C) and liver injury (ALT, AST, and ALP). This evaluation aimed to assess the progression of disease injury in model animals and the therapeutic effects of ESEE on disorders of glycolipid metabolism and their resultant body injury. The results of the blood biochemical test (Fig. 2) revealed that all lipid metabolism indexes of control animals on a normal diet remained within the normal physiological range and showed no abnormalities. In contrast, the lipid metabolism indexes of the model control animals significantly worsened under the influence of a high-fat and high-fructose diet. After the induction of this diet, the serum cholesterol indexes (CHOL, HDL-C, and LDL-C) noticeably increased compared to the control group following a normal diet in the same cycle. However, the serum total TG level exhibited a noticeable decline while following the model diet.

Fig. 2. The Effect of ESEE on the Changes of ALT, AST, ALP, GLU, TG, TC, HDL-C and LDL-C in Model Mice Blood (n = 10)

(A) ALT content. (B) AST content. (C) ALP content. (D) GLU content. (E) TG content. (F) TC content. (G) HDL-C content. (H) HDL-C content. Data are reported as means ± S.E.M. (^### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the model group).

ESEE demonstrated significant inhibitory effects on the elevated responses of all cholesterol indicators in the serum of the model animals. Additionally, the levels of CHOL, LDL-C, and HDL-C in the serum of animals in each treatment group decreased to some extent following ESEE intervention (Figs. 2F–H). Interestingly, the serum total TG levels of animals in the treatment group exhibited minimal changes in comparison to those of the model group. Furthermore, these levels displayed a gradual decrease when compared to the normal group, indicating a downward trend (Fig. 2E).

Concomitant with the continuous feeding of a high-fat, high cholesterol, and high fructose diet, the model animals developed evident symptoms of lipid metabolism disorder syndrome. Therefore, the study aimed to evaluate the degree of liver injury mediated by the progression of lipid metabolism disorder in model animals and assess the effects of ESEE on liver injury in these animals by analyzing the changes in serum liver function indexes (ALT and AST, and ALP) in each experimental group. The results of the serum liver function index tests (Figs. 2A, B) indicated that the model control animals, who were continuously fed a high-fat, high cholesterol, and high fructose diet for 16 weeks, exhibited clear signs of hepatic damage. These animals showed a significant increase in serum ALT and AST levels compared to the normal control animals during the same time period. However, the serum ALP level showed no significant change. ESEE demonstrated an ameliorative effect on the progression of liver injury in the model animals, following the established treatment protocol. The activities of their serum liver enzymes all exhibited a decline in response, and under similar conditions, the levels of ALT and AST in the serum of animals treated with ESEE significantly decreased. At the same time, the ESEE treatment group showed no significant effect on the serum ALP level of the model animals. Its level remained consistent with that of the model group and the normal group.

Effects of ESEE on the Contents of Total Triglyceride and Total Cholesterol in Liver of Model Animals

The three treatment groups administered with compound ESEE demonstrated significant improvements in hepatic fat deposition in disease model animals. In order to assess the impact of ESEE on liver lipid metabolism in experimental animals, the levels of total triglyceride and total cholesterol in the liver were measured using lipid assay kits. The measurement of liver total triglyceride (Fig. 3A) revealed a significant increase in liver triglyceride content in the model control animals. However, ESEE exhibited clear inhibitory effects on the elevation of hepatic triglyceride content in model animals during the prescribed treatment period. The levels of total triglyceride in the liver of animals in 30 and 90 mg/kg treatment group were significantly lower compared to those in the model group at the same time. These findings were consistent with the results obtained from the diagnostic scoring of hepatocellular steatosis by histopathology of the liver. The findings from the liver total cholesterol detection (Fig. 3B) revealed that ESEE exhibited a remarkable inhibitory effect on the heightened levels of liver total cholesterol in the animal models. Furthermore, all treatment groups demonstrated a notable reduction in liver total cholesterol levels compared to the model control group.

Fig. 3. The Effect of ESEE on Hepatic Triglyceride and Total Cholesterol Content in Model Mice

(A) TG content in liver tissue. (B) TC content in liver tissue. (C) Representative histological images of the ORO staining (Scale bars = 100 µm). Data are reported as means ± S.E.M. (^### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the model group).

The results of ORO staining showed that compared with normal control animals, the liver of model control animals showed obvious ORO staining positive signal (bright red) in the hepatocytes of the central vein and the confluence region, with clear boundaries between large and small lipid droplets, and the positive staining signal became deeper with the increase of the degree of steatosis. The livers of animals in all dose groups of the test product ESEE showed obvious ORO positive staining signals, and the positive signals decreased to a certain extent with the increase of the administered dose (Fig. 3C), which was consistent with the results of the histopathological detection of the liver.

Effects of ESEE on Histopathological Features of Liver and on the Progression of Liver Fibrosis in Model Animals

After 12 weeks of Western diet induction, the livers of model control animals showed obvious NASH lesions, which were characterized by a continuous increase in the degree of diffuse steatosis, mixed degeneration of hepatocytes with large fat droplets/small fat droplets, and obvious inflammatory infiltration in the confluent zone, with obvious hepatocyte necrosis and fibroplasia around the foci of inflammatory infiltration in the confluent zone, and a continuous deepening of hepatic fibrosis. In the model control group, there were obvious fibroplasia in the confluent area, some fibers were bridged, and a large number of hepatocytes with steatosis and inflammatory infiltration appeared along the edge of fibroplasia, which was consistent with the characteristics of liver histopathological lesions in clinical NASH patients; at the same time, the effect of hepatic lipid deposition was obvious, with the accumulation of lipid droplets in the hepatocytes, and the staining of oil-red was positive (Data not shown).

Western diet Induced C57BL/6J mice NASH/NAFLD Model with ESEE treatment for 4 weeks, Liver histopathological staining and diagnostic results, as shown in Fig. 4A, revealed that the livers of the normal control animals exhibited normal architecture, clear hepatic lobular architecture, distinct cytoplasmic and nuclear boundaries of hepatocytes, uniform staining, and the absence of any noticeable inflammatory cell infiltrations. On the other hand, model control animals fed a high-fat, high-fructose, high-cholesterol diet continuously developed histopathologic lesions typical of nonalcoholic fatty liver disease/hepatitis, which were characterized by hepatocellular steatosis (mixed macro- and micro-vesicular steatosis), inflammatory response mediated by neutrophil and lymphocyte infiltration in the hepatic tissues, hepatocellular punctate necrosis around the foci of inflammatory infiltration, and perisinusoidal fibrosis triggered by extracellular matrix deposition. Fibrotic process. Comparatively, when compared to the model group, treatment with compound ESEE led to a partial reduction in liver cell swelling and degeneration. Moreover, there was a noticeable improvement in fat vacuole accumulation and inflammatory cell infiltration, with the most prominent effect observed at the high dose of ESEE.

Fig. 4. Effects of ESEE on Histopathological Features of Liver and on the Progression of Liver Fibrosis in Model Animals

Representative liver images and histological images of the H&E (Scale bars = 500 µm) (A) and Sirius red staining (Scale bars = 100 µm) (B). Results of pathologic diagnostic scores of hepatic cellular steatosis (C), histopathologic scoring of hepatic inflammatory infiltration (D), histopathological scoring of hepatocellular swelling and degeneration (E), liver nonalcoholic steatohepatitis disease activity (F) and liver fibrosis scores (G) in animals of all experimental groups. Data are reported as means ± S.E.M. (^### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the model group).

The experimental results (Figs. 4C–F) showed that the progression of fatty injury induced by lipid accumulation was not significant in the livers of control animals fed a normal diet. However, the hepatocytes of model control animals developed steatosis characterized by a mixture of large and vesicles. Additionally, inflammatory infiltrate foci dominated by neutrophils were evident within hepatic lobules. The NAS score was significantly higher in the livers of model control animals compared to normal animals. The disease pathology in these animals was characterized by a predominance of hepatocyte large and vesicular mixed steatosis, inflammatory infiltrates dominated by neutrophils, and hepatocyte necrosis. In comparison, the progression of intrahepatic fat deposition in the ESEE treated animals was significantly reduced. Most animals exhibited mild steatosis in their livers, with the most significant improvement observed in the group treated with ESEE (90 mg/kg). As hepatocellular steatosis improved, the level of intrahepatic inflammatory activity gradually decreased in all treated animals. Furthermore, no obvious swelling and degeneration of hepatocytes were observed in the livers of animals treated with ESEE (90 mg/kg). Considering all the histopathological scores together, the mean liver NAS score of the animals treated with ESEE (90 mg/kg) showed a significant reduction compared to the model control group. It is worth noting that this reduction was primarily achieved by improving scores for swelling degeneration and lobular inflammation. Overall, the compound ESEE demonstrated a clear ameliorative effect on hepatocyte steatosis and hepatitis response in the diseased animals.

The liver samples subjected to Sirius red staining revealed that half of the animals in the model control group displayed hepatocyte necrosis caused by notable infiltration of inflammatory cells (Fig. 4B). Additionally, there was accumulation of extracellular matrix in the hepatocyte space adjacent to the inflammatory necrosis lesion, resulting in sinus fibrosis surrounding the liver tissue. However, the fibers did not form distinct connections. Based on the grading of fibrosis extent and morphology, the model control animals exhibited mild perisinusoidal fibrotic changes, graded as S1 lesions. Under the prescribed treatment schedule, ESEE exhibited a certain degree of ameliorative effect on the progression of liver fibrosis in the model animals. Notably, animals treated with ESEE at doses of 90 mg/kg in three dose groups showed significant alleviation of histological changes in the liver. None of these animals displayed obvious fibrotic characteristics, and the mean histopathological score of liver fibrosis was zero points (Fig. 4G).

Effects of ESEE on NLRP3 Signaling and Genes Involved in Inflammation and Fibrosis

Previous studies have found that NASH lesions caused by the Western diet are associated with activation of the NLRP3 signaling pathway. As shown in Figs. 5A–C, the Western diet-induced NASH mouse model increased the expression of NLRP3 and cleaved-Caspase-1 while downregulated the expression of proteins related to the NLRP3 signaling pathway after administration of ESEE.

Fig. 5. Effects of ESEE on NLRP3 Signaling and Genes Involved in Inflammation and Fibrosis

(A) Representative Western blot images for the protein expression of NLRP3, Cleaved Caspase-1 and Caspase-1. (B, C) Quantitative data of the protein expression of NLRP3 and Cleaved Caspase-1, β-actin/Caspase-1 were used as internal control proteins.). (D, E) mRNA levels of inflammatory cytokines (TNF-α and IL-1β). (F) mRNA levels of fibrosis-related genes (Col1a1). Data are shown as means ± standard deviation (S.D.) (n = 3). ^## p < 0.01, ^### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the model group.

The mRNA expression levels of inflammatory factors (TNF-α and IL-1β) and fibrosis-related genes (Col1a1) were detected by qPCR. The expression levels of these genes were significantly increased in the model group compared with the normal group. In the detection of inflammatory signaling genes, ESEE medium- and high-dose treatments were found to have a significant ameliorating effect on TNF-α and IL-1β (Figs. 5D, E). The mRNA of Col1a1, a gene associated with fibrosis, was decreased in all three ESEE treatment groups (Fig. 5F). These findings suggest that ESEE has the effect of inhibiting fibrosis and improving inflammation.

DISCUSSION

NAFLD/NASH is a metabolic liver injury disease closely associated with insulin resistance and genetic predisposition. A high-fat, high cholesterol, and high fructose diet, which mimics the typical Western dietary structure, has been commonly used as a diet-induced model for nonalcoholic fatty liver disease/hepatitis in experimental animals. This diet increases the dietary intake of lipids, leading to an increase in the amount of free fatty acids in the blood. As a result, excess free fatty acids cause disorders in body fatty acid metabolism. This, in turn, mediates intracellular triglyceride accumulation in the liver and induces insulin resistance, thus acting as the first hit to the liver.¹⁷⁾ Disorders involving fatty acid metabolism and insulin resistance increase the vulnerability of hepatocytes to various damaging factors and inflammation. This leads to the production of numerous reactive oxidative free radicals, triggering hepatic oxidative stress and resulting in the development of NASH.^18,19)

Currently, the most suitable model for studying the clinical human pathogenesis of NAFLD/NASH is the rat/mouse model induced by a high-fat, high fructose diet. This model primarily focuses on increasing the intake of lipids and sugars in the diet of experimental animals. This abnormal energy metabolism leads to the accumulation of excessive triglycerides and free fatty acids in the liver. Consequently, the hepatic oxidative stress response is activated, generating a significant amount of reactive free radicals that mediate the process of hepatic inflammation. Ultimately, this results in the development of metabolic syndrome, accompanied by obesity, insulin resistance, hyperglycemia, and elevated liver enzymes. This model not only replicates the histopathological changes observed in human NAFLD/NASH, including hepatocyte steatosis, inflammation in the intralobular and confluent zones, hepatocyte injury (ballooning and apoptosis), and fibrosis, but also induces obesity and metabolic disorder syndrome (insulin resistance). Therefore, it currently stands as the ideal animal model for studying the pathogenesis and pharmacodynamic evaluation of NAFLD/NASH. In this study, we employed a high-fat, high cholesterol, and high fructose diet to induce a murine NAFLD/NASH model. This model exhibits pathophysiological features similar to human NASH, including insulin resistance, disrupted serum lipid metabolism, abnormal serum liver enzymes, hepatocyte steatosis, hepatocyte injury, intrahepatic inflammatory response, and perisinusoidal fibrosis.

In the present study, we demonstrated that the compound ESEE exhibits inhibitory effects on the pathological progression of NASH. Following four weeks of continuous cycles of ESEE treatment, we assessed the therapeutic effects of ESEE on a mouse model of NAFLD/NASH induced by a high-fat, high fructose diet. These effects were evaluated based on changes in animal body weight, blood biochemical lipid metabolism, liver function, liver histopathology, liver fibrosis, and related pathological scores. Our findings indicate that ESEE can slow down the process of disturbed glycolipid metabolism in the animal model, significantly reduce the levels of cholesterol and lipids in the serum, and have noticeable effects on improving lipid metabolism. We also observed that NASH is often associated with circulating biochemical disorders, particularly with the levels of ALT and AST, crucial markers of liver injury.²⁰⁾ The serum ALT and AST levels in the treatment groups with ESEE showed a decrease compared to the model control group over the same period, suggesting an improvement in liver damage. Moreover, NAFLD is characterized by the accumulation of triglyceride-rich lipid droplets within liver cells, leading to liver injury caused by oxidative stress and inflammation.²¹⁾ Quantification of hepatic triglyceride and total cholesterol levels in liver tissue homogenates confirmed that ESEE exhibited dose-dependent inhibitory effects on their elevation. Using the nonalcoholic fatty liver disease activity score (NAS) scale, we found that ESEE treatment significantly attenuated the progression of intrahepatic fat deposition in animals. Most livers showed mild steatosis, and the treatment effectively ameliorated the degree of hepatocellular steatosis in the model animals. Moreover, the level of intrahepatic inflammatory activity gradually decreased in animals of all treatment groups, as ESEE not only improved hepatocyte steatosis but also slowed down the intrahepatic inflammatory process to some extent. Additionally, ESEE had a significant ameliorative effect on the progression of hepatic fat deposition and inflammatory response in the model animals. The histopathological scores of intrahepatic steatosis and inflammatory infiltration were significantly decreased in animals treated with each dose compared to the model control group during the same period. Furthermore, ESEE exhibited an ameliorative effect on the progression of liver fibrosis, as seen in the decreased liver fibrosis score and incidence in the treated animals compared to the model control animals. This improvement was statistically significant.

NLRP3 knockout reverses NASH in high cholesterol diet-fed mice due to lack of inflammasomes. NLRP3 expression was significantly increased in the liver tissues of NASH patients. In addition, the NLRP3 pathway plays an important role in the regulation of inflammation during the development of NASH.²²⁾ Studies have also shown that the pathological process of NASH can be mitigated by inhibiting the NLRP3 pathway. Activated NLRP3 forms a complex called the inflammasome. In the inflammasome, Caspase-1 is activated and involved in the regulation of inflammatory response. Activated Caspase-1 promotes the production and release of IL-1β, which is an important inflammatory mediator. Our data show that the Western diet induced NASH mouse model activates the NLRP3 signaling pathway and downstream protein or gene expression, and these inflammatory and fibrotic processes are reversed after treatment with ESSS.

Our findings strongly support that ESEE can significantly inhibit the progression of Western diet-induced NASH in mice, and its molecular mechanism may be related to ESEE targeted inhibition of NLRP3 activation. However, it is important to note that this study has certain limitations. Without in vitro mechanistic studies examining the genes and proteins involved, direct assessment of fibrotic markers becomes challenging. Additionally, it is possible that the current findings may be influenced by the combined beneficial effects of ESEE on cardiovascular and metabolic systems, in addition to liver function.

CONCLUSION

In summary, the results of this study demonstrate that a novel anthraquinone, ESEE, effectively protects against diet-induced increases in NASH-associated lipids and hepatitis. Additionally, it induces a robust antifibrotic effect that significantly ameliorates liver injury in a murine model of nonalcoholic fatty liver disease/hepatitis, aligning with the clinical pathogenesis profile. Hence, ESEE may potentially provide an effective therapeutic approach for treating fibrotic NASH. Additionally, it is crucial to evaluate whether ESEE can be used as therapeutic agents alone or in combination.

Acknowledgments

This work was financially supported by Programs Foundation for Leading Talents in National Administration of Traditional Chinese Medicine of China “Qihuang scholars” Project.

Conflict of Interest

The authors declare no conflict of interest.

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