Research ArticleExperimental Studies

Oral Administration of Mulberry (Morus alba L.) Leaf Powder Prevents the Development of Hepatocellular Carcinoma in Stelic Animal Model (STAM) Mice

KOJI WAKAME, KEISUKE SATO, MASAFUMI KASAI, EMI KIKUCHI, KIHIRO SHIMIZU, AKO KUDO, KEN-ICHI KOMATSU and AKIFUMI NAKATA
Anticancer Research August 2022, 42 (8) 4055-4062; DOI: https://doi.org/10.21873/anticanres.15902

Abstract

Background/Aim: We examined the inhibitory effect of mulberry leaf (ML) (Morus alba L.) administration on the development of hepatocellular carcinoma (HCC) in stelic animal model (STAM) mice. This STAM mouse model of nonalcoholic steatohepatitis (NASH) closely resembles the progression from NASH to HCC in human clinical practice. Materials and Methods: Streptozotocin (STZ, 200 μg) was administered to C57L/6J mice that were fed a high-fat diet (HFD; STAM mice) with 1% ML ad libitum. After sacrificing, the liver and blood were collected. Biochemical tests of plasma and histologic examination of the liver were performed. Results: Pathologic examination of all (6/6) liver samples of the STAM mice showed HCC. On the contrary, in STAM mice that received ML, fat deposition and adenoma were observed in 6/6 and 2/6 of the liver samples, respectively, but there was no HCC. Conclusion: Administration of ML in STAM mice inhibited the progression from nonalcoholic hepatitis (NASH) to HCC. ML may be effective in preventing the development of HCC.

Key Words:

Mulberries (Morus alba L.) grow wild in Japan and other Asian countries, and their cultivation has flourished along with the development of silk production (1). Mulberry leaves (ML) and fruits have been used for centuries as food and in folk medicine, whereas the root bark has been used as a Chinese herbal medicine (i.e., So-haku-hi) for cough relief and its anti-inflammatory effects (2). The major component of ML is 1-deoxynojirimycin, which has been reported to inhibit the α-glucosidase effect on the intestinal tract, thereby, suppressing the increase in postprandial blood glucose level (3). In our previous study, we found that administration of ML to a mice model of metabolic syndrome suppressed the development of fatty liver and that induction of the lipin1 gene in the liver is one of the mechanisms that promoted fat metabolism (4).

Nonalcoholic hepatitis (NASH) is a lifestyle-related disease that is caused by excessive intake of carbohydrates and fats (5). Fatty liver disease can develop over time into nonalcoholic fatty liver disease (NAFLD), NASH, cirrhosis, and hepatocellular carcinoma (HCC) (6). In human clinical cases, the progression of NASH to HCC is only a few percent. Nevertheless, prevention and treatment of these fatty liver-related diseases are important, because the estimated number of NAFLD cases in Japan is about 20 million (7, 8). The prevention and treatment of NASH include dietary caloric restriction, exercise therapy, and pharmacotherapy, such as vitamin E, antidiabetic drugs, and antihyperlipidemic drugs (9). In recent years, blood glucose and triglyceride levels have been controlled by intake of healthy food for the purpose of protecting the liver function (10, 11).

In this study, we aimed to determine whether ML can inhibit the progression of NASH to HCC in this mouse model. Some of these NASH mice models were developed by a methionine–choline-deficient diet (12), high fructose–cholesterol diet (13), and knockout genes (14). However, these mouse models had several problems before they fully developed HCC, for example, the long time to develop NASH and low incidence of NASH/HCC. Therefore, we used stelic animal model (STAM) mice, which were developed by Fujii et al. (15). These were shown to induce a condition that is very similar to the development of NAFLD/NASH in humans. Furthermore, maintaining these STAM mice on HFD assured a 100% probability of developing HCC, and administration of ML prevented the development of HCC.

Materials and Methods

Animal treatment. The ML powder (lot 1604) was obtained from Health Age Co., Ltd., Tokyo, Japan. The STAM mice were purchased from SMC Laboratories, Inc., Tokyo, Japan and maintained on the HFD and administered ML (Figure 1). Fourteen-day pregnant C57BL/6 mice were purchased from Charles River Japan, Inc., Kanagawa, Japan. The NASH to HCC pathology was induced by a single subcutaneous injection of 200 μg streptozotocin (STZ, Fujifilm-Wako, Tokyo, Japan) to male mice at two days after birth. At four weeks of age, the mice were given a diet containing high amounts of fat (HFD 32, CLEA Japan, Tokyo, Japan) ad libitum and housed in individual cages under conventional conditions with a 12-h light–dark cycle and at 23±1°C temperature with 55±15% humidity.

Figure 1.
Figure 1.

The experimental designs for the STAM and STAM+ML groups of mice. STZ and HFD were administered to the STAM group, whereas STZ and HFD plus 1% ML powder were administered to the STAM+ML group. After sacrificing all mice, blood and liver samples were obtained for each examination. STAM: Stelic animal model; ML: mulberry leaf; STZ: streptozotocin; HFD: high-fat diet.

The mice were divided into the following three groups (n=6 per group): 1) normal mice (Normal group), 2) STAM (STAM group), and 3) STAM plus 1% ML powder in HFD (STAM+ML group). We created two identical mouse groups, one for biochemical experiments and the other for life extension observations. This study conformed to the Guiding Principles for the Care and Use of Experimental Animals of Hokkaido University of Science (2018). The protocol approval number was 2019-006.

Plasma biochemical examinations and liver histologic analysis. At week 8, all 12-week-old mice were placed under isoflurane anesthesia, and blood and liver samples were obtained. Each liver sample was fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into thin 5-μm sections that were stained with hematoxylin & eosin (HE) solution. HE-stained liver tissues were histologically examined using an Olympus AX70 light microscope (Olympus Co., Ltd., Tokyo, Japan), which was equipped with 10× ocular and 4× objective lenses (40×). Liver histology was examined to determine the state of NAFLD, NASH, and HCC by evaluating and quantifying the grading score (G score) and the NAFLD activity score (NA score). For the G score, microscopic findings of fatty deposits, altered hepatocellular foci, hepatocellular adenomas, and tumor development were judged based on an evaluation scale (Table I). The NA score was calculated as the cumulative value of each evaluation criterion of fatty deposition, cell balloon-like degeneration, and intralobular inflammation (Table II). These morphological analyses were performed using a microscope, on 10 microscopic fields per section.

View this table:
Table I.

G score evaluation form.

View this table:
Table II.

NA score evaluation form.

Blood samples from the heart of the mice were collected in an EDTA-containing plasma separator tube (BD Japan Co., Ltd., Tokyo, Japan). Each plasma sample was obtained from whole blood, separated by centrifugation at 12,000 × g/min for 10 min, and stored at –80°C. The plasma levels of aspartate aminotransferase (AST, mg/dl); alanine aminotransferase (ALT, mg/dl); lactate dehydrogenase (LDH, IU/l); triglyceride (TG, mg/dl); total cholesterol (T-CHO, mg/dl); high-density lipoprotein cholesterol (HDL-C, mg/dl); low-density lipoprotein cholesterol (LDL-C, mg/dl); cholesterol ester ratio (E/T, %); total bilirubin (T-BIL, mg/dl); cholinesterase (ChE, IU/l); total protein (TP, g/dl), and albumin (ALB, g/dl) were measured by Oriental Yeast Nagahama Life Science Laboratory, Inc., Tokyo, Japan.

Statistical analysis. These biochemical results were expressed as mean±standard error of the mean (S.E.). Statistical tests were performed using BellCurve for Excel ver.2.00 software (Social Survey Research Information Co., Ltd., Tokyo, Japan). One-way ANOVA followed by Tukey’s method were used to compare groups. The survival duration was compared between the STAM and STAM+ML groups using the Kaplan–Meier method. p-Values <0.05 were considered statistically significant. Normal vs. STAM: *p<0.05, **p<0.01, Normal vs. STAM+ML; †p<0.05, ††p<0.01, STAM vs. STAM+ML; §p<0.05, §§p<0.01.

Results

Length of survival. In the STAM group, the first case of death occurred at the age of 12 weeks, and all mice died by the age of 22 weeks. In the STAM+ML group, the first case of death occurred at the age of 15 weeks, and all mice died by the age of 30 weeks. There was a significant difference in the survival duration between the two groups (p=0.039) (Figure 2).

Figure 2.
Figure 2.

The survival curves of the STAM and STAM+ML groups. The STAM+ML group showed a significant survival benefit, compared with that of the STAM group (p=0.039). STAM: Stelic animal model; ML: mulberry leaf.

Change in body weight and histologic analysis of the liver. The mean body weight of each group is shown in Table III. In all groups, body weight increased as the age of the animals increased. At 12 weeks, the mean body weight was significantly lower in the STAM group than that in the normal group (p=0.004) but there was no difference between the STAM+ML and normal groups.

View this table:
Table III.

Changes in body weight in each group at 6, 10, and 12 weeks.

At the time of sacrifice, the liver was observed to have several HCC lesions on gross examination (Figure 3A) and microscopic observation on HE staining (Figure 3B). Histologic examination of the liver revealed fatty deposits, ballooning degeneration of cells, and intralobular inflammation as indicated by the NA score and fatty deposits, altered hepatocellular foci, hepatocellular adenoma, and hepatocellular carcinoma as indicated by the G score. The NA score was highest in the STAM group, followed by the ML and normal groups (Figure 4). Based on the G score, fatty deposits, altered hepatocellular foci, and HCC were observed in both the STAM and ML groups, but HCC was observed only in the STAM group (Figure 5). All mice from the STAM group showed HCC (6/6). In the STAM+ML group, fat deposition (6/6) and adenoma (2/6) were observed in the liver samples.

Figure 3.
Figure 3.

Gross findings and microscopic examination of the liver in each group. There are several hepatocellular carcinoma lesions seen on the STAM group (white open circle: tumors) (A). The liver specimens were fixed with formaldehyde and stained with HE for microscopic examination (B). The G and NA scores were determined on these images. There are fatty deposits in both the STAM and STAM+ML groups, but hepatocellular carcinoma findings are seen only in the STAM group (black open circle: carcinoma). G score: Grading score; NA score: NAFLD activity score; HE: hematoxylin and eosin; STAM: stelic animal model; ML: mulberry leaf.

Figure 4.
Figure 4.

Changes in the NA score of the liver in each group at 12 weeks. The maximum NA score was 8 points. Values are expressed as means±S.E. p-Values <0.05 were considered statistically significant. Normal vs STAM: **p<0.01, Normal vs. STAM+ML: ††p<0.01, STAM vs. STAM+ML: §§p<0.01. STAM: Stelic animal model; ML: mulberry leaf; NA score: NAFLD activity score.

Figure 5.
Figure 5.

Changes in the G score of the liver in each group at 12 weeks. The maximum G score was 13 points. Values are expressed as means±S.E. p Values <0.05 were considered statistically significant. Normal vs STAM: **p<0.01, Normal vs. STAM+ML: ††p<0.01, STAM vs. STAM+ML: §§p<0.01. STAM: Stelic animal model; ML: mulberry leaf; G score: grading score.

Plasma biochemical examinations. As shown in Table IV, the biochemical parameters AST, LDH, HDL-C, LDL-C, ET ratio, and T-BIL were not significantly different among the groups, whereas the ALT, TG, T-CHO, ChE, TP, and ALB were significantly different at each group.

View this table:
Table IV.

Changes in biochemical parameters after 12 weeks in each group.

Discussion

The mulberry plant (leaves and fruits) contains many components, such as vitamins (16), minerals (17), flavonoids, flavonols, and anthocyanins (18-21). Mulberries also possess anti-obesity, anti-hypolipidemic (22, 23), antidiabetic (24, 25), anti-bacterial (26, 27), anti-oxidant (28), anti-Inflammatory (29), anti-cancer (30, 31), and hepatoprotective (32) functions. Based on these reports, we believe that ML has the potential to offer protection against liver damage in STAM mice.

The STAM mouse model is known to have a 100% probability of developing HCC and is similar to human NAFLD/NASH in terms of disease progression and biochemical and pathologic features (33, 34). Currently, the multiple concurrent hits theory proposed by Tilg et al. (35) is a well-accepted explanation of the pathogenesis of NAFLD/NASH. This theory proposed that several hits, such as endoplasmic reticulum stress, release of adipokines from the gut, and adipose tissue, diet, and genetic factors, occur in parallel rather than sequentially to induce NAFLD/NASH (36, 37). Thus, hyperglycemia was first induced by STZ, and diabetes, hyperlipidemia, and oxidative stress were assumed to occur in parallel to lead to the development of NASH in the STAM mice. In addition, continuous intake of HFD stimulates oxidative stress, which leads to the transition from NASH to HCC (33).

The reason for the similar body weight between the STAM mice and the mice in the normal group could be the result of restricted growth secondary to the development of severe diabetes, NASH, and HCC (38). In the present study, two evaluation methods were used to make an accurate pathologic diagnosis of the STAM mice liver samples. The G score criteria for pathological diagnosis were used and indicated the degree of activity in the liver tissue by quantifying the necrosis and inflammatory findings (39). The NA score evaluation quantified the degrees of fatty liver and parenchymal inflammation, as well as the frequency of appearance of balloon hepatocytes (40). In particular, the liver of mice in the STAM group showed many HCC lesions, but ML administration completely inhibited HCC development.

It has been reported that the biochemical parameters in the serum of the STAM model, change with progression to end-stage HCC (41). In particular, in the HCC model, the blood levels of ChE, TP, and ALB were low but the levels of AST, ALT, LDH, and T-BIL were high because of chronic liver damage (15). Considering that liver diseases progress from inflammation to fibrosis to cancer, we can understand the changes in the levels of these markers during inflammation, hepatocyte destruction, and chronic and severe liver damage (15, 41).

Based on the blood biochemical examinations, ALT levels were higher in the STAM+ML group compared to the other two groups and did not differ between the normal and STAM groups. The reason for the high ALT level in this STAM+ML group is unclear. Further analysis will be performed in the future.

The STAM group showed low levels of ChE which can also be decreased by end-stage HCC. TP levels were low in the STAM group, likely because of a worsening nutritional status with the progression of liver damage, but it was high in the ML group. Considering the results of the reversal of the decrease in TP levels by ML, ML may be involved in the prevention of worsening liver function and nutritional status (42).

In addition, the TG level was higher in the STAM group than that in the STAM+ML group. We have previously reported that ML suppressed the increase of TG in the blood and increased the expression of the lipin1 gene in the liver of mice fed the HFD (4). Therefore, we believe that ML promotes lipid metabolism in the livers of STAM mice.

Conclusion

In this study using a STAM mouse as a NASH model, we confirmed a 100% probability of developing HCC, which was observed to be associated with decreased liver function and nutritional status. Administration of ML inhibited the progression to HCC and prolonged the life span of mice. In the future, we plan to investigate in detail the mechanism of HCC prevention by ML.

Acknowledgements

The Authors would like to thank Enago (www.enago.jp) for the English language review. Preparation of ML samples and detailed information on ML components were provided by Health Age Co., Ltd., Tokyo, Japan. The authors also thank SMC Laboratories, Inc., Tokyo, Japan for the technical support of STAM mice models. This study was funded by the Grant-in-Aid for Scientific Research (C, 19K11648).

Footnotes

  • Authors’ Contributions

    A Nakata and K Wakame designed the research protocol. M Kasai provided test samples. E Kikuch, K Shimizu, A Kudo, and K Sato analyzed the physical data for evaluation of adverse events. K Komatu and M Kasai performed statistical analysis. K Wakame and A Nakata wrote the manuscript. K Komatsu and K Sato reviewed and edited the manuscript. All Authors read and approved the final version of the manuscript.

  • Conflicts of Interest

    The Authors have no conflicts of interest directly relevant to the content of this article.

  • Received May 20, 2022.
  • Revision received June 7, 2022.
  • Accepted June 14, 2022.

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Oral Administration of Mulberry (Morus alba L.) Leaf Powder Prevents the Development of Hepatocellular Carcinoma in Stelic Animal Model (STAM) Mice
KOJI WAKAME, KEISUKE SATO, MASAFUMI KASAI, EMI KIKUCHI, KIHIRO SHIMIZU, AKO KUDO, KEN-ICHI KOMATSU, AKIFUMI NAKATA
Anticancer Research Aug 2022, 42 (8) 4055-4062; DOI: 10.21873/anticanres.15902
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