Rice Bran Attenuates Liver Fibrosis Caused by Vitamin E Deficiency in the Pre-Aging Stage

ABSTRACT

Vitamin E (VE) is a fat-soluble vitamin and well-known as an antioxidant. VE deficiency is associated with various oxidative stress- and aging-related pathologies and sufficient VE intake is thought to help prevent these conditions. However, it has been unclear how VE deficiency in the pre-aging stage affects the liver. In the present study, 11-month-old mice were fed a VE-restricted diet for 3 months, when age-related changes began to appear, to examine the effects of VE deficiency on the liver. Compared to the livers of mice fed a standard diet, those fed a VE-deficient diet showed metabolic dysfunction-associated steatohepatitis (MASH)-like findings including fibrosis. Mice fed the VE-deficient diet mixed with 2% rice bran (RB) showed milder fibrosis than those fed the VE-deficient diet alone. These results suggest that VE deficiency in the pre-aging stage may cause MASH-like changes in the liver, particularly fibrosis, and that RB is an effective means of supplying VE.

Vitamin E (VE) is fat-soluble and is known to have antioxidant capacity.¹ VE deficiency and the loss of its transport protein (αTTP: α-tocopherol transfer protein) are known to cause oxidative stress, which can result in cognitive impairment and ataxia.^{2, 3} Oxidative stress is expected to affect the nervous system and organs throughout the body including the liver during aging.^4,5,6 Under oxidative stress, the liver has been reported to present metabolic dysfunction-associated steatohepatitis (MASH)/nonalcoholic steatohepatitis (NASH)-like findings, such as fat accumulation and infiltration of inflammatory cells.^{5, 7, 8} However, it has been unclear how VE deficiency in the pre-aging stage affects the liver.

Rice bran (RB), a byproduct of the rice milling process, is known to be rich in VE. Brown rice retains the components of RB and can therefore be a valid food source of VE.^{9, 10}

In the present study, we investigated the effects of VE deficiency in the liver and the effects of RB supplementation using pre-aging mice.

MATERIALS AND METHODS

Animals

C57BL/6 mice were obtained from CLEA Japan, Inc, (Tokyo, Japan). The mice were housed under standardized conditions of temperature (25 °C), humidity (approximately 50%), and light (06:00–18:00), and were allowed free access to food and water. The mice used in this study were treated in accordance with the Guidelines for Animal Experimentation of Tottori University (No.14-Y-03, 18-Y-02). VE-deficient feed (Funabashi Farm, Chiba, Japan), normal feed, and RB were prepared. The VE-deficient and normal feeds were in powder form similar to RB. To investigate the effects of RB, mice were fed three types of feed (Fig. 1A). The diets were as follows: (i) normal diet alone (14 months) (n = 2), (ii) normal diet (11 months) plus VE-deficient diet (VEdef) (3 months) (n = 2), and (iii) normal diet (11 months) plus a mixture of VEdef and RB at a concentration of 2% (w/w) (n = 2).

Fig. 1.

 Protocol of VEdef diet model and histological findings. Experimental protocol After being fed a normal diet for 11 months, mice were divided into three groups as follows: normal diet, VEdef, and VEdef + RB. (B–D) Upper panels show HE staining, middle panels show PSR staining, and lower panels show MTC staining. The collagen fibers were stained red and blue with PSR and MTC, respectively. The left three panels show low-magnification images, and the right three panels show high-magnification images. Scale bar = 10 μm. (B) Liver sections from mice fed a normal diet. No significant findings, such as accumulation of lipid droplets, cell infiltration, or fibrosis, were observed. (C) Liver sections from mice treated with VEdef. HE staining shows lipid accumulation (arrows) and cell infiltration (triangles). PSR and MTC staining revealed increased collagen fibers (red and blue staining, respectively). (D) Liver sections from mice fed VEdef and 2%RB.

Histological study

Hematoxylin and eosin (HE), Masson’s trichrome (MTC), and Picro-Sirius Red (PSR) staining were performed on paraffin-embedded sections of mouse liver tissue. The liver sections were perfusion-fixed in 4% paraformaldehyde (Wako) and embedded in paraffin. Paraffin sections (5 μm) were deparaffinized by immersing the slides in xylene three times, followed by rehydration in graded ethanol solutions. HE and MTC staining were performed according to standard protocols. PSR staining was performed using a Picro-Sirius Red Stain Kit (ScyTec Laboratories, Inc., Köln, Germany). The images were captured using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). Lipid droplets in HE staining and fibrosis in Picro-Sirius Red stain were semiquantified using ImageJ software.

RESULTS

To investigate the effects of VE deficiency during the pre-aging stage, 11-month-old mice were fed a VEdef for 3 months, when age-related changes began to appear, to examine the effects of VE deficiency on the liver and RB administration.

In the livers of mice fed the normal diet, there was almost no accumulation of lipid droplets, infiltration of inflammatory cells, or fibrosis (Fig. 1B). In contrast, mild MASH/NASH-like changes were observed in the livers of mice fed VEdef (Fig. 1C). HE staining (Fig. 1C, upper panels) revealed the accumulation of lipid droplets and infiltration of inflammatory cells. The amount of lipid droplets in VEdef and VEdef + RB was 3.50-fold and 2.45-fold, respectively, compared with livers on a normal diet. PSR (Fig. 1C, middle panels) and MTC staining (Fig. 1C, lower panels) showed fibrosis, particularly around the intrahepatic bile duct. The mice fed the VEdef diet mixed with 2% RB showed milder fibrosis than those fed the VEdef diet alone (Fig. 1D, middle and lower panels). The amount of liver fibrosis in VEdef and VEdef+RB was 5.66-fold and 1.60-fold, respectively, compared with livers on a normal diet. Fat accumulation and inflammatory cell infiltration were mild (Fig. 1D, upper panels).

DISCUSSION

VE is a fat-soluble vitamin and a well-known antioxidant present in the cell membrane that protects cells and organs against oxidative stress.¹ VE deficiency is associated with various oxidative stress- and aging-related pathologies, and sufficient VE intake is thought to help prevent these conditions. Furthermore, VE exerts various effects through mechanisms other than its antioxidant capacity.^11,12,13 In our previous study, we showed that 2%RB supply sufficient vitamin E to VEdef diet.¹⁴

There are 8 VE derivatives, such as α-, β-, γ-, and δ-tocopherol and α-, β-, γ-, and δ-tocotrienol.¹ Although the concentrations of tocopherols and tocotrienols in RB vary according to the rice species and milling techniques, RBs contain abundant tocopherols and tocotrienols.^{9, 10} In our previous report, RB used in this study included predominately α-tocopherol, γ-tocopherol, and δ-tocopherol. The levels of tocotrienols were low in RB.¹⁴ Dietary RB is reportedly useful for intestinal regulation by decreasing low-density lipoprotein levels and lowering blood pressure.¹⁵ We previously investigated the effects of dietary RB on neuronal abnormalities induced by VE deficiency, such as cerebellar ataxia.¹⁴

In the present study, 11-month-old mice were fed a VEdef for 3 months, when age-related changes began to appear, to examine the effects of VE deficiency on the liver and RB administration. (Fig. 1) Compared to the livers of mice fed a standard diet, those fed a VEdef showed fat accumulation, infiltration of inflammatory cells, and fibrosis. Mice fed the VEdef mixed with 2% RB showed milder fibrosis than those fed the VEdef alone. Mild fat accumulation and inflammatory cell infiltration were also observed. It has been reported that oxidative stress occurs in the liver of mice with aging, resulting in MASH/NASH-like changes such as fat accumulation, inflammation, and fibrosis.¹⁶ Vitamin E deficiency also enhances oxidative stress in the liver and induces various metabolic changes.^{17, 18} Considering that VE has antioxidant properties and that the administration of VE-rich RB improved this condition, it is thought that the oxidative stress induced by VE deficiency promoted liver fibrosis in this model. This study revealed that VE deficiency during the pre-aging period promoted MASH/NASH-like changes and fibrosis in the liver. Furthermore, this suggests that the intake of RB or brown rice is a promising method for supplying VE and can alleviate VE deficiency-related hepatic dysfunction. Adequate VE intake during the early stages of aging is important for preventing liver fibrosis.

One limitation is that the effects of other components contained in rice bran, such as vitamin B1, were not examined in this study, and this is an issue that needs to be investigated in the future.