Chronic intake of high-dose of blueberry leaf extract does not augment the harmful effects of ethanol in rats

Reagents

BLEx was prepared as a hot water extract by Bizen Chemical Co. Ltd (Okayama, Japan). Briefly, blueberry leaf powder was extracted in 16 parts of hot water (95–100 ° C) for 30 min twice. Then, the extract was filtered and heat sterilized. Finally, the extract was dried with a spray dryer, producing a powder. Ethanol was purchased from the Wako Pure Chemical Industries (Osaka, Japan). The oxidized form of β-nicotinamide adenine dinucleotide hydrate (NADH), 4-methylpyrazole, 2,4-dinitrophenylhydrazine, and trifluoroacetic acid (TFA) were purchased from the Tokyo Chemical Industry (Tokyo, Japan).

Ethanol intake model

The animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the University of Miyazaki (Animal Experiment Committee of Miyazaki University: 2017-014-2) and in compliance with the Law Concerning the Protection and Control of Animals (Japan Law No. 105), Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain (Notification no. 88 of the Ministry of the Environment, Japan), and The Guidelines for Animals Experimentation (the Japanese Association for Laboratory Animal Science). The room temperature was maintained at 22–24 °C and the animals were housed under a 12-h light/dark cycle (09:00–21:00).

Sprague-Dawley (SD) rats (all male, 7 weeks old, total 20) were purchased from Japan SLC (Hamamatsu, Japan) and acclimatized for 1 week. The rats were divided into 4 groups (5 per group): normal liquid diet group (NLD), normal liquid diet + BLEx group (NLD + BLEx), alcohol liquid diet group (ALD), and alcohol liquid diet + BLEx group (ALD + BLEx). The rats were fed with a Lieber-DeCarli liquid diet (Lieber & DeCarli, 1982). The composition of the liquid diet is shown in Table 1. Ethanol intake in the ALD group was gradually increased from 1% on day 1 to 5% over 7 days. 3% BLEx liquid diet was mixed with dry diet and the rats were fed for 5 weeks. After the end of the experiment, the rats were sacrificed using a mixture medetomidine (Kyoritsu Seiyaku Corporation, Tokyo, Japan), midazolam (Astellas Pharma Inc., Tokyo, Japan), and butorphanol (Meiji Seika Pharma Co., Ltd., Tokyo, Japan).

Serum biochemical tests

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were estimated using Wako Transaminase CII-Test Kit (Wako). Serum total protein and albumin level and albumin/globulin (A/G) ratio were estimated using A/G B-Test Wako Kit (Wako).

Liver aldehyde dehydrogenase (ADH) and alcohol dehydrogenase
(ALDH) activities

The liver tissues (50 mg) were homogenized using 250 µl of homogenized buffer (0.25 M sucrose, 5 mM Tris, 0.5 mM ethylenediaminetetraacetic acid (EDTA)-2Na, and 2 mM 2-mercaptoethanol). The homogenates were centrifuged (13, 000 × g for 10 min at 4 °C) and the supernatant was collected. Ten-fold diluted supernatant (25 µl) was mixed with ADH reaction assay buffer (225 µl, 5 mM NADH, 5 mM ethanol, 9 mM glycine, and 120 mM sodium pyrophosphate (pH 8.8)) or ADH negative assay buffer ((225 µl, 5 mM NADH, 1 mM 4-methylpyrazole, 9 mM glycine, and 120 mM sodium pyrophosphate (pH 8.8)) in a 96-well plate. To determine ALDH activity, 25 µl of 10-fold diluted supernatant was mixed with 225 µl of ALDH reaction assay buffer [0.5 mM NADH, 10 mM acetaldehyde, 0.1 mM 4-methylpyrazole, 2 µM rotenone, and 60 mM sodium pyrophosphate (pH 8.8)] or 225 µl ALDH negative assay buffer (0.5 mM NADH, 0.1 mM 4-methylpyrazole, 2 µM rotenone, and 60 mM sodium pyrophosphate (pH 8.8)) in a 96-well plate. The change in absorbance (340 nm) was measured by a plate reader at 0, 3, 6, 9, 12, and 15 min. The protein level was estimated by using a commercial Pierce^® Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo). The liver ADH and ALDH activities were calculated by the following equation: ADH and ALDH activity = (ΔA/min × V × D)/(6. 3 × d × v), where, ΔA/min is the change in absorbance at 340 nm, V is the final volume, D is the dilution rate, 6.3 is the molecular extinction coefficient at 1 mM of NADH at 340 nm, d is the optical path length, and v is the sample volume.

Fractionation of the liver microsomal fraction

The liver tissues (2 g) were homogenized using 10 ml of homogenized buffer (250 mM sucrose, 75 mM nicotinamide, 2.5 mM EDTA-2Na, 20 mM 2-mercaptoethanol, and 50 mM potassium phosphate buffer (pH 7.4)). Following that, the homogenates were centrifuged at 12, 425 × g for 20 min at 4 °C and the supernatants were ultracentrifuged at 109, 572 × g for 1 h at 4 °C. After the supernatant was discarded, the pellets were redissolved in 2 ml of dissolution buffer (5 mM (±)-dithiothreitol and 20 mM potassium phosphate buffer (pH 7.4)).

Western blot

The microsomal fraction was lysed with 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 2% Triton X-100, 2 mM EDTA, 50 mM NaF, and 30 mM Na₄P₂O₇ with 1/50 volume of a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). The protein levels were estimated using a BCA protein assay reagent (Pierce, Rockford, IL). The lysate containing 1 µg of protein was denatured and separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto Hybond^®-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). The nonspecific sites were blocked by incubating the membrane with 3% non-fat dried milk in Tris-buffered saline and 0.1% Tween-20 (T-TBS) for 60 min at a room temperature. The antibodies were diluted with Can Get Signal solutions 1 and 2 (TOYOBO, Tokyo, Japan). CYP2E1 specific polyclonal antibody was purchased from Proteintech Group, Inc. (IL, USA). Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Cell Signaling Technology^® (Cell Signaling Technology, Inc., MA, USA). Following each antibody binding reaction, the membranes were washed with T-TBS. The proteins on the membrane were detected using ImageQuant LAS 4000 (GE Healthcare). The band intensity was quantified using ImageQuant TL (GE Healthcare) and the representative blot patterns are shown.

Isolation of RNA, reverse-transcription of RNA, and real-time polymerase chain reaction (PCR)

The total RNA was isolated from the liver using TRIzol reagent (Thermo Fisher Scientific Inc., MA, USA). Rever Tra Ace qPCR RT Kit (TOYOBO) was used to synthesize cDNA. Real-time PCR was performed with an Agilent AriaMx Real-Time PCR System (Agilent Technologies, Inc., CA, USA) using THUNDERBIRD^® SYBR^® qPCR Mix (TOYOBO). The temperature was controlled according to the manufacturer’s instructions. The primer sequences for the real-time PCR are listed in Table 2. To quantify mRNA expression, the data obtained by real-time PCR were analyzed according to the Praffl method. Expression of β-actin, a housekeeping gene was estimated to normalize the expression of the target genes.

Estimation of liver carbonyl protein

The liver carbonyl protein assay was performed according to the method described by Colombo et al. (Colombo et al., 2016). The liver tissues (100 mg) were homogenized using 1 ml phosphate buffered saline (PBS), the homogenates were centrifuged (12,000× g for 15 min at 4 °C), and the supernatant was collected. The protein level in the supernatant was determined using a BCA protein assay reagent. The supernatant was diluted to a concentration of 1 mg/ml. 500 µl of the diluted supernatant was mixed with 100 µl of 10 mM 2,4-dinitrophenylhydrazine (DNPH)-(2N) HCl and incubated for 1 h at a room temperature with shaking.

The final sample was mixed with ice-cold 20% perchloric acid (600 µl) and then incubated for 15 min on ice. Following that, the samples were centrifuged (10,000× g for 5 min at 4 °C) and the pellets were collected. The pellets were mixed with ice-cold 20% perchloric acid (500 µl) and again centrifuged (10,000× g for 5 min at 4 °C). Subsequently, the pellets were collected and mixed with 1:1 (v/v) ethanol-ethyl acetate. They were again centrifuged (10,000× g for 5 min at 4 °C) and the final pellets were collected. This process of mixing ethanol-ethyl acetate and centrifugation was repeated.

The obtained pellets were mixed with 250 µl of 0.2% (w/v) SDS in 20 mM Tris–HCl (pH 6.8) and incubated at 95 °C for 10 min. The pellets were then homogenized by ultrasonication and the protein content was determined by BCA protein assay. Western blot was performed with the sample. The samples containing 1 µg of protein were denatured. Anti-DNPH was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

Estimation of liver triglyceride (TG) and total cholesterol (T-Chol) levels

The liver tissues (200 mg) were homogenized using 1 ml of PBS, the homogenates were centrifuged (12, 000 × g for 15 min at 4 °C), and the supernatant was collected. Liver TG and T-Chol levels were determined using the Triglyceride E-Test Wako (Wako) and the Cholesterol E-Test Wako (Wako), respectively.

Statistical analysis

Initially, the data were analyzed using two-way analysis of variance (ANOVA) to identify the effects of alcohol, BLEx, and their interaction. When the interaction was significant, the data were analyzed using the Tukey-Kramer test. The analyses were conducted using Statcel3 software (OMS Publishing, Saitama, Japan). A p value of <0.05 was considered as statistically significant.

Effects of BLEx on body weight, food intake, and liver weight

As shown in Fig. 1, final body weight and food intake in the alcohol intake groups were significantly lower than the non-alcohol intake groups. No significant difference was, however, noted between the non-BLEx intake and BLEx intake groups. The liver weight was unchanged by alcohol and BLEx intake.

Effects of BLEx on indices of alcoholic liver injury

The levels of serum AST, ALT, albumin, total protein, AST/ALT ratio, and A/G ratio in rats are shown in Fig. 2. As shown in Figs. 2A–2C, the serum ALT level in alcohol intake groups was significantly higher than the non-alcohol intake groups. The serum AST level and the ratio of AST/ALT, however, were not changed by alcohol and BLEx. As shown in Figs. 2D–2F, no significant difference was found among the experimental groups.

Effects of BLEx on level or expression of protein and mRNA related to alcohol metabolism in the liver

The liver ADH and ALDH activities in the rats are shown in Figs. 3A and 3B. The liver ADH activity was unchanged by alcohol and BLEx (Fig. 3A). The liver ALDH activity, however, significantly increased in the alcohol intake groups as compared to the non-alcohol intake groups. However, the ALDH activity was not changed by BLEx intake (Fig. 3B).

The liver ADH1 and ALDH2 mRNA expression in the rats are shown in Figs. 3C and 3D. Liver ADH1 mRNA expression was significantly changed by BLEx intake. Liver ALDH2 mRNA expression was not changed by alcohol and BLEx. Liver CYP2E1 protein and mRNA expression levels are shown in Figs. 3E–3G. As shown in Figs. 3E and 3F, liver CYP2E1 protein expression significantly increased in the ALD and ALD + BLEx groups as compared to the NLD and NLD + BLEx groups, respectively. No significant difference in the liver CYP2E1 protein expression was, however, noted between the ALD and ALD +BLEx groups. As shown in Fig. 3G, the liver CYP2E1 mRNA expression was significantly higher in the alcohol intake groups as compared to the non-alcohol intake groups. However, the CYP2E1 mRNA expression was not changed by BLEx.

Effects of BLEx on liver carbonyl protein level and HO-1 mRNA expression in the liver

As shown in Fig. 4A, the liver carbonyl protein level was not changed by alcohol and BLEx. As shown in Fig. 4B, the HO-1 mRNA expression in the liver was significantly increased in the alcohol intake groups as compared to the non-alcohol intake groups.

Effects of BLEx on liver TG and T-Chol levels in the liver

As shown in Figs. 5A and 5B, the TG and T-Chol levels in the liver were not changed by alcohol and BLEx.