Yu-Ping Wang, Ming-Liang Cheng, Bao-Fang Zhang, Mao Mu,Ming-Yu Zhou, Jun Wu and Cheng-Xiu Li

Guiyang, China

Effect of blueberry on hepatic and immunological functions in mice

Yu-Ping Wang, Ming-Liang Cheng, Bao-Fang Zhang, Mao Mu,Ming-Yu Zhou, Jun Wu and Cheng-Xiu Li

Guiyang, China

(Hepatobiliary Pancreat Dis Int 2010; 9: 164-168)

blueberry;Nrf2;HO-1;Nqo1;T-cell function

Introduction

Blueberry (Vaccinium spp.) is a fl owering plant of the family Ericaceae. Although blueberry is native to North America, it is now grown in the Southern Hemisphere in Australia, New Zealand, and South American countries. Since the beginning of this century, it has been successfully introduced and cultivated in China.Blueberry is among the functional foods called superfruits,having the favorable combination of nutrient richness and antioxidant properties. Accumulated evidence suggests that it has health bene fi ts and versatility for manufacturing popular consumer products.[1]Blueberry ranks the fi rst in the fi fteen functional foods listed by authoritative nutritionists in the United Kingdom. In view of its rich nutrient ingredients, the Human Nutrition Research Center of the United States has made a series of studies on blueberry, indicating that it contains anthocyanins, polyphenols, and fl avonoids, and appears to have the highest antioxidant capacity among fruits and vegetables.[2,3]

Oxidative stress and cellular immunity dysfunction are important in the pathogenesis of liver diseases.[4]Nrf2 is an important transcription factor regulating antioxidative stress reactions. Since both HO-1 and Nqo1 are major antioxidative enzymes that are controlled by Nrf2,[5,6]we intended to determine whether blueberry increases the antioxidative capability of the liver by induction of Nrf2, HO-1, and Nqo1, and by modulation of liver superoxide dismutase (SOD) and malondialdehyde(MDA) levels in mice.

Antioxidative responses increase immune function by scavenging free radicals and stimulating immunecells. CD3+T lymphocytes in peripheral blood represent the level of cellular immune function as a whole.The percentages of CD4+lymphocytes and CD8+T lymphocytes and the ratio of CD4+/CD8+conveniently estimate the immune state. In this study, we measured the differentiation antigens of CD3+, CD4+and CD8+in peripheral blood of mice by fl ow cytometry and spleen lymphocyte proliferation by the MTT assay to further determine the effects of blueberry on cellular immune function.

MethodsReagents and animal treatments

Blueberry (Rabbiteye Blueberry) was obtained from the Blueberry Production Field of Majiang, Guizhou.Blueberry was harvested freshly in July, then stored at-20 ℃ until use, and homogenized to prepare blueberry juice. One milliliter of blueberry juice was equivalent to about 2 g of dried blueberry. All other chemicals were reagent grade.

Male Kunming mice, 6-8 weeks old and weighing 18-22 g, were obtained from the Experimental Animal Center of Guiyang Medical College, Guiyang, China. All animal studies complied with the Animal Care and Use Guidelines of China.

The study was divided into three parts. In experiment 1, healthy mice were randomly divided into a blueberry group (n=10) and a normal saline control group(n=8). Mice in the blueberry group were administered blueberry juice (0.6 g dried blueberry/10 g body weight), once a day by gavage for 21 days, while mice in the control group were treated with normal saline.At the end of the experiment, mice were sacri fi ced to collect the liver for total RNA isolation. Antioxidation parameters (SOD and MDA) were determined, and the liver index was measured. In experiment 2, forty healthy mice were randomly divided into four groups: a normal control group (A), a low-dose blueberry-treated group(B), a medium-dose blueberry-treated group (C) and a high-dose blueberry-treated group (D), with 10 mice in each group. One mouse died in group B. Mice in groups B, C, and D were treated with blueberry juice at 0.4, 0.6,and 0.8 g/10 g body weight, respectively, once a day by gastrogavage for 35 days, while those in group A were treated with normal saline. At the end of the experiment,all mice were anesthetized, weighed, and their blood was collected into anticoagulant tubes. The thymus and spleen were removed and weighed. In experiment 3, the same treatments were performed as in experiment 2 (10 mice per group except for 9 in group B) and at the end of this experiment, the MTT colorimetric assay was used to measure spleen lymphocyte proliferation.

Liver index (LI) was calculated according to the formula:[7]

LI =[liver weight (g)/body weight (g)]×100%

Thymus index (TI) was calculated according to the formula:[8]

TI =[thymus weight (g)/body weight (g)]×100

Spleen index (SI) was calculated according to the formula:[8]

SI=[spleen weight (g)/body weight (g)]×100

Measurement of liver homogenate SOD and MDA

Liver homogenates were prepared. The SOD activity was measured with the xanthine oxidase method and MDA content with the thiobarbituric acid method,according to the manufacturer's instructions (Jiancheng Biologic Co., Nanjing, China).

Real-time RT-PCR analysis

Total RNA was extracted from liver tissues with Trizol reagent (Invitrogen, USA) and puri fi ed on RNeasy columns (Tiangen, China), followed by reverse transcription with MMLV reverse transcriptase and oligodT primers. The forward and reverse primer sequences for selected genes were designed with ABI Primer Express software (Foster City, CA, USA) (Table 1). The SYBR Green DNA PCR kit (Applied Biosystems, Cheshire, UK)was used for real-time RT-PCR analysis. The Ct values of the genes of interest were fi rst normalized with β-actin of the same sample, and then the relative differences between the groups were calculated and expressed as relative increases, setting controls as 100%.

Flow cytometric analysis of differentiation antigens of CD3+, CD4+ and CD8+

Anticoagulated blood from groups A, B, C, and D was treated with hemolysin and made into a lymphocyte suspension (1×106/ml), which was marked (FITC antimouse CD3+, PE anti-mouse CD4+, PE anti-mouseCD8+; eBioscience, USA) away from light. Differentiation antigens of CD3+, CD4+, and CD8+were measured by fl ow cytometry. Data analysis was made using a FACSCalibur fl ow cytometer (Becton Dickinson, USA) and Cell Quest software.

Table 1. Primer sequences for real-time RT-PCR analysis

MTT assay for proliferation

Spleens from each group were removed and gently squeezed with a syringe in RPMI-1640 (hyclone, USA).The cells were fi ltered through 200 sieve-meshes and were resuspended in 8.3 ml/L Tris-NH4Cl to lyse erythrocytes, then washed and resuspended in RPMI-1640. One hundred microliters of cell suspension was seeded in 96-well culture plates (Corning, USA)with 2×106cells/ml, 6 wells per sample. To 3 wells,100 μl RPMI-1640 containing 10% fetal bovine serum (controls) was added, and cell culture medium containing concanavalin A (Con A, fi nal concentration,5 μg/ml; Sigma, USA) (treatment) was added to the other three. The fi nal volume of each well was 200 μl. Cells were incubated at 37 ℃ in a 5% CO2air atmosphere.After culture for 72 hours, 100 μl supernatant was discarded, then 20 μl MTT (5 mg/ml; Sigma, USA)was added to each well and the culture continued for 4 hours. After that, 150 μl DMSO was added to each well.The optical density (OD) of the samples was measured at 570 nm with an ELISA reader (BioTek, USA). The stimulation index (SI) was calculated for each sample as absorbance values for cells with mitogen divided by absorbance values for cells without mitogen.[9,10]

Statistical analysis

The data were expressed as mean±SD. For comparison between two groups, Student's t test was used. For comparisons among three or more groups, data were analyzed using a one-factor analysis of variance(ANOVA). All statistical analyses were conducted using SPSS version 13.0 for Windows, and a P value ≤0.05 was considered statistically signi fi cant.

Results Effect of blueberry on LI, SOD, and MDA in liver homogenate

Blueberry had no effect on LI (P＞0.05),but increased the activity of SOD and decreased the activity of MDA of liver homogenate (P＜0.05) (Table 2).

Effects of blueberry on mRNA expression of Nrf2,HO-1, and Nqo1 in the liver of mice

Compared to the normal saline control group,the expressions of Nrf2 mRNA, HO-1 mRNA, and Nqo1 mRNA in the blueberry group were signi fi cantly elevated (Fig. 1).

Table 2. LI, SOD, and MDA in the liver of mice with and without blueberry treatment

Fig. 1. Effects of blueberry on the expression of Nrf2 mRNA,HO-1 mRNA, and Nqo1 mRNA in liver tissues of mice. *: P＜0.05,compared with the control group.

Fig. 2. Effect of blueberry on TI and SI in mice. *: P＜0.05, compared with group A.

Effect of blueberry on SI and TI in mice

The SI in groups B, C, and D was higher than that in group A (P＜0.05) (Fig. 2). However, the differences of TI in the various groups were not statistically signi fi cant.

Effects of blueberry on T lymphocytes in peripheral blood of mice

The frequency of CD3+and CD4+and the ratio of CD4+/CD8+in peripheral blood were all increased by treatment with blueberry (P＜0.05), but the frequency of CD8+was not changed signi fi cantly (Table 3).

Table 3. Effects of blueberry on T lymphocytes in peripheral blood of mice

Table 4. Effects of blueberry on T lymphocytes proliferation of the spleen

Effects of blueberry on T lymphocyte proliferation

The stimulation index of groups B, C, and D was higher than that in group A (P＜0.05) (Table 4).

Discussion

In the present study, blueberry had no detectable impact on liver index, and had the ability to increase the production of anti-oxidant SOD and the expression of Nrf2, HO-1, and Nqo1 in the liver of mice, suggesting that blueberry induces the expression of HO-1 and Nqo1 through the Nrf2-ARE pathway. The Nrf2 pathway is an important defense system against oxidative stress, and blueberry is bene fi cial to the liver by activating these antioxidant components. The SI in groups with different dosages of blueberry was signi fi cantly higher than that in the control group, suggesting a bene fi cial effect of blueberry on immune organs. Blueberry also increased the amounts of CD3+and CD4+and the CD4+/CD8+ratio,suggesting a bene fi cial effect of blueberry on cellular immune function.

Blueberry has been shown to have bene fi cial effects on human retinal cells, brain cells, and tumor cells.[11-14]Blueberry also increases the antioxidant capacity (SOD and glutathione peroxidase) of whole blood in children with type 1 diabetes.[15]Polyphenolics of blueberry enhance red blood cell resistance to oxidative stress in vitro and in vivo.[16]A blueberry-enriched diet provides cellular protection against oxidative stress, and reduces kainate-induced learning impairment in rats.[17]The present study showed that blueberry is also effective in activating hepatic antioxidant components and modulating cellular immune function.

Nrf2 is an important transcription factor for the antioxidant system and has a protective role against oxidative stress. The activity of Nrf2 is controlled by the protein Kelch-like ECH-associated protein 1 (Keap1).Keap1 holds Nrf2 in the cytoplasm of the cell when no oxidants are present. When oxidative stress occurs,Keap1 no longer interacts with Nrf2, which moves into the nucleus, links to a binding site (5'-GCT GAG TCA-3')in ARE and promotes the expression of antioxidant components against oxidative stress.[18]HO-1 is one of the important antioxidant enzymes regulated by Nrf2.Similarly, Nqo1 is a cytosolic enzyme regulated by Nrf2. Nqo1 also defends against intracellular oxidative stress by scavenging superoxide and maintaining the reduced form of endogenous antioxidants including alpha-tocopherol-hydroquinone and coenzyme Q.[19]Nrf2 knockout rats are quite sensitive to liver injury induced by CCl4and paracetamol.[20,21]Oleanolic acid induces the activation of the Nrf2 pathway to produce hepatoprotective effects.[22]The capability of blueberry to induce the Nrf2 pathway may have implications for its bene fi cial effects on the liver.

Enhancement of antioxidant capacity could have a positive impact on cellular immune function, possibly through scavenging free radicals and activating immune cells.[23]The thymus and spleen are important immune organs,[24]and the increased spleen index suggest a bene fi cial effect of blueberry on immune function. In further investigation, the subsets of T cells should be analyzed. CD3+T lymphocytes represent the level of cellular immunity as a whole, while CD4+lymphocytes and CD8+T lymphocytes represent helper T lymphocytes and T suppressor lymphocytes, respectively. Acute liver failure is often accompanied by disturbance of cellular immune function.[25]Cellular immune dysfunction usually occurs in patients with chronic hepatitis B[26-28]and immunode fi ciency due to HCV, a major factor that leads to chronic hepatitis C.[29,30]Immunotherapy also affects T lymphocyte subsets.[31]In this study, the results showed that blueberry also had bene fi cial effects on the frequency of CD3+, CD4+and the CD4+/CD8+ratio, suggesting that cellular immune function is enhanced by blueberry.

In conclusion, this study demonstrated that blueberry induces the expression of Nrf2, HO-1, and Nqo1 in the liver of mice, which is bene fi cial to the liver antioxidant system. In addition, blueberry also has bene fi cial effects on T-cell immune function.

Acknowledgment

The authors thank Dr. Liu J for his help in the experimental research.

Funding: This study was supported by a grant from the Foundation of High Level Talented Specialists of Guizhou province, China(TZJF-200850).

Ethical approval: Not needed.

Contributors: CML proposed the study. WYP wrote the fi rst draft and analyzed the data. All authors contributed to the design and interpretation of the study. CML is the guarantor.

Competing interest: No bene fi ts in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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BACKGROUND: Conventional drugs used in the treatment and prevention of liver diseases often have side effects, therefore research into natural substances are of signi fi cance. This study examined the effects of blueberry on liver protection and cellular immune functions.

METHODS: To determine the effects of blueberry on liver protective function, male mice were orally administered blueberry (0.6 g/10 g) or normal saline for 21 days. Hepatic RNA was extracted by Trizol reagent, and the expression of Nrf2, HO-1, and Nqo1 was determined by real-time RTPCR. Superoxide dismutase (SOD) and malondialdehyde(MDA) in liver homogenate were determined, and liver index was measured. To assess the effects of blueberry on cellular immune function, male mice

blueberry (0.4, 0.6, or 0.8 g/10 g) for 35 days, and the percentages of CD3+, CD4+,and CD8+T lymphocyte subgroups in peripheral blood were detected by fl ow cytometry, the index of the thymus and spleen was measured, and lymphocyte proliferation in the spleen was determined by MTT assay.

RESULTS: Blueberry treatment signi fi cantly increased the expression of Nrf2, HO-1, and Nqo1, the important antioxidant components in the liver. Hepatic SOD in the blueberry group was higher and MDA was lower than that in the control group(P＜0.05). Blueberry also increased the index of the spleen and enhanced the proliferation of lymphocytes of the spleen(P＜0.05). The percentages of the CD3+and CD4+T lymphocyte subsets and the CD4+/CD8+ratio were also increased by blueberry (P＜0.05).

CONCLUSIONS: Blueberry induces expression of Nrf2, HO-1,and Nqo1, which can protect hepatocytes from oxidative stress.In addition, blueberry can modulate T-cell function in mice.

Author Af fi liations: Department of Infectious Diseases (Wang YP, Cheng ML,Zhang BF, Mu M, Zhou MY and Wu J), and Department of Pharmacology(Li CX), Guiyang Medical College, Guiyang 550004, China

Ming-Liang Cheng, MD, Department of Infectious Diseases, Guiyang Medical College, Guiyang 550004, China (Tel: 86-851-6752795; Fax: 86-851-6741623; Email: chengml@21cn.com)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.

April 29, 2009

Accepted after revision February 21, 2010