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Kim, Ahn, Kim, Lee, Kim, Kim, and Shin: Hepatoprotective effect of fermented black radish (Raphanus sativus L. var niger) in CCl4 induced liver injury in rats

Abstract

Oxidative stress is one of common cause of fatty changes in the liver. Antioxidant capacity was confirmed in various vegetables including black radish (Raphanus sativus L. var niger). Fermentation of vegetables using Lactobacillus plantarum has been known to generate bioactive components. This study was conducted to determine if fermented black radish (FBR) ameliorates oxidative liver injury induced by CCl4 in rats. To accomplish this, FBR (250 and 500 mg/ kg) was orally administered to rats for 7 consecutive days, single CCl4 (1.5 mL/kg) treatment or no treatment orally. Serum chemistry at 24 hours after CCl4 injury showed that FBR (500 mg/kg) significantly reduced the level of both alanine aminotransferase and aspartate aminotransferase in CCl4 exposed rats. Moreover, FBR treatment significantly increased radical-scavenging effects in livers with the reduction of lipid peroxidation in CCl4 exposed rats. Histopathologic findings including Kupffer cell activation in the liver of each group matched those of serum chemistry. Collectively, black radish, through fermentation, exerts hepatoprotective capacity in CCl4 induced liver injury in rats through anti-oxidation.

INTRODUCTION

Raphanus sativus L.var niger (black radish), a variety of radish belonging to the Brassicaceae family, has been used worldwide in cuisine as well as in traditional medicine to treat metabolic disorders including hyperlipidemia [7], as well as for respiratory, alimentary [1, 15] and urinary system disorders [19]. The biological effects of black radish have been attributed to its components including anthocyanin, polyphenols, glucosinolate and its metabolite, isothiocyanate, each of which is closely associated with anti-oxidative effects [6]. Previous studies have shown that alcoholic extract of black radish attenuates bleomycin-induced pulmonary fibrosis via decreasing transforming growth factor beta1 level [5], and that aqueous extract of black radish induces detoxification enzymes in the cultured HepG2 human hepatoma cell line [11]. Moreover, white radish has been known to protect livers from attack by CCl4 [14] and by a mycotoxin zearalenone [19], while both ethanol- and aqueous-extracted radish leaves are involved in the hepatoprotection in CCl4 induced liver injury [22].
Radishes, including black radish, contain fibers, proteins, minerals and chemically diverse components, that are affected by processing [9]. Previous studies have shown that either ethanol- [5] or aqueous-extract [11] of black radish have been used for bleomycin-induced pulmonary fibrosis and the human hepatoma HepG2 cell. Alternatively, fermentation using microorganism including Lactobacillus spp. has been applied for the dissociation of fibers in vegetables and to release bioactive substances, leading to health benefits [17, 20].
Carbon tetrachloride (CCl4) has long been used as an oxidative chemical [16] for the induction of liver injury in animals through oxidation and Kupffer cell activation [8, 18]. Upon intra-peritoneal administration of CCl4 in rats, oxidative injury is preferentially found in the liver with fatty changes [3, 18]. Additionally, the CCl4 injury model is regarded as an appropriate model for the assessment of anti-oxidatives, including fruit extracts [3], fermented resources [4] and the single compound allyl isothiocyanate [2].
This study was conducted to determine if fermented black radish (FBR) ameliorates CCl4 induced liver injury in rats.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats weighing 200–250 g (6-7 weeks old) were used for all experiments (OrientBio, Kyunggido, Korea). Animals were maintained at a controlled temperature of 25°C–28°C under 12-h light/dark cycles and fed a standard diet and water ad libitum. All experimental procedures were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals at Jeju National University in Jeju City, Korea (permit number: 2016-0040).

Fermentation of black radish

A slightly modified method for vegetable fermentation was used in this experiment. Black radish obtained from a local farm in Jeju-do, Korea, was cleaned, and sterilized at 95 °C for 15 min to disinfect the surface, after which it was ground using a food mixer. The mixture was then mixed with distilled water (1:1 suspension) and autoclaved for 15 min at 121°C. The seed culture of Lactobacillus plantarum (Korean Culture Center of Microorganisms, KCCM) was incubated in De Man, Rogosa and Sharpe (MRS) agar for 24 h at 37 °C, and propagated in MRS broth under the same conditions. The organism was then used to give concentration of 0.7-1.0% in black radish suspension for 48 h in a shaking incubator. The fermentation was stopped by heating at 95°C for 15 min. Finally, the FBR was freeze-dried, pulverized, and packaged in a vacuum aluminum foil bag and stored at 4 °C until use. The fermentation yield was approximately 15% (w/w).

Total phenolic content

The total phenolic compound was measured by the Folin-Denis method as previously described [21]. Briefly, each sample (200 μL) was mixed with distilled water (1800 μL), then amended with Folin-Ciocalteaus’s phenol reagent (200 μL). The resulting solution was then mixed well and incubated for 5 min at room temperature. Next, 400 μL of 2M Na2CO3 was added to the previous solution, and finally diluted to 4 mL. After 1 Hour of incubation in the dark at room temperature, the absorbance was measured at 725 nm using an ELISA reader. The total phenolic content was calculated based on comparison to a standard curve generated using gallic acid (Sigma Chemical Co., St Louis, MO, USA).

DPPH radical scavenging activity

DPPH radical scavenging activity was determined previously described [12]. Briefly, the mixture solutions containing the same volume of samples and 0.4 mM DPPH solution were incubated at room temperature for 30 min. The absorbance of the reaction mixture was then measured at 517 nm. Ascorbic acid was used as a positive control.

Experimental groups

Rats were divided into five groups of five animals each (n = 5). The normal control group was orally administered tap water, while rats in the FBR group were orally administered FBR emulsified in water at a dose of 250 mg/kg and 500 mg/kg body weight for seven consecutive days prior to CCl4 injection.

CCl4 -induced hepatotoxicity

Three hours after final administration of FBR, a 1:1 (v/v) mixture of CCl4 and sterile olive oil was orally administered (1.5 mL/kg). Rats were fasted for 24 h after CCl4 administration and then sacrificed. At the time of sacrifice, blood samples and liver tissues were collected for serum chemistry and tissue examination, respectively.

Histopathological examination

After euthanasia, blood samples were collected, and liver tissues were fixed in 4% paraformaldehyde solution and routinely processed for paraffin embedding. Sections (5 µm) of paraffin-embedded liver were deparaffinized and stained with hematoxylin and eosin.

Serum chemistry

Serum sample prepared at the time of sacrifice, were subjected to biochemical analysis as described in our previous paper [2]. To determine the extent of liver damage, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a FUJI DRI-CHEM 4200 (FUJIFILM, Tokyo, Japan).

Measurement of liver superoxide dismutase and catalase activities

To examine the superoxide dismutase (SOD) and catalase activity (CAT), liver tissues (n = 5 animals in each group) were immediately frozen and then homogenized using a glass-Teflon homogenizer with cold lysis buffer. The biochemical parameters, including SOD and CAT, were determined by commercial assay kits (Abcam, Cambridge, UK).

Measurement of lipid peroxidation in the liver

Lipid peroxidation determined by measuring the level of thiobarbituric acid-reactive substances (TBARS) as shown in our previous paper [2] is one of markers for lipid peroxidation in the liver.

Immunohistochemistry

For detection of activated Kupffer cells/macrophages, liver tissue was subjected to immunohistochemistry using an ABC Elite kit (Vector Labs, Burlingame, CA, USA) according to the manufacturer’s recommendations, with the primary rabbit anti-ionized calcium binding protein-1 (Iba-1) (Iba-1; 1:800; Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 1 h. The peroxidase reaction was developed using a diaminobenzidine (DAB) substrate kit (Vector Labs). The sections were counterstained with hematoxylin before mounting. Semi-quantitative analysis was then performed using ImageJ software (NIH, Bethesda, MD, USA). Iba-1 immunostaining sections were captured by 200× magnification using a digital camera (Olympus DP72) attached to a light microscope (Olympus BX53/U-LH 100HG, Olympus Corp., Tokyo, Japan). Finally, the Iba-1 immunolabeling-positive area of the total area [(positive area/total area) × 100 (%)] was determined, and the results were shown as means ± the standard error of the mean (SEM).

Statistical analysis

The data were presented as the means ± standard error. Groups were compared by one-way analysis of variance followed by the Student–Newman–Keuls post hoc test for multiple comparisons. In all cases, a p < 0.05 was considered to indicate significance.

RESULTS

Black radish fermentation

Powder of FBR in water suspension was orally administered to rats at a volume of approximately 1-2 mL each time. In a preliminary experiment, we have tested a different dose of FBR, and chosen daily 500 mg/kg in a highest concentration of FBR. FBR contains a variety of components including fibers and ingredients as well as cell walls of Lactobacillus plantarum. Among these, the total phenolic contents in FBR were increased (4.49 ± 0.01 mg GAE/g) compared with those of non-fermented black radish (2.98 ± 0.00 mg GAE/g) (Table 1). The DPPH radical scavenging activity in FBR (83.4 ± 2.0%) was also higher than that of non-fermented black radish (61.2 ± 2.5%) (Table 1). Therefore, we used the FBR, which has more polyphenols and better DPPH radical-scavenging activity.
Table 1
Total polyphenol contents and DPPH concentration in the black radish with or without fermentation
Total polyphenol content (mg GAE/g) DPPH radical scavenging activitya(%)
Fermented black radish 4.49 ± 0.01 83.4 ± 2.0
Black radish 2.98 ± 0.00 61.2 ± 2.5

a Sample concentration was 10 mg/mL.

Serum chemistry

In the control rats, the levels of serum ALT and AST were in normal ranges (51.5 ± 2.59 U/L and 99 ± 16.59 U/L, respectively; Table 2). A significant increase in both ALT (314.25 ± 19.13 U/L) and AST (582.5 ± 32.60 U/L) was detected in vehicle treated CCl4 treated rats (p < 0.001 vs. the normal control). Pretreatment with a high dose of FBR (500 mg/kg) for 7 days significantly reduced the levels of both ALT (146.5 ± 32.09 U/L) and AST (180.75 ± 25.38 U/L) (p<0.001 vs. CCl4 group). The ameliorative effect of FBR at 250 mg/kg was also recognized in ALT (262.75 ± 28.86 U/L) (p<0.05) (Table 2).
Table 2
Effects of pretreatment with FBR on biochemical parameters in the sera and livers of rats injected with carbon tetrachloride
ALT AST SOD CAT
(U/L) (U/L) (U/mg) (U/mg)
Normal 51.5 ± 2.59 99 ± 16.59 4.17 ± 0.68 15.4 ± 2.0
Vehicle+ CCl4 314.25 ± 19.13*** 582.5 ± 32.60*** 3.02 ± 0.48** 9.45 ± 3.3**
FBR 250 mg/kg + CCl4 262.75 ± 28.86# 453.5 ± 64.52 5.23 ± 1.03## 22.8 ± 0.32##
FBR 500 mg/kg + CCl4 146.5 ± 32.09### 180.75 ± 25.38### 5.81 ± 1.44## 18.01 ± 3.29##

Values are means ± SEM.

** p < 0.01,

*** p < 0.001vs. Normal control group.

# p < 0.05,

## p < 0.01,

### p < 0.001, vs. CCl4 treated rats.

Abbreviations. ALT: alanine aminotransferase; AST: aspartate aminotransferase; FBR: fermented black radish.

FBR ameliorates the down-regulation of radical- scavenging enzymes

To examine the anti-oxidant and radical-scavenging effects of FBR on acute CCl4 liver injury of rats, we investigated the activities of SOD and CAT in liver tissues. Both SOD and CAT levels were significantly decreased in the livers of rats treated with vehicle compared to normal control groups. However, pretreatment with FBR significantly reduced the levels of SOD and CAT (Table 2).

Histopathology

Oral administration of CCl4 induced infiltration of inflammatory cells, as well as vacuolar changes in hepatocytes in the liver of rats (Fig. 1B), while no inflammatory cells were found in the livers of rats treated with vehicle (Fig. 1A). Pretreatment with FBR 250 mg/kg (Fig. 1C) and 500 mg/kg (Fig. 1D) followed by CCl4 exposure significantly reduced vacuolar changes in the livers of rats.
Fig. 1
A representative photos of liver sections from control and CCl4-injured rats with or without FBR pretreatment. (A) Normal control; (B) vehicle-treated CCl4 group; (C) FBR (250 mg/kg)-treated CCl4 group; (D) FBR (500 mg/kg)-treated CCl4 group. A–C: Hematoxylin-Eosin staining. CV, central veins. Scale bars in A–D represent 50 µm.
JPVM_41_143_fig_1.tif

FBR reduces lipid peroxidation

To investigate whether FBR modulates lipid peroxidation in the liver, MDA levels were measured in the livers of CCl4 injured rats with or without FBR pretreatment at 500 mg/kg, which was found to be the most effective dose. The level of MDA increased significantly in vehicle treated CCl4 injured rat liver (0.207 ± 0.031 μM/mg) compared with that of the normal control (0.121 ± 0.015 μM/mg), while FBR pretreatment significantly reduced MDA levels (0.136 ± 0.021 μM/mg) compared to those of vehicle-treated CCl4- injured rats (P<0.01) (Fig. 2).
Fig. 2
Bar graphs of MDA level of liver of rats with or without FBR treatment. MDA level was significantly increased in vehicle treated rats with CCl4- injury compared with that of normal control (P<0.01). FBR pretreatment in rats with CCl4 treatment significantly reduced the elevated level of MDA (p<0.01). #, p < 0.01 vs. normal control. *, p < 0.01 vs. vehicle treated CCl4 group.
JPVM_41_143_fig_2.tif

FBR reduces Kupffer cells/macrophages activation

Kupffer cells/macrophages activation is an important indicator of inflammation in CCl4-injured experiments [2]. Some Iba-1-positive Kupffer cells were detected along the sinusoids of the rat liver in the normal control group (Fig. 3A), in which no infiltration of inflammatory cells was seen. Activation of Kupffer cells and infiltration of inflammatory cells (Fig. 3B, Fig. 3arrowheads) were detected in the CCl4–injured rats (Fig. 3B), while FBR pretreatment reduced the numbers of Iba-1-positive cells in the 250 mg/kg (Fig. 3C) and 500 mg/kg treated rats (Fig. 3D). Semi-quantitative analysis of Iba-1 immunoreactivity in liver tissues using the ImageJ software supported the histopathological findings (Fig. 3E). The proportion of the Iba-1-immunoreative area in the CCl4 control was increased significantly (4.97 ± 0.57%; p < 0.01) relative to the normal group (2.60 ± 0.25%), while the Iba-1-immunoreactive area was reduced significantly in the 250 mg/kg (3.95 ± 0.55%; p < 0.05) and 500 mg/kg (3.83 ± 0.20%; p < 0.01) FBR pretreatment groups, relative to the CCl4 control group.
Fig. 3
Immunohistochemical staining of Iba-1 in the CCl4 injured rat liver with or without FBR treatment. (A) Normal control, (B) Vehicle + CCl4, (C) FBR 250mg/kg + CCl4. (D) FBR 500mg/kg + CCl4. (E) Bar graph shows the semi-quantitative analysis of Iba-1 immunoreactivity. Scale bars in (A–D) are 50 μm. Values in (E) are means ± SEM, n = 5 animals in each group. **p < 0.01, vs. control group. #p < 0.05, ##p < 0.01, vs. CCl4-injured group.
JPVM_41_143_fig_3.tif

DISCUSSION

This is the first confirmation that oral uptake of black radish that has been subjected to Lactobacillus fermentation is associated with hepatoprotection in rats exposed to CCl4. In fermented black radish, the level of polyphenolic compounds, which serve as an alternative marker for antioxidation from plants, was approximately 4.49 ± 0.01 mg GAE/g, indicating that the fermented black radish was partly involved in protection of hepatocytes.
We found that FBR protected the liver in a dose dependent manner. Specifically, pretreatment with FBR at 500 mg/kg was shown to significantly reduce the level of both ALT and AST elevated by CCl4, suggesting that fewer hepatocytes are destroyed in FBR treated groups. In addition, both SOD and CAT, which were representative enzymes in the liver injury experiment, were significantly increased by FBR pretreatment in the CCl4-inujured rats when compared with those of the vehicle-treated CCl4 groups. Even at low doses (250 mg/kg), the ameliorative effect of CCl4 injured liver was verified (Table 1). We postulate that anti-oxidative components in FBR play a role in the protection of hepatocytes subjected to oxidative attack, as does alcohol extract of radish [19] and protease digested white radish [14]. This is because radish with or without color contains a variety of bioactive substances, including anthocyanins, polyphenols, isothiocyanates, and quercetin [9, 10, 22].
Lipid peroxidation is a marker of cell death [2]. In the present study, MDA was minimally detected in the liver of control rats, while CCl4 exposure in rats elevated the MDA level in the liver, suggesting that CCl4 stimulates lipid peroxidation in the cell membrane, leading to cell death. The present study revealed that FBR partially inhibited the lipid peroxidation caused by CCl4 in this model.
Kupffer cells/macrophages activation is an important indicator of liver injury. Additionally, the activation of and/or increases in macrophages and Kupffer cells has been associated with CCl4-induced liver injury [2] and suggested to play critical roles in chronic ethanol induced liver injury [23]. In the present study, we semi-quantitatively compared the activation of Kupffer cells/macrophages among group. In a semi-quantitative analysis of Kupffer cells/macrophages, we found an increased number of Iba-1-positive cells in CCl4-induced liver injury, while pretreatment with FBR (250 and 500 mg/kg) suppressed the activation of Kupffer cells and infiltration of macrophages.
We postulate that ingredients from FBR synergistically protect hepatocytes in this experiment with dead Lactobacillus plantarum. This is because it is well known that nano-sized Lactobacillus plantarum, even when dead, improve the immune response in cultured mouse splenocytes as well as in RAW 264.7 cells [13]. Collectively, black radish, fermented by Lactobacillus spp., exerted a hepatoprotective effect, possibly through antioxidation. Accordingly, fermented radish could be an alternative to the radish diet.

ACKNOWLEDGEMENTS

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant number: 316006-05-1-HD040).

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