Immunostimulatory Effects of Red Beet Ferment Extract on Cyclophosphamide-Induced Immunosuppression in Wistar Rats

Abstract

Red beet extract contains a variety of bioactive compounds, including antioxidant and immune-stimulating properties. Red beets are rich in nutrients and have been reported to help mitigate cardiovascular disease, while also possessing potent antioxidant capacity. In this study, we sought to determine the immune-enhancing effects of red beet fermented extract. The immunosuppressive drug cyclophosphamide (Cy) and red beet fermented extract were orally administered to Wistar rats once daily for four weeks. Blood analysis was performed to measure the expression of immune cytokines. We also examined the immunostimulatory effects of red beet fermented extract by measuring the activity and viability of spleen cells and the expression of immune cytokines. The expression of immune cytokines in the blood and spleen cells was increased in the red beet fermented extract treatment group compared to that in the negative control group, which induced immunosuppression of cytokines. It also increased the activity of NK cells and improved the viability of spleen cells. In conclusion, red beet fermented extract increased the expression of immune cytokines and increased the activity of adipocytes and NK cells, confirming its immunostimulatory effect. Therefore, red beet fermented extract has the potential to be utilized as a nutraceutical ingredient to enhance immunity.

1. Introduction

The immune system is an important physiological process that protects our bodies from pathogens and abnormal cells. It identifies and eliminates foreign cells or substances that have invaded the body. Immunity is divided into innate immunity, which is present at birth, and acquired immunity, which develops after exposure to a pathogen. Innate immunity is a natural defense mechanism that an organism possesses at birth that provides immediate protection by quickly identifying and eliminating invading pathogens or abnormal cells [1,2,3]. Innate immunity is primarily based on the structure of the skin, mucous membranes, and chromosomal DNA, and the main components of innate immunity include physical barriers such as the skin and mucous membranes, natural killer cells, and inflammatory responses [4,5,6].

Acquired immunity, on the other hand, develops after birth as the body is exposed to pathogens, providing a specific response to foreign invaders and allowing the body to adapt continuously. Adaptive immunity includes antibody-mediated and cell-mediated immune responses, which develop through interactions with pathogens [7,8]. The main adaptive immune cells include B cells and T cells, which are responsible for recognizing and destroying pathogens. Adaptive immunity also has a memory function, allowing it to respond quickly when re-exposed to previously encountered pathogens [9,10].

Fermented natural products have been used since ancient times to promote health, and natural raw materials are fermented with beneficial microorganisms or enzymes to produce a variety of bioactive substances. The fermentation process not only increases the bioavailability of nutrients present in the raw material but also creates new beneficial components that positively impact health. Fermented natural products are particularly rich in prebiotics, which promote the growth of beneficial bacteria and inhibit harmful bacteria, improving the balance of the gut microbiome and aiding digestion [11,12,13]. Fermentation also increases the absorption of nutrients, such as vitamins, minerals, and amino acids, optimizing nutritional status [14].

The most beneficial aspect of fermented natural products is their potent antioxidant activity, which protects against cell damage, reduces inflammation, and contributes to anti-aging and chronic disease prevention [15]. In particular, research has shown that fermentation can promote the activation of immune cells, with a study showing that a fermented schisandra extract administered to mice increased the proliferation of B and T cells by 30% and 22%, respectively, and significantly enhanced cytokine secretion and NK cell activity, resulting in an enhanced immune response [16]. This suggests that the metabolites produced during fermentation may potentiate immunostimulatory effects.

Red beet (Beta vulgaris subspecies) is a root vegetable with a distinctive red color that is rich in betalains and anthocyanins and has excellent antioxidant and anti-inflammatory effects [17,18]. Red beets contain high amounts of essential nutrients, such as vitamin C, potassium, magnesium, and iron, and are low in calories and fat, making them a favorable food for health management and weight control [19,20]. In particular, the nitrates contained in red beets help dilate blood vessels and improve blood flow, which lowers blood pressure and promotes cardiovascular health [21]. In addition, antioxidant components such as betacyanin and betanin protect cells from oxidative stress, reduce inflammation, and contribute to the prevention of chronic diseases [22,23]. These properties make red beets an important raw material for the development of functional foods and pharmaceuticals [24,25]. Previous studies have shown that red beets are effective as anti-anemic, anti-ischemic, anti-inflammatory, antioxidant, and anticancer agents and are also beneficial for optimizing intestinal peristalsis and lipid metabolism [19]. Red beets have also been reported to exert immune effects by protecting immune cells [26]. These studies show that red beets contribute to immune regulation through a variety of physiological activities.

Previous studies have fermented red beets with various additives such as salt, sugar, garlic, and bacteria to enhance their antioxidant and anti-inflammatory effects and improve the bioavailability of their bioactive components [27,28,29]. Meanwhile, other studies have identified anti-inflammatory and immune-enhancing effects of betalain, the active ingredient in red beet [30,31,32]. However, the in vivo immunomodulatory effect of red beet fermented extract in immunosuppression-induced rats is unknown.

In this study, we aimed to evaluate the immune-enhancing effects of fermented red beet by spontaneously fermenting only red beet at low temperatures using a simplified process, in contrast to conventional fermentation methods. Through this low-temperature spontaneous fermentation, we aimed to identify the effects of red beet’s unique components on immune regulation and analyze the immune activity of fermented red beet extract. To address our research objectives, we evaluated the immune-enhancing effects of red beet fermented extract in a cyclophosphamide-induced immunosuppressed Wistar rat model. Natural fermented extracts are known to provide various beneficial effects due to their inclusion of diverse bioactive compounds. Numerous studies have also reported their antioxidant effects, immune-boosting properties, improved digestion and absorption, and anti-inflammatory and antimicrobial activities. In this study, we hypothesized that the fermentation process of red beet, the raw material used in this research, would break down and transform its active components, thereby enhancing their bioavailability and efficacy. Furthermore, we predicted that bioactive compounds produced during fermentation would modulate and strengthen the immune system or exert alleviating effects.

2. Materials and Methods

2.1. Preparation of Red Beet Fermented Extract

Red beet raw materials were provided by Care Innovation (Jeju, Republic of Korea), powdered, and dissolved in distilled water. to prepare an aqueous solution. The aqueous red beet extract was fermented at 4 °C for 7 days. The fermented aqueous solution was centrifuged at 9000 rpm for 30 min and freeze-dried using a freeze-dryer (LP80, Ilshin Bio, Jeonju, Republic of Korea) to form a powder for use in the experiments.

2.2. Cell Line and Culture

The AR42J cell line was obtained from the American Type Culture Collection (ATCC). The reagents required for cell culture were purchased from Gibco (Waltham, MA, USA). The cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antifungal solution. The cells were maintained at 37 °C in an incubator containing 5% CO₂.

2.3. Experimental Groups and Sample Administration

Wistar rats were purchased from OrientBio (Seongnam, Republic of Korea) and allowed to adapt for 7 days. The temperature in the housing room was maintained at 24 ± 2 °C, relative humidity was 50 ± 5%, light/dark cycle was maintained at 12 h, and a standard diet and water were provided ad libitum. After a one-week acclimatization period, the rats were divided into six groups: normal, negative control, red beet fermented extract 30 mg/kg, 100 mg/kg, 300 mg/kg, and positive control, with 10 rats per group. The normal group was not administered any of the agents. The negative control group was treated with Cy at a dose of 5 mg/kg to induce immunosuppression, but no samples were treated. After inducing immunosuppression, low (30 mg/kg), medium (100 mg/kg), and high (300 mg/kg) concentrations of red beet fermented extract were treated, while the positive control group was treated with the herbal medicine preparation Hemohim (purchased from Kolma BNH., Seoul, Republic of Korea) at a dose of 1000 mg/kg. All treatments were administered at the same time once daily for 4 weeks. All animal experiments in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUV approval number: iv-RB-17-2304-10).

2.4. Organ Weights

All experimental animals were weighed once before sample administration and once a week thereafter at the same time. At the end of the experiment, the animals were anesthetized using isoflurane (Isoflurane, 99.9%, USP, North Bethesda, MD, USA). The anesthetic concentration during induction was set at 2–2.5% inhaled oxygen, and the anesthetic concentration during maintenance was set at 1.5–1.8%. After inhalational anesthesia, blood was drawn from the abdominal vena cava, and after euthanasia using CO₂, the abdominal cavity was opened to remove and weigh the spleen and thymus. The spleen and thymus indices were calculated using the following formula: Spleen index or thymus index (%) = (spleen or thymus weight/body weight) × 100%.

2.5. Complete Blood Cell (CBC) Count Analysis

At the end of the 4-week Cy and red beet fermented extract administration period, blood samples were collected from the abdominal vena cava of the experimental animals and analyzed. A hematology analyzer (BC-2800, Mindray, Bath, UK) was used to measure white blood cells (WBC), granulocytes (GRA), lymphocytes (LYM), and medium-sized cells (MID). For biochemical analysis, the collected blood was centrifuged at 3000 rpm for 10 min to separate the plasma. The separated serum was used for the measurement of interleukin-2 (IL-2; Elisa kit, Mybiosource, San Diego, CA, USA), interleukin-6 (IL-6; Elisa kit, San Diego, CA, USA), tumor necrosis factor-alpha (TNF-α; Elisa kit, R&D Systems, Minneapolis, MN, USA), interferon-gamma (IFN-γ; Elisa kit, R&D Systems, Minneapolis, MN, USA), nitric oxide (NO; Elisa kit, Mybiosource, San Diego, CA, USA), and aspartate aminotransferase (AST) for biochemical assays: Aspartate aminotransferase, ALT: Alanine aminotransferase).

2.6. Nitric Oxide (NO) Production

0.5 mL of rat serum was placed in a centrifuge tube, mixed with buffer, centrifuged at 4 °C, 10,000× g for 10 min, and the supernatant was used to measure nitric oxide using an ELISA kit (Mybiosource, San Diego, CA, USA) according to the manufacturer’s instructions.

2.7. Flow Cytometric Analysis of Splenocyte

Splenocytes isolated from each Wistar rat were treated with erythrocyte lysis buffer (Sigma, Burbank, CA, USA) for 3 min to remove erythrocytes. Cells were then incubated with 0.2 μg of FITC-anti-mouse CD4 (BioLegend, 201505, San Diego, CA, USA) and 0.2 μg of PE-anti-mouse CD8 (BioLegend, 200608) antibodies to a concentration of 1 × 10⁶ cells/100 μL. After a 1-h reaction at 4 °C, the cells were washed twice (350× g, 4 °C, 5 min) and fixed with 70% ethanol for 10 min. Cell suspension and washing were performed using a cell staining buffer (BD FACS Calibur, Temecula, CA, USA), and CD4+ and CD8+ lymphocytes were analyzed using a flow cytometer (BD FACS Calibur, Temecula, CA, USA) and expressed as percentages of total lymphocytes.

2.8. Splenocyte Viability Analysis

Splenocytes isolated from each Wistar rat were cultured at 1 × 10⁶ cells, 100 μL/well, in 5% CO₂ for 24 h at 37 °C. After incubation, 10 μL of WST-1 solution was added to the cell culture medium and incubated at 37 °C for 2 h. The absorbance was then measured using a multidetection reader (Infinite 200, TECAN Group Ltd., Männedorf, Switzerland). Lipopolysaccharide (LPS), a known immunostimulant, was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell proliferation assay (%) = (absorbance of sample treated group/absorbance of control group) × 100

2.9. Natural Killer (NK) Cell Activity Assay

Splenocytes isolated from each Wistar rat were seeded in 96-well plates at a density of 4 × 10⁵ cells/well. AR42J cells were used as target cells, and the effector-to-target cell ratio was set to 20:1. The cells were then incubated at 37 °C in 5% CO₂ for 24 h. After incubation, 10 μL of lysis solution was added to each well and incubated for 5 min at room temperature. The cells were then centrifuged at 600× g for 5 min to precipitate the cells. The supernatant (10 μL) was transferred to a new 96-well plate, and 100 μL of the Lactate Dehydrogenase (LDH) reaction mixture was added to each well. The plate was gently mixed and allowed to react in the dark for 30 min at room temperature. The absorbance was measured using a plate reader. LDH was measured using an EZ-LDH kit (DOGEN, DG-LDH1000, Seoul, Republic of Korea), and the formazan formed by the oxidation of NAD in the reaction solution was measured at 450 nm (reference wavelength 600 nm).

2.10. Analysis of Cytokine Production in Splenocyte

Splenocytes isolated from each Wistar rat were seeded in 48-well plates at a density of 4 × 10⁶ cells/well. LPS at a final concentration of 10 μg/mL was added to each well and incubated at 37 °C in 5% CO₂ for 24 h. After incubation, supernatants were harvested and analyzed for IL-2, IL-6, TNF-alpha, and IFN-gamma using ELISA kits (Mybiosource, San Diego, CA, USA) according to the manufacturer’s instructions.

2.11. Western Blot Analysis

Spleen tissues were homogenized in ice-cold RIPA buffer containing protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Samples were centrifuged at 4 °C and 15,000 rpm for 10 min. The total protein concentration was calculated using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Total protein (10–30 µg per lane) was separated by 8% or 10% SDS-poly-acrylamide gels and transferred to PVDF paper (Whatman, GE Healthcare, Freiburg, Germany). The membranes were blocked with 5% skim milk and then incubated with the indicated primary antibody overnight at 4 °C. The blots were incubated with the antibodies against Erk (Extracellular signal-regulated kinase), P38, JNK (c-Jun N-terminal kinase), NFkB (Nuclear factor kappa-light-chain-enhancer of activated B cells), and GABDH, which were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The blot was quantified by enhanced chemiluminescence detection (Bio-Rad, Hercules, CA, USA) with LAS 500 mini (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). GABDH is an internal control that ensures the loading is equal [33].

2.12. Statistical Analysis

All experimental results were calculated as the mean ± standard deviation (mean ± SD). Statistical analysis for statistical significance between groups was performed by one-way analysis of variance (ANOVA) followed by Duncan’s post hoc comparison and was determined to be significant when p < 0.05 (SPSS V12., SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Organ Weights

The spleen and thymus were significantly reduced in weight by immunosuppression with Cy. However, contrary to expectations, we found no significant changes in the weight of the spleen and thymus compared to the pitch control in the experimental group administered Cy with low, moderate, and high concentrations of erythematous fermented extract, requiring further study (Figure 1).

3.2. Complete Blood Cell (CBC) Count Analysis

The experiment was performed by collecting blood from the abdominal veins of Wistar rats. White blood cell counts slightly increased in the low- and medium-concentration red beet fermented extract groups compared to those in the negative control group. Granulocyte counts showed a small increase in the low, medium, and high red beet fermented extract concentration groups. The increase in lymphocyte count was low only in the medium-concentration group of red beet fermented extract, while monocyte count did not increase compared to that in the control group (Table 1).

The expression levels of immune-related cytokines (IL-2, IL-6, TNF-α, and IFN-γ) in the serum were measured. For IL-2, TNF-α, and IFN-γ, the expression levels of immune-related cytokines (IL-2, TNF-α, and IFN-γ) were lower than those in the negative control group due to immunosuppression by Cy. The expression levels of immune-related cytokines (IL-2, TNF-α, and IFN-γ) were higher than those in the negative control group in all red beet fermented extract treatments. (Figure 2A,C,D) In IL-6, immunosuppression was not achieved by Cy and the efficacy of red beet fermented extract could not be confirmed. (Figure 2B) As a result, red beet fermented extract significantly upregulated the expression of immune-related cytokines.

3.3. Nitric Oxide (NO) Production

As a result of Cy administration, there was some immunosuppressive effect due to a slight decrease in the NO concentration; however, there was no strong immunosuppressive effect. In addition, no significant change in NO concentration was observed in the experimental group administered low-, medium-, and high-concentration red beet fermented extract compared to the negative control group, so further research is needed (Figure 3).

3.4. Flow Cytometry Analysis of Splenocyte

We detected T lymphocyte subpopulations CD4+ and CD8+ in splenocytes by flow cytometry (Figure 4A). Analysis of helper T cells (a type of leukocyte) in splenocytes showed that the expression of CD4+ was significantly suppressed in the negative control group compared to that in the normal group by the administration of Cy. The expression of CD4+ was significantly improved in the red beet fermented extract low- and intermediate-concentration groups compared to that in the negative control group (Figure 4B). Analysis of CD8+ cytotoxic T cells (a type of lymphocyte) in splenocytes revealed no immunosuppressive effect in the Cy group, which was contrary to expectations and warrants further investigation (Figure 4C).

3.5. Splenocyte Viability Assay

Splenocyte viability was reduced by Cy treatment, and no significant difference was observed after administration of red beet fermented extract. (Figure 5A) In response to LPS treatment, we observed a significant decrease in cell viability due to the occurrence of an inflammatory response and the promotion of cell death, and no significant change was observed with the administration of red beet fermented extract, requiring further study (Figure 5B).

3.6. Natural Killer (NK) Cell Activity Assay

To assess splenic NK cell activity, AR42J cells were used to evaluate cytotoxic activity. The negative control inhibited splenic NK cell activity. Red beet fermented extract treatment stimulated NK cell activity. NK cell activity increased in a dose-dependent manner from low to high concentrations of red beet fermented extract groups. Red beet fermented extract significantly enhanced NK cell activity overall (Figure 6).

3.7. Analyzing Cytokine Production in Splenocyte

We measured the expression of immune-related cytokines (IL-2, IL-6, TNF-α, and IFN-γ) in splenocytes. IL-2, TNF-α, and IFN-γ levels were lower than those in the negative controls. The expression levels of immune-related cytokines (IL-2, TNF-α, IFN-γ) were significantly increased compared to negative controls following treatment with red beet fermented extract, especially at low concentrations of red beet fermented extract. The results showed that we regulated the expression of immune-related cytokines in splenocytes immunosuppressed by Cy (Figure 7A,C,D). In IL-6, immunomodulation was not achieved by Cy, and the efficacy of red beet fermented extract was not confirmed (Figure 7B). This issue will be resolved in further studies.

3.8. Western Blot Analysis

Erk, P38, JNK, and NFkB proteins play crucial roles in cell signaling pathways and are deeply involved in the regulation of inflammatory responses. These proteins regulate inflammatory responses through the MAPK and NFkB pathways, modulating the expression of inflammatory cytokines and amplifying immune responses. Induction of Cy reduced the expression of Erk, P38, and NFkB proteins in the negative control group compared to that in the normal group. The expression levels of Erk, P38, and NFkB were significantly upregulated in a concentration-dependent manner by treatment with the red beet fermented extract. No significant changes were observed for JNK. Red beet fermented extract showed good improvement in the expression of inflammation-regulating proteins in the spleen immunosuppressed by Cy (Figure 8).

4. Discussion

Red beet is known to have immunostimulatory and anti-inflammatory effects, and these properties may be useful in immunosuppressive conditions [22,26]. In the present study, we evaluated the immune-enhancing effects of red beet fermented extract on Cy-induced immunosuppression in Wistar rats.

Cy is known to damage intracellular DNA, leading to cell death and a subsequent reduction in the number and function of immune cells. It is also recognized as a substance that suppresses immune responses during the treatment of autoimmune diseases and organ transplantation. This study highlights that the most beneficial effect of red beet fermented extract is its ability to mitigate the reduction in natural killer (NK) cell activity, which plays a crucial role in innate immune responses. Some results suggest that red beet fermented extract exhibits superior data values at low concentrations, indicating that it provides sufficient immune-enhancing effects, even at low doses.

Cy treatment decreased the expression of IL-2, TNF-α, and IFN-γ. The decreased expression of IL-2, TNF-α, and IFN-γ was increased by red beet fermented extract treatment, and the effect was greater at medium and high concentrations of red beet fermented extract. The findings on the proteins involved in the MAPK and NFkB signaling pathways suggest that red beet extract influences the signaling process, which is presumed to amplify immune responses through inflammatory cytokines.

In addition, NK cells, important cells of the innate immune system, are responsible for directly eliminating infected or tumor cells, and activated NK cells can express a number of secreted molecules that increase immune responses [34]. Cy treatment inhibited NK cell activity, but NK cell activity was significantly increased after treatment with red beet fermented extract, showing that red beet fermented extract can promote NK cell activity and improve immune function under immunosuppressive conditions. Red beet contains natural antioxidants, such as betaine and anthocyanins, which are presumed to enhance their efficacy through the fermentation process. These antioxidants are believed to neutralize free radicals, thereby mitigating cellular damage and contributing to the prevention of inflammation by supporting the production of anti-inflammatory cytokines. Through this study, we aimed to reaffirm the findings demonstrated in our research and the benefits of red beet suggested by previous studies. Based on these considerations, we emphasize the importance of utilizing easily accessible red beet and upgrading its immune-enhancing properties through a simple processing method.

Author Contributions

Conceptualization, H.-M.H., Y.-M.P., H.-Y.L., D.-Y.S., J.-G.K., J.-S.B., M.-J.K. and M.-S.K.; Methodology, H.-M.H., Y.-M.P., H.-Y.L., D.-Y.S. and J.-G.K.; Formal analysis, Y.-M.P., H.-Y.L. and J.-S.B.; Investigation, H.-M.H., D.-Y.S., J.-G.K., J.-S.B., D.-S.H. and H.-J.Y.; Resources, D.-S.H., H.-J.Y. and M.-J.K.; Data curation, M.-J.K. and M.-S.K.; Writing—original draft, J.-J.L. and I.-A.L.; Writing—review & editing, J.-J.L. and I.-A.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All animal experiments in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUV approval number: iv-RB-17-2304-10).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors.

Acknowledgments

This work was supported by the “Food Functionality Evaluation Program” under the Ministry of Agriculture, Food and Rural Affairs and the “The Research Program” of the Korea Food Research Institute under the Ministry of Science and ICT.

Conflicts of Interest

Author Hai Min Hwang, Young Mi Park, Hak Yong Lee, Dong Yeop Shin, Jae Gon Kim were employed by the company INVIVO Co., Ltd. Author Dae Sik Haam was employed by the company Baeryeo Innovation. The remaining authors declare that the re-search was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

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Figures and Table

Figure 1. Effect of red beet fermented extract on spleen and thymus weight and index in immunosuppressed Wistar rats (A) Spleen weight, (B) Spleen index, (C) Thymus weight, and (D) Thymus index values. All values are expressed as mean ± SD. Different letters in the same index indicate statistically significant differences at p [less than] 0.05.

Figure 2. Effect of red beet fermented extract on cytokine production in serum (A) IL-2, (B) IL-6, (C) TNF-α, and (D) IFN-γ levels in immunosuppressed Wistar rats. All values are expressed as mean ± SD. Different letters in the same indicator indicate statistically significant differences at p [less than] 0.05.

Figure 3. Effect of oral administration of red beet fermented extract on nitric oxide production in immunosuppressed Wistar rats. All values are expressed as mean ± SD. All values are expressed as mean ± SD. Different letters in the same index indicate statistically significant differences at p [less than] 0.05.

Figure 4. Effects of red beet fermented extract on flow cytometric analysis of splenocytes from immunosuppressed Wistar rats. (A) Representative flow cytometry analysis. Percentage of (B) CD4+ and (C) CD8+ T cell subsets. All values are expressed as mean ± SD. Different letters in the same index indicate statistically significant differences at p [less than] 0.05.

Figure 5. Effect of red beet fermented extract on splenocyte viability in immunosuppressed Wistar rats (A) Cy-induced values, (B) Cy and LPS-induced values. All values are expressed as mean ± SD. Different letters in the same index indicate statistically significant differences at p [less than] 0.05.

Figure 6. Effect of red beet fermented extract on the activity of splenic NK cells in immunosuppressed Wistar rats. All values are expressed as mean ± SD. Different letters for the same indicator indicate statistically significant differences at p [less than] 0.05.

Figure 7. Effect of red beet fermented extract on cytokine production in splenocytes of immunosuppressed Wistar rats (A) IL-2, (B) IL-6, (C) TNF-α, and (D) IFN-γ. All values are expressed as mean ± SD. Different letters in the same indicator indicate statistically significant differences at p [less than] 0.05.

Figure 8. Effects of red beet fermented extract on protein expression of Erk, P38, JNK, and NFkB in the spleen of immunosuppressed Wistar rats. (A) Protein expression analysis of Erk, P38, JNK, and NFkB using western blot; (B) Erk, (C) P38, (D) JNK, and (E) NFkb. All values are expressed as mean ± SD. Different letters in the same indicator indicate statistically significant differences at p [less than] 0.05.

Table 1

Hematologic analysis values of Wistar rats treated orally with red beet fermented extract for 4 weeks are presented as mean ± standard deviation (SD). Blood analysis results of normal, negative control, and low, medium, high, and positive concentration groups of red beet fermented extract are shown. WBC (white blood cells), GRA (granulocytes), LYM (lymphocytes), MID (monocytes). All values are expressed as mean ± SD. Different letters in the same index indicate statistically significant differences at p < 0.05.

Group	×103 Cell/μL
	WBC	GRA	LYM	MID
Normal	6.20 ± 0.27 c	1.19 ± 0.05 c	4.87 ± 0.23 c	0.13 ± 0.01 b
Negative Control	2.06 ± 0.16 a	0.68 ± 0.04 a	1.23 ± 0.12 a	0.07 ± 0.00 a
Red Beet Fermented Extraction-Low	2.21 ± 0.17 a	0.82 ± 0.06 ab	1.25 ± 0.11 a	0.07 ± 0.01 a
Red Beet Fermented Extraction-Medium	2.44 ± 0.10 ab	0.88 ± 0.07 b	1.41 ± 0.10 ab	0.07 ± 0.01 a
Red Beet Fermented Extraction-High	2.06.0.19 a	0.80 ± 0.05 ab	1.10 ± 0.16 a	0.06 ± 0.00 a
Positive Control	2.80 ± 0.14 b	0.78 ± 0.05 ab	2.82 ± 0.18 b	0.07 ± 0.00 a

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