Traditional Korean Doenjang Restores Splenic and NK Cell Function in Cyclophosphamide-Induced Immunosuppressed Rats

Abstract

Fermented soybean-based foods contain diverse bioactive compounds with recognized health benefits. Among them, doenjang is widely consumed in East Asia and has been associated with protective effects against several disorders, including immunosuppression. This study evaluated the immunoenhancing effects of doenjang sourced from four regions of Korea in cyclophosphamide (CP)-induced immunosuppressed rats. Four-week doenjang administration restored spleen weight and improved hematological parameters, including white blood cell, lymphocyte, neutrophil, and monocyte counts. Additionally, doenjang intake enhanced immune function, as evidenced by increased splenic natural killer cell activity, increased splenocyte proliferation under lipopolysaccharide- and concanavalin A-stimulated conditions, and higher levels of interleukin (IL)-2, IL-12, interferon-γ, and immunoglobulin G. Furthermore, the suppressed phosphorylation of mitogen-activated protein kinases/nuclear factor kappa B signaling was recovered, accompanied by improved splenic structure. Collectively, our findings demonstrate that the regional varieties of doenjang effectively mitigate CP-induced immune dysfunction, indicating their potential as functional dietary interventions.

1. Introduction

Doenjang, a traditional fermented soybean paste, has been consumed in Korea for centuries as both a protein source and seasoning agent [1]. It is produced by fermenting soybean bricks, known as “Meju,” in brine, a process similar to those used to make Japanese “Miso” and Indonesian “Tempeh” [2,3,4]. During fermentation, soy proteins are hydrolyzed into various bioactive compounds, including organic acids, amino acids, minerals, and peptides [1,5]. This transformation is facilitated by beneficial microorganisms such as Aspergillus spp. and Bacillus subtilis, which enhance the bioavailability of essential nutrients such as amino acids, vitamins, and minerals [6,7]. Doenjang is a staple condiment in Korean cuisine, renowned for its rich umami flavor and versatility [8,9]. Recently, it has attracted increasing scientific attention owing to its potential health-promoting effects. Studies have shown that doenjang exhibits a broad range of bioactivities, including antioxidant, anti-obesity, and anti-cancer effects [9,10]. These benefits are largely attributed to the high levels of phenolic compounds and bioactive peptides produced during fermentation. Doenjang is increasingly recognized not only as a traditional food but also as a functional fermented product with promising applications in health and nutrition.

Fermented soybean products such as doenjang have been shown to possess higher levels of phenolic compounds and proteins, as well as strong antioxidant activity, than their non-fermented counterparts [11,12]. These enhanced nutritional and functional properties are key factors contributing to the positive health effects of fermented soybean foods [13]. In particular, several studies have reported that doenjang consumption may help reduce levels of low-density lipoprotein cholesterol (LDL-c), commonly referred to as “bad cholesterol,” thereby improving overall lipid profiles [14,15,16]. These findings support the potential role of doenjang as a functional food for promoting cardiovascular health and managing dyslipidemia. Beyond its role as a traditional seasoning, soybean paste is increasingly being recognized as a functional food because it produces various physiologically active compounds during fermentation that may contribute to health promotion in modern populations [10]. Previous studies have primarily focused on bioactive compounds (such as isoflavones, genistein, etc.) (Figure S1) or microorganisms isolated from doenjang, demonstrating their anti-inflammatory, antioxidant, and health-promoting properties [17,18,19,20]. However, the immunomodulatory effects of doenjang have not yet been elucidated. In this study, we aimed to evaluate whether doenjang intake modulates immune function under immunosuppressed conditions. Using a cyclophosphamide (CP)-induced immunosuppression model, we investigated the changes in key immune parameters following 4 weeks of doenjang supplementation to determine its potential to restore or enhance immune responses.

2. Results

2.1. High-Performance Liquid Chromatography (HPLC) Analysis of Doenjang

As shown in Table 1 and Figure 1, the isoflavone contents of doenjang S1–S4 were compared. The total isoflavone content was highest in S4 (Gosung-gun) at 72.07 ± 0.69 mg/100 g, which was markedly higher than that of S1–S3. S2 (Asan-si) showed a moderately higher total isoflavone content (44.67 ± 1.71 mg/100 g) compared with S1 and S3. Daidzein content was highest in S4, whereas daidzin was detected only in S1. Genistein was detected at low levels in S1 and S3 but was not detected in S2 or S4. In contrast, genistein was present in doenjang S1–S4, with higher levels observed in S2 and S4. Glycitein was also detected in doenjang S1–S4 and was markedly higher in S4. These results indicate that isoflavone composition and content vary substantially among doenjang, likely reflecting differences in fermentation characteristics.

Table 1.

HPLC analysis of doenjang.

	Doenjang	S1 Commercial	S2 Asan-si	S3 Jeju-si	S4 Gosung-gun
Ingredient
Total isoflavone (mg/100 g)		38.54 ± 0.63	44.67 ± 1.71	38.92 ± 1.32	72.07 ± 0.69
Daizein (mg/100 g)		14.03 ± 0.42	20.39 ± 0.36	14.09 ± 0.24	24.60 ± 0.56
Daidzin (mg/100 g)		3.08 ± 0.24	ND	ND	ND
Genistein (mg/100 g)		1.05 ± 0.09	ND	1.05 ± 0.09	ND
Genistin (mg/100 g)		2.26 ± 0.25	4.74 ± 0.62	2.30 ± 0.39	5.25 ± 0.20
Glycitein (mg/100g)		18.12 ± 0.12	19.54 ± 0.79	18.32 ± 0.43	42.22 ± 0.38

ND: not detect.

Figure 1.

HPLC analysis of doenjang (S1-S4). (A) S1, a commercial brand (made in Korea); (B) S2, Asan-si (Chungcheongnam-do Province, Korea); (C) S3, Jeju-si (Jeju Special Self-Governing Province, Korea); and (D) S4, Gosung-gun (Gyeongsangnam-do Province, Korea).

2.2. Body Weight and Immune-Related Tissue Weights

CP, an alkylating agent belonging to the nitrogen mustard class, is known for its immunosuppressive effects and is widely used in the treatment of various cancers [21]. Because CP suppresses not only tumor cells but also immune cells, it negatively affects the immune system and often induces body weight (BW) loss [22,23]. In this study, we monitored changes in BW as an indicator of overall health status. CP, doenjang, and HemoHIM were administered orally, and BW was measured once weekly throughout the experimental period (Figure 2). The results showed that the CP-treated groups (vehicle and CP with doenjang) exhibited a significant decrease in BW compared with the normal group, indicating its immunosuppressive effects. However, administration of either doenjang or HemoHIM did not result in a significant increase in BW (Figure 2A). These interventions did not appear to mitigate CP-induced BW reduction. After autopsy, thymus and spleen weights were measured. Thymus weight, which was reduced by treatment of CP, was significantly increased in the S3 group compared with the CP-treated group (vehicle), while the S4 group also exhibited an increasing trend (Figure 2B). Similarly, the spleen weight tended to increase in all treated groups; however, consistent with the thymus results, only the S3 group exhibited a significant increase (Figure 2C). These results suggest that doenjang intake does not affect BW but increases spleen weight, thereby influencing spleen-associated immunity.

Figure 2.

Effects of doenjang on body weight (BW) and immune-related tissues. (A) BW (black line, normal group; red line, vehicle group (only CP); gray line, S1 group; orange line, S2 group; green line, S3 group, light green line, S4 group; and indigo, positive group); (B) Thymus weight (mg); and (C) Spleen weight (mg). Statistical differences among groups at the same time point were determined by one-way ANOVA followed by a post hoc multiple comparison test. Different letters (a–d) indicate statistically significant differences (p < 0.05).

2.3. Complete Blood Count (CBC) in Whole Blood

In an immunosuppressed conditions, alterations in WBC composition serve as important indicators of immune status [24]. A reduction in total WBC count generally reflects suppressed hematopoietic activity and an overall decline in immune competence [25]. Accordingly, CBC analysis was performed immediately after the autopsy (Figure 3). Total WBC counts showed a non-significant increase in the S2 group, whereas all other doenjang supplementation groups exhibited significant increases (Figure 3A). Lymphocyte counts were significantly elevated in the S3 group (Figure 3B). Neutrophil counts increased across all groups, with significant increases observed in the S1 and S4 groups (Figure 3C). No changes were observed in eosinophil counts (Figure 3D). Finally, monocyte levels in the S3 group were comparable to those in the CP-treated group (vehicle), whereas the S1 and S2 groups showed no significant increases (Figure 3E). Notably, a significant increase in monocyte count was observed in the S4 group. Collectively, these results indicate that doenjang intake enhanced immune cell populations, with significant increases in total WBCs, lymphocytes, neutrophils, and monocytes, depending on the group. Among the groups, S4 exhibited the most consistent immune-enhancing effects.

Figure 3.

Effects of doenjang on the whole-blood of CP-induced immunosuppressed rats. (A) White blood cells; (B) lymphocytes; (C) neutrophils; (D) eosinophils; and (E) monocytes. Statistical differences among groups at the same time point were determined by one-way ANOVA followed by a post hoc multiple comparison test. Different letters (a–e) indicate statistically significant differences (p < 0.05).

2.4. Splenocyte Proliferation and Splenic Natural Killer Cell Activity

Increases in splenocyte proliferation and natural killer (NK) cell activity indicate recovery of both adaptive and innate immunity, suggesting enhanced overall immune function under immunosuppressed conditions [26,27]. To evaluate the effects of traditional doenjang administration on splenocyte proliferation in a CP-induced immunosuppressed rats, humoral (LPS-induced) and cellular (ConA-induced) immune responses were assessed (Figure 4). The proliferation of LPS-treated splenocytes was significantly reduced by treatment of CP compared with the normal group, whereas all S1–S4 groups exhibited increased proliferation. Although the S2 and S3 groups did not show statistically significant differences, they showed increased proliferation relative to the CP-treated group (vehicle, Figure 4A). In the ConA-treated splenocytes, all groups showed a significant increase in proliferation relative to the CP-treated group (vehicle, Figure 4B). These results suggest that traditional doenjang administration can restore the splenocyte proliferative capacity in both humoral and cellular immune responses under CP-induced immunosuppression. Splenic NK cell activity was also increased by doenjang intake compared with that in the CP-treated group (vehicle, Figure 4C). These results indicate a potential immunomodulatory effect of doenjang, contributing to the recovery of overall immune function.

Figure 4.

Doenjang activates cell proliferation and NK cell activity in the spleen of CP-induced immunosuppressed rats. (A) Cell proliferation by LPS; (B) cell proliferation by ConA; and (C) NK cell activity. Statistical differences among groups at the same time point were determined by one-way ANOVA followed by a post hoc multiple comparison test. Different letters (a–d) indicate statistically significant differences (p < 0.05).

2.5. Serum Cytokine and Immunoglobulin G Levels

The immune status can be evaluated by assessing serum levels of cytokines (interleukin (IL)-2, IL-12, and interferon (IFN)-γ) and immunoglobulin G (IgG). Previous studies have reported that CP decreases the levels of these immune markers [22]. Therefore, we examined the effects of doenjang on the serum levels of IL-2, IL-12, IFN-γ, and IgG. IL-2, IL-12, and IFN-γ levels were markedly decreased in the CP-treated group relative to the normal group (Figure 5). The intake of doenjang (S1–S4) or HemoHim ameliorated these reductions, with the S2 and S4 groups exhibiting a tendency toward recovery, whereas the doenjang (S1 and S3)- and HemoHim-treated groups exhibited significant increases compared with the CP group (Figure 5A–C). The level of IgG was reduced in the CP-treated group (vehicle) compared with the normal group, confirming the immunosuppressive effect of CP. (Figure 5D) In contrast, doenjang (S1–S4)- and HemoHim-treated groups showed significantly restored serum IgG levels relative to the CP-treated group (vehicle), indicating a beneficial effect on humoral immunity (Figure 5D). Collectively, these results suggest that doenjang effectively alleviated CP-induced immunosuppression by restoring cytokine secretion and IgG production.

Figure 5.

Doenjang increases the level of cytokines in CP-induced immunosuppressed rats. Level of (A) IL-2, (B) IL-12, (C) IFN-γ, and (D) IgG in serum. Statistical differences among groups at the same time point were determined by one-way ANOVA followed by a post hoc multiple comparison test. Different letters (a–d) indicate statistically significant differences (p < 0.05).

2.6. MAPKs/NFkB Pathway in the Spleen

To determine whether doenjang intake modulates immune-related signaling pathways, we first examined the phosphorylation of MAPKs (including Erk, JNK, and p38) and the NF-κB pathway in the spleen (Figure 6). The reduced phosphorylation of these pathways is a well-established indicator of immunosuppression, particularly in CP-induced immunosuppressed Wistar rats [22,23]. Previous studies have shown that CP-induced immunosuppressed rats exhibit markedly reduced phosphorylation of MAPKs/NF-κB pathway, whereas supplementation with immunoenhancing agents (such as Cheonggukjang and HemoHim) restores these signaling activities to nearly normal levels [22]. As shown in Figure 6, we evaluated the effect of doenjang intake on CP-induced suppression of MAPKs and NF-κB pathway phosphorylation in the spleen. In the CP-treated group (vehicle), the phosphorylation levels of both MAPKs and NF-κB were markedly reduced compared with those in the untreated group (Figure 6A–E). Notably, 4 weeks of doenjang supplementation effectively restored the phosphorylation of these signaling pathways to levels comparable to those observed in the untreated group (Figure 6A–E). These findings suggest that 4 weeks of doenjang intake effectively restored phosphorylation levels comparable to those in the untreated group, thereby supporting the activation of immune-related signaling pathways.

Figure 6.

Doenjang increases immune-related signal pathways in the spleen of CP-induced immunosuppressed rats. Phosphorylation (A) of (B) Erk, (C) JNK, (D) p38, and (E) NFκB. Statistical differences among groups at the same time point were determined by one-way ANOVA followed by a post hoc multiple comparison test. Different letters (a–c) indicate statistically significant differences (p < 0.05).

2.7. Histology of Spleen Tissue

CP caused severe splenic damage [28]. This study investigated whether doenjang intake could improve histological changes in CP-induced splenic injury (Figure 7). Four weeks of CP treatment caused severe splenic damage including white pulp atrophy, marginal zone (MZ) collapse, and lymphoid depletion. In contrast, the doenjang-treated groups showed improved histological features compared with the CP-treated group (vehicle). S2 exhibited slight red pulp condensation but no white pulp atrophy, whereas S1, S3, and S4 exhibited reduced red pulp condensation, diminished white pulp atrophy, and clearer MZ structures. The positive group also showed a distinct separation of red and white pulps, with markedly decreased cellular condensation. Among the doenjang groups, S3 and S4 exhibited the most comparable improvements compared with the positive group. These findings indicate that doenjang may exert protective effects against CP-induced immunosuppression by improving splenic integrity.

Figure 7.

Doenjang improves the histological shape of the spleen in immunosuppressed rats. RP, red pulp; WP, white pulp; and MZ, marginal zone (arrow); H&E stain magnification, 10×; scale bar = 100 μm.

3. Discussion

Immunosuppression arises from diverse pathological and environmental factors, including malnutrition, aging, chronic disease, infection, and the administration of immunosuppressive agent such as CP [29,30,31]. Among these, CP-induced immunosuppression represents a clinically relevant condition characterized by profound impairment of both innate and adaptive immune compartments. Such disruption not only compromises leukocyte production and cytokine balance but also predisposes the host to opportunistic infections and delayed immune recovery [31,32]. To prevent immunosuppression and promote immune competence, an appropriate nutritional status should be maintained; in particular, diets rich in high-quality proteins, essential micronutrients, and antioxidants are crucial for supporting immune cell proliferation, redox homeostasis, and overall immunological integrity [33,34]. Given the increasing clinical need for supportive strategies that preserve immune competence during immunosuppressive therapy, identifying safe and nutritionally accessible immunomodulatory interventions remains an important research priority. The present study was designed to determine whether traditionally fermented doenjang could restore systemic immune function in a CP-induced immunosuppressed rat. Rather than merely improving isolated immune indices, our findings demonstrate a coordinated recovery of immune homeostasis, encompassing restoration of immune organ mass, normalization of circulating leukocyte subsets, recovery of Th1-associated cytokine production, enhancement of splenic NK cell activity, and reactivation of MAPKs/NF-κB signaling pathways. Collectively, these integrated responses suggest that fermented doenjang contributes to the re-establishment of functional immune competence under immunosuppression conditions.

Fermentation of soybeans generates diverse bioactive compounds, including isoflavone aglycones, peptides, and antioxidant metabolites, which may exert immunomodulatory effects. Isoflavones have been reported to regulate cytokine secretion, enhance NK cell activity, and modulate inflammatory signaling in various cellular systems [35,36,37]. However, direct in vivo evidence linking specific doenjang-derived components to systemic immune restoration remains limited. Although Cheonggukjang prepared from similar raw materials has shown immunological benefits [22], the present study extends these observations to regionally fermented doenjang preparations. Importantly, our data indicate that the intake of doenjang restored leukocyte populations and increased IL-2, IL-12, and IFN-γ levels, reflecting reactivation of the Th1-driven cellular immune axis, which is essential for cytotoxic and macrophage-mediated host defense. The concomitant increase in serum IgG further suggests recovery of humoral immunity, implying partial normalization of B cell function and adaptive immune responsiveness [38,39].

The spleen plays a central role in coordinating systemic immune responses, and CP-induced structural disruption of splenic structure is closely associated with impaired lymphocyte proliferation and diminished host defense [40]. In the present study, doenjang was associated with restoration of splenic morphology, enhanced splenocyte proliferative responses to LPS and ConA stimulation, and increased NK cell cytotoxic activity. These findings indicate functional recovery of both innate and adaptive immune compartments. Moreover, activation of MAPKs and NF-κB signaling pathways in splenic tissues suggests re-engagement of intracellular signaling networks critical for cytokine production, immune cell maturation, and coordinated immune activation. Importantly, this signaling recovery was observed in parallel with histological normalization, supporting the concept of structural–functional coupling in splenic immune restoration [22,23,28].

In this study, a critical consideration is whether such immune restoration could interfere with the chemotherapeutic efficacy of CP. CP exerts its primary antitumor effects through DNA alkylation and cytotoxicity in rapidly proliferating cells [21]. The current study was conducted in a non-tumor-bearing model and focused specifically on immune tissue recovery; therefore, no direct conclusions can be drawn regarding potential interactions with CP’s antineoplastic activity. However, the observed effects were confined to immune and immune-related tissues and reflected normalization rather than excessive immune activation. Thus, the findings suggest supportive immune recovery rather than broad cytoprotective effects against CP. Future studies employing tumor-bearing models will be required to determine whether dietary doenjang influences the therapeutic index of CP. From a translational perspective, doenjang differs fundamentally from standardized herbal formulations such as HemoHIM. Doenjang is a culturally embedded dietary component consumed frequently in Korean foods, making it a practical and accessible food-based immunomodulatory strategy rather than a pharmacological intervention [8,10]. The significance of this study therefore lies not only in demonstrating immune recovery comparable to that of established herbal products but also in providing scientific evidence supporting the immunological value of traditionally fermented foods as functional dietary resources. Several limitations should be acknowledged. First, the active components responsible for the observed immunological effects were not isolated or quantified. Second, the study utilized an acute CP-induced immunosuppression model without evaluation in chronic or tumor-bearing conditions. Third, pharmacodynamic interactions between doenjang and CP were not directly assessed. Addressing these limitations through component-specific analyses, mechanistic dissection, and translational studies will be essential to clarify the long-term immunomodulatory and clinical relevance of doenjang.

In conclusion, traditionally fermented doenjang significantly restored systemic immune competence in CP-induced immunosuppressed rats through coordinated recovery of immune cell populations, cytokine balance, splenic structure, and intracellular signaling pathways. These findings support the concept that food-based fermentation products may serve as adjunct nutritional strategies to mitigate immunosuppression and promote immune homeostasis. Further mechanistic and translational investigations are warranted to define the active constituents and evaluate their clinical applicability.

4. Materials and Methods

4.1. Preparation of Doenjang

The traditional Korean doenjang was provided by the Microbial Institute for the Fermentation Industry (MIFI, Sunchang, Republic of Korea). The four types of doenjang used were as follows: S1, a commercial brand (made in Korea); S2, Asan-si (Chungcheongnam-do Province, Republic of Korea); S3, Jeju-si (Jeju Special Self-Governing Province, Republic of Korea); and S4, Gosung-gun (Gyeongsangnam-do Province, Republic of Korea). Doenjang (S1–S4) was prepared according to the recipes for each region of Korea. They were finely ground in distilled water (DW; 150 and 350 g) using a blender for 60 s. After drying, the weight of the dried product was measured (S1:154.9 ± 5.0 mg/mL, S2:124.3 ± 0.8 mg/mL, S3:119.9 ± 1.3 mg/mL, and S4:128.0 ± 0.4 mg/mL); it was orally administered daily at 1000 mg/kg based on the dry weight.

4.2. HPLC Analysis of Doenjang

Standard solutions of daidzein, daidzin, genistein genistin, glycitein, and glycitin were prepared by dissolving 5 mg of each compound in methanol and diluting to 50 mL. Standards were prepared at concentrations of 0.1, 0.5, and 1 mg/kg via appropriate dilution of the standard solutions and were used to generate calibration curves for quantification. All samples are sonicated for 30 min and then filtered through a membrane filter of 0.45 μm. The HPLC analysis was performed using an Agilent 1260 series high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector (DAD). Chromatographic separation was achieved on a Capcellpak UG120 C18 column (250 mm × 4.6 mm, 5 μm particle size). The detection wavelength was set at 260 nm. The mobile phase consisted of solvent A (0.1% acetic acid in water) and solvent B (methanol containing 0.1% acetic acid), and separation was carried out using a gradient elution program as follows: 70% A and 20% B at the initial condition, changed to 60% A and 50% B at 20 min, followed by 30% A and 70% B at 35 min, and returned to the initial condition (70% A and 20% B) at 40 min. The flow rate was maintained at 0.8 mL/min, the injection volume was 5 μL, and the column oven temperature was set at 40 °C.

4.3. Animals and Oral Administration

All the experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals. This study was approved by the Institutional Animal Care and Use Committee of INVIVO Co., Ltd. (IV-RB-17-2502-07, 26 February 2025, Nonsan, Republic of Korea). The experimental groups were divided based on previous studies [22,23]. Male Wistar rats (5-week-old) were purchased from Orient BIO Co. (Gyeonggi-do, Republic of Korea) and divided into seven groups (normal, vehicle, S1, S2, S3, S4, and positive). The rats were orally administered CP (5 mg/kg) to induce immunosuppression [22,23]. Concurrently, the rats received either doenjang (1000 mg/kg) or the HemoHIM herbal preparation (1000 mg/kg, positive group; purchased from Kolmar BNH Co., Ltd., Sejong, Republic of Korea) via oral gavage for 4 weeks. The BW of the animals were monitored weekly during the experimental period. During autopsy, the weights of the thymus and spleen were immediately measured.

4.4. CBC Analysis

After induction of inhalation anesthesia, whole blood was collected from the vena cava and placed into tubes containing ethylenediaminetetraacetic acid (EDTA). Parameters were subsequently measured without delay using an automated hematology analyzer (BC-5000VET; Mindray, Bath, UK).

4.5. Levels of Cytokines and IgG in Serum

Serum separation was performed in accordance with previously reported methods [22]. Briefly, whole blood was allowed to stand at 25 °C for 30 min and were then centrifuged at 3000 rpm for 15 min at 4 °C. Serum concentrations of IL-2, IL-12, IFN-γ, and IgG were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits (IL-2: MBS269718, Mybiosource; IFN-γ: ab239425, Abcam; IgG: ab189578, Abcam, Cambridge, UK). The absorbance was measured using a Sunrise microplate reader (Tecan, Männedorf, Switzerland).

4.6. Primary Cell Culture and Proliferation of Splenocytes

Splenic tissues were mechanically dissociated and filtered through a 70 μm cell strainer (SPL Life Sciences, Gyeonggi-do, Republic of Korea) to obtain splenocytes. The cells were washed with RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), and red blood cells were removed using a lysis buffer (Sigma-Aldrich, Carlsbad, MO, USA). Splenocytes were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin in a 5% CO₂ incubator. After 24 h of stabilization (5 × 10⁵ cells/well), cell proliferation was assessed using a WST-1 assay kit (ITSBio, Seoul, Republic of Korea) and measured with an ELISA plate reader.

4.7. Splenic NK Cell Activity

AR42J cells (CRL-1492; American Type Culture Collection, Manassas, VA, USA) were used as target cells for the evaluation of splenic NK cell activity. Primary splenocytes were obtained from each experimental group (normal, vehicle, fore-type doenjang-treated and positive) and served as effector cells. Effector and target cells were co-incubated in 96-well plates at an effector-to-target ratio of 25:1 under standard culture conditions (37 °C, 5% CO₂) for 24 h. Following incubation, the viability of AR42J cells was determined using a WST-1 assay kit, and absorbance was measured with an ELISA plate reader. NK cell activity was expressed as the relative survival rate of AR42J cells compared with that of the normal group.

4.8. Tissue Lysis and Western Blot Analysis

Splenic tissues were homogenized in PRO-PREP Protein Extraction Solution (iNtRON, Cat No. 17081) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein (10 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with primary antibodies against phospho-Erk, Erk, phospho-p38, p38, phospho-JNK, JNK, phospho-NFκB, NF-κB, and β-actin (Cell Signaling Technology, Danvers, MA, USA). Protein bands were visualized using a Western blot imaging system (Azure Biosystems c300) and quantified with ImageJ 1.53k software (National Institutes of Health, Bethesda, MD, USA).

4.9. Histological Analysis of the Spleen

Briefly, the spleens were excised from Wistar rats, weighed, and immersed in 10% neutral-buffered formalin for fixation. After 48 h of fixation, the tissues were embedded in paraffin and sectioned into 4 μm slices using a microtome (Thermo Fisher Scientific, Waltham, MA, USA). Tissue sections were stained with hematoxylin and eosin (H&E) and imaged using a Motic EasyScan One digital slide scanner (Motic, Hong Kong).

4.10. Statistical Analysis

All data are expressed as the mean ± standard error of the mean, and differences between groups were analyzed using one-way analysis of variance (Duncan’s multiple-range test). All statistical analyses were performed using the SPSS software (version 23.0; IBM Corp., Armonk, NY, USA). Each value represents the mean of at least three independent experiments for each group. Statistical significance was set at p < 0.05.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052492/s1.

Author Contributions

Investigation, H.Y.L., Y.M.P., D.Y.S., H.M.H., S.H.C., S.J.L., and J.G.K.; Validation, Y.M.P.; Data curation, Y.M.P. and D.Y.S.; Formal analysis, G.S.H., H.-J.Y., M.S.R., J.W.S., D.-Y.J., and J.S.B.; Writing—draft, H.Y.L., J.S.B., and J.G.K.; Writing—review and editing, J.G.K. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All animal experiments were approved by the Institutional Animal Care and Use Committee of INVIVO Co., Ltd. (IV-RB-17-2502-07, 26 February 2025).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available upon request.

Conflicts of Interest

The authors declare no conflicts by INVIVO Co., Ltd. The authors declare that the research was conducted in the absence of commercial or financial relationships that could be construed as potential conflicts of interest.

Funding Statement

This work was supported by “Functional research of fermented soybean food (safety monitoring)” under the Ministry of Agriculture, Food, and Rural Affairs and partly by the Korea Agro-Fisheries and Food Trade Corporation in 2025.