Anti-Inflammatory Effects of Goat Whey Protein in Concanavalin-A Induced Hepatitis

Abstract

Background/Objectives: Immune-mediated hepatitis, including autoimmune hepatitis, remains a formidable clinical challenge characterized by the rapid destruction of the liver parenchyma. While whey proteins are well-regarded for their anti-inflammatory properties, goat whey possesses a distinct bioactive profile, offering superior digestibility and reduced allergenicity compared to their bovine counterparts. This study investigated the hepatoprotective potential and underlying immunological mechanisms of lyophilized goat whey (LGW) in a Concanavalin A (ConA)-induced model of acute hepatitis. Methods: BALB/c and C57BL/6 mice were administered LGW orally (1 g/kg/day) for five consecutive days prior to a ConA challenge. Liver injury was quantified via serum transaminase levels and histopathological evaluation. The cytokine profiles and the phenotype of liver mononuclear cells (MNCs) were analyzed using ELISA and flow cytometry, respectively. Results: LGW pretreatment significantly attenuated ConA-induced hepatitis in both mouse strains, markedly reducing serum transaminase levels and preserving hepatic architecture. Mechanistically, LGW triggered a fundamental shift in the hepatic immune microenvironment by suppressing the pro-inflammatory Th1/Th17 axis (evidenced by decreased IFN-γ and IL-17) while concurrently upregulating the anti-inflammatory cytokine IL-10. Furthermore, LGW induced a tolerogenic phenotype in hepatic dendritic cells (CD11c⁺CD206⁺), which directly correlated with a significant expansion of regulatory T cells (Tregs). This strain-independent protection suggests that LGW modulates fundamental, early-stage immune signaling pathways within the liver. Conclusions: Our findings demonstrate that LGW exerts potent hepatoprotection by effectively reprogramming the hepatic immune microenvironment toward a tolerogenic state. These results position LGW as a promising, safe, and effective functional food candidate for the prevention and adjunct management of immune-mediated inflammatory liver diseases.

1. Introduction

Liver diseases characterized by immune-mediated inflammation, most notably autoimmune hepatitis (AIH), represent a formidable global health burden. Due to their intricate pathogenesis and the relentless progression toward cirrhosis and hepatic failure, these conditions necessitate constant investigation into more effective management strategies [1]. To simulate the acute phase of human AIH, the Concanavalin A (ConA)-induced hepatitis model has become the established gold standard. This model triggers a rapid, T-cell-dependent inflammatory cascade, marked by a massive surge of pro-inflammatory cytokines—specifically TNF-α, IL-17, and IFN-γ—which ultimately precipitates extensive hepatocyte necrosis [1,2,3].

Current clinical management of AIH primarily relies on corticosteroids and azathioprine. In cases of drug intolerance or refractory disease, second-line agents like mycophenolate mofetil, cyclosporine, or infliximab are employed [4]. However, these pharmacological interventions often represent a double-edged sword; their efficacy is highly variable among individuals, and long-term administration is frequently hampered by severe systemic adverse effects. Consequently, there is a growing clinical demand for personalized treatment approaches and the exploration of safer, alternative therapeutic agents that could augment or even substitute traditional regimens [4].

In recent years, the paradigm of experimental immunonutrition has gained significant momentum, exploring functional foods and dairy-derived bio-actives as prophylactic adjuncts to conventional therapy. Among these, whey proteins have emerged as a focal point of interest due to their multifaceted biological activities. Previous research has established that whey supplementation can suppress the expression of pro-inflammatory mRNA (TNF-α, IL-1β) and attenuate liver damage across diverse experimental models, including lipopolysaccharide-induced injury, hepatic steatosis, and chronic ethanol-induced damage [5,6,7,8,9].

While the majority of studies have historically focused on bovine whey, scientific interest has recently pivoted toward the unique immunomodulatory potential of goat milk derivatives [10,11,12]. Lyophilized goat whey (LGW) possesses a distinct biochemical signature that differentiates it from its bovine counterpart. It is notably enriched with bioactive oligosaccharides that closely mimic the complexity of human milk, providing superior prebiotic and anti-infective properties [13,14]. Furthermore, LGW contains a high concentration of medium-chain triglycerides (MCTs) and specific bioactive proteins, such as lactoferrin and α-lactalbumin, which exist in structural conformations that offer enhanced digestibility but also potent signaling cues for the innate immune system [15,16,17].

Despite these promising attributes, the specific capacity of LGW to modulate hepatic immune cell populations and prevent immune-mediated injury remains largely unexplored. Dendritic cells (DCs) serve as the primary gatekeepers of hepatic immunity, and their ability to undergo a phenotypic shift toward a tolerogenic state is a critical determinant in maintaining immune homeostasis and preventing catastrophic tissue destruction [18].

Consequently, the present study was designed to investigate the hepatoprotective and immunomodulatory effects of LGW within the ConA-induced hepatitis framework. Our primary objective was to determine whether oral pretreatment with LGW could effectively “prime” the hepatic microenvironment by inducing a regulatory phenotype in dendritic cells and promoting the expansion of anti-inflammatory T cells. By bridging the gap between nutritional science and advanced hepatic immunology, this study aims to position LGW as a viable functional food candidate for the prevention of immune-mediated inflammatory liver diseases.

2. Materials and Methods

2.1. Experimental Animals

Male BALB/c and C57BL/6 mice, 8 to 10 weeks old and weighing 20 to 22 g, were used in the experiments. The mice were bred and maintained at the Animal Facility of the Faculty of Medical Sciences, University of Kragujevac (founder breeding pairs originally purchased from the Military Medical Academy, Belgrade, Serbia). They were housed in standard polycarbonate cages (up to 5 animals per cage) under controlled conditions (12 h light/dark cycle, temperature 22 ± 2 °C, humidity 50 ± 10%) with ad libitum access to food and water. Mice were transferred from the breeding colony to the experimental animal housing unit, where they underwent a 7-day acclimation period under identical environmental conditions prior to the initiation of the study. Upon transfer to the experimental animal facility, mice were housed individually in standard polycarbonate cages to ensure precise monitoring and controlled experimental conditions. No attrition or removal of animals occurred during the study; all animals that entered the experiment were included in the final analysis. All experimental procedures were approved by the Ethics Committee of the Faculty of Medical Sciences, University of Kragujevac (protocol number 01-8686/1) and were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

The LGW was provided by ‘Koza Nostra’ (Gunjaci, Serbia). This specific product is characterized by a high concentration of bioactive oligosaccharides, lactoferrin, and branched-chain amino acids (BCAAs). The standardized lyophilization process preserves the integrity of protein fractions and maintains a specific lipid profile dominated by medium-chain triglycerides along with highly bioavailable minerals (calcium, magnesium, phosphorus, potassium, zinc) and B-complex vitamins.

The total number of mice was 136: 24 BALB/c mice and 112 C57Bl/6 mice. Mice were randomly assigned to experimental groups, using a simple randomization method. A total of 24 BALB/c mice (three groups with 8 mice per group) were assigned to the following groups: 1. Untreated mice; 2. ConA mice: mice received a single intravenous injection of ConA (12.5 mg/kg in 200 µL saline) via the tail vein; and 3. LGW + ConA mice: mice were pretreated with oral LGW (1 g/kg/day dissolved in 100 µL distilled water) via oral gavage for five days prior to the ConA challenge. C57BL/6 mice were also randomly assigned to experimental groups using a simple randomization method. A total of 112 C57BL/6 mice were used in the study. The animals were assigned to the following groups: (1) Untreated control (n = 20): 8 mice were used for histology, 6 for ELISA, and 6 for flow cytometry; (2) LGW group (n = 20): mice received LGW (1g/kg/day dissolved in 100 µL distilled water via oral gavage for five consecutive days); 8 mice were used for histology, 6 for ELISA, and 6 for flow cytometry; (3) ConA group (n = 36): mice received a single intravenous injection of ConA (12.5 mg/kg in 200 µL saline) via the tail vein, for biochemical and histological studies; 24 mice were used (n = 8 per each of the three time points, for histology, 8 h and 24 h time points were analyzed). An additional 12 mice were used for ELISA (n = 6), and flow cytometry (n = 6); (4) LGW + ConA group (n = 36): mice were pretreated with oral LGW for five days prior to the ConA challenge. For biochemical and histological studies, 24 mice were used (n = 8 per each of the three time points), while 12 mice were used for ELISA (n = 6), and flow cytometry (n = 6).

The sample size (n = 6 and n = 8 per group) was chosen based on our previous experience with the ConA-induced hepatitis model to ensure sufficient statistical power. To ensure unbiased results, the investigators were blinded during the histological scoring and biochemical analyses.

At the designated time points, mice were euthanized, and blood was collected for serum transaminase (AST, ALT) detection. Liver tissue was harvested for histological examination and the quantification of cytokines at both the protein and mRNA levels. Additionally, liver mononuclear cells (MNCs) were isolated for comprehensive flow cytometric analysis. For assessing the effects of LGW on the functional states of liver immune cells, we used two groups of C57BL/6 mice: untreated and LGW-treated. These mice were sacrificed 24 h after the last LGW dose, which was the time when animals in previous experiments received a ConA injection.

2.2. Biochemical Analysis of Serum Transaminases

Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 2, 8, and 24 h post-ConA injection. Analysis was performed via spectrophotometry using an automated biochemistry analyzer (Olympus AU 400; Olympus Diagnostica GMBH, Hamburg, Germany).

2.3. Cytokine Quantification

Liver tissue samples (200 mg) were homogenized in 1 mL of phosphate-buffered saline (PBS) and centrifuged at 14,000× g for 10 min at 4 °C. The resulting supernatants were harvested for cytokine quantification. Concentrations of TNF-α, IFN-γ, IL-10, and IL-17 were determined using mouse DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

2.4. Histological Evaluation

Liver samples, harvested 8 and 24 h after ConA administration, were fixed, paraffin-embedded, cut on a microtome and stained with hematoxylin and eosin (H&E). H&E-stained liver sections were examined under light microscopy (BX51; Olympus (Tokyo, Japan) and Leica DM2500 (Wetzlar, Germany)) equipped with a Leica Flexacam i5 digital camera. Histopathological scoring was performed blindly by two independent examiners based on the following criteria: 0, no inflammation; 1, inflammation without necrosis; 2, mild focal necrosis (scattered necrotic hepatocytes); 3, severe focal necrosis (groups of dead hepatocytes); and 4, diffuse or bridging necrosis (15, 16).

2.5. Isolation of Hepatic Mononuclear Cells and Flow Cytometry

Liver-infiltrating MNCs were isolated as previously described [19]. Mononuclear cells isolated from the liver were resuspended in FACS buffer (PBS with 5 mM EDTA and 0.2% BSA) and incubated with fluorochrome-conjugated antibodies against mouse CD3, CD4, CD8, CD69, CD25, FoxP3, CD86, CD206, and CD11c. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate, ionomycin and Golgi Stop (BD Biosciences, San Jose, CA, USA). Following stimulation, cells were fixed, permeabilized, and stained with anti-mouse IFN-γ, IL-17 and IL-10 antibodies. Data acquisition was performed on a FACSCalibur Flow Cytometer (BD Biosciences), and results were analyzed using FlowJo software V9 (Tree Star, Phoenix, AZ, USA).

2.6. Statistical Analysis

Data are expressed as mean ± standard deviation (SD). Normality was assessed using the Shapiro–Wilk test. Differences between groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons post hoc test, or the Kruskal–Wallis test where appropriate. For comparisons between two groups, the Student t-test was employed. Statistical significance was defined as p < 0.05. Exact p-values are reported in Section 3 where applicable. All analyses were conducted using SPSS 20.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Lyophilized Goat Whey Protein Attenuates Liver Damage Induced by ConA

To explore the hepatoprotective potential of LGW, we utilized a ConA-induced hepatitis model in BALB/c mice, which exhibit minimal variation in liver indices and serum markers of hepatic damage [20]. Our results demonstrate that oral administration of LGW alleviates immune-mediated hepatitis. Specifically, five days of LGW pretreatment prior to intravenous ConA challenge significantly attenuated liver damage (Figure 1). Histological analysis 24 h post-ConA application revealed an absence of necrotic fields in LGW-pretreated mice, with only sparse immune cell infiltration (Figure 1A). In contrast, the ConA-only group exhibited confluent areas of necrosis, significant mononuclear infiltration, and hemorrhages (Figure 1A). Biochemical analysis of serum liver enzymes confirmed these histopathological findings. Serum ALT levels were significantly lower in the LGW + ConA group compared to the ConA-only group 24 h after ConA injection (Figure 1B). Notably, there was no significant increase in ALT levels in LGW + ConA mice compared to untreated controls, indicating near-complete protection (Figure 1B).

LGW treatment exerted similar protective effects in C57BL/6 mice, a strain characterized by high sensitivity and stability in the ConA model, developing lesions that closely mimic human AIH. No signs of liver injury were observed following 5 days of LGW treatment alone (Figure 2A). Furthermore, LGW pretreatment significantly reduced liver tissue necrosis and inflammation, as reflected by lower histological scores at both 8 h and 24 h post-ConA injection compared to the ConA-only group (Figure 2B). Livers from LGW-pretreated mice showed only minimal necrotic foci and sporadic inflammatory cells, whereas ConA-injected mice displayed extensive necrosis and intralobular infiltration around the central veins (Figure 2A). Furthermore, serum ALT levels were significantly reduced in the LGW-pretreated group 8 h post-ConA application (Figure 2C).

3.2. LGW Modulates Cytokine Production in Liver Tissue

The attenuation of inflammatory liver damage in LGW-pretreated C57BL/6 mice correlated with a significant decrease in intrahepatic pro-inflammatory cytokine production. Concentrations of TNF-α, IFN-γ, and IL-17 in liver homogenates were significantly lower in LGW + ConA mice compared to those challenged with ConA alone (Figure 3). Conversely, the level of the anti-inflammatory cytokine, IL-10, was significantly higher in the LGW + ConA group (Figure 3). Interestingly, LGW administration alone significantly enhanced IL-10 levels compared to untreated controls, suggesting a basal immunomodulatory effect (Figure 3).

3.3. LGW Enhances Regulatory T Cells in ConA Hepatitis

Since inflammatory cytokines produced by recruited lymphocytes drive hepatocyte necrosis [1], we analyzed the phenotype of liver mononuclear cells in Th1-biased, C57BL/6 mice [19] using flow cytometry. LGW + ConA mice exhibited significantly higher percentages of IL-10-expressing CD4⁺ and CD8⁺ T cells (Figure 4A). In contrast, the ConA-only group showed significantly higher frequencies of IFN-γ-producing CD4⁺ cells (Figure 4B) and IL-17-expressing CD4⁺ and CD8⁺ cells (Figure 4C) 12 h after ConA injection.

Furthermore, the percentages of CD3⁺CD4⁺ T cells and activated CD3⁺CD4⁺CD69⁺ cells were significantly lower in the LGW + ConA group compared to the ConA-only group (Figure 5A). Conversely, the percentages of regulatory CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ cells were significantly higher in the LGW + ConA group (Figure 5B). No difference in the percentage of CD3⁺CD4⁺ T cells was detected between the LGW-only, LGW + ConA, and untreated mice, suggesting that LGW does not independently induce T cell influx into the liver. Additionally, IL-10 expression within the CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ populations was markedly higher in the LGW + ConA group compared to all other groups (Figure 5C).

3.4. LGW Treatment Induces a Regulatory Phenotype in Hepatic Dendritic Cells

As T cell differentiation depends on the phenotype of antigen-presenting cells, we examined the effect of LGW on hepatic DCs. While CD11c⁺ cell percentages remained unchanged across groups (Figure 6A), LGW + ConA mice displayed a significantly higher proportion of alternatively activated (CD206-expressing) CD11c⁺ cells (Figure 6B). Although CD86 expression (a marker of classical activation) was slightly elevated in the LGW + ConA group, the ratio of classically to alternatively activated DCs (DC1/DC2) was lower (Figure 6B). Furthermore, oral LGW induced a significant increase in regulatory, IL-10-expressing, CD11c⁺ cells (Figure 6C). These data suggest that oral LGW primes the liver by inducing a regulatory DC phenotype, which may shift T cell differentiation away from pro-inflammatory Th1/Th17 subsets, thereby preventing liver injury.

4. Discussion

In the present study, we investigated the hepatoprotective potential of LGW using a ConA-induced hepatitis model, which serves as a well-established representative of human immune-mediated liver injury [1]. Our results demonstrate that five-day oral administration of LGW significantly attenuates hepatic damage, as evidenced by the preservation of tissue architecture and a marked reduction in serum transaminase levels. These findings suggest that LGW does not merely act as a passive nutritional supplement but functions as a potent biological response modifier that reprograms the hepatic immune microenvironment toward a tolerogenic state.

A critical aspect of our experimental design was the selection of the LGW dose (1 g/kg/day). This dose was selected according to the observed effects of LGW in rodent models in studies with a similar design [10] and justified through preliminary pilot experiments, which indicated that while 0.25 g/kg provided partial protection, the 1 g/kg dose consistently achieved a significant reduction in transaminase levels without inducing any observable adverse effects. To ensure clinical relevance, we performed a dose translation using the body surface area normalization method [21,22]. The murine dose of 1 g/kg corresponds to a human equivalent dose (HED) of approximately 81.1 mg/kg, which translates to a daily intake of 5.7 g for a 70 kg adult. This is a physiologically achievable amount in human functional nutrition, comparable to standard supplemental protein intake. Furthermore, the absence of clinical toxicity, behavioral changes, or histopathological alterations in the LGW-only group reinforces its high safety profile as a functional food candidate.

While previous research has highlighted the ability of whey proteins to mitigate liver inflammation in models of hepatic steatosis [23] or CCl₄-induced injury, our work specifically addresses the complexities of acute immune-mediated hepatitis. The pathogenesis of ConA-induced injury is driven by the rapid activation of T lymphocytes and the massive release of pro-inflammatory cytokines, including IFN-γ, TNF-α, and IL-17, which orchestrate hepatocyte destruction [1]. Our data show that LGW treatment dramatically suppressed these pro-inflammatory pathways while concurrently upregulating the anti-inflammatory cytokine IL-10 within the liver tissue. IL-10 plays a pivotal role in limiting the collateral damage of the immune response and is essential for the induction of regulatory T cells [24].

The core mechanistic contribution of this study is the characterization of the hepatic DC-Treg axis. We observed that LGW significantly increased the frequency of tolerogenic CD11c⁺CD206⁺ DCs and IL-10-expressing DCs. In the unique immunological landscape of the liver, these regulatory DCs are crucial for maintaining homeostasis [25]. This shift toward a regulatory phenotype likely serves as the primary signal for the expansion of both CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ Tregs, effectively establishing immune tolerance and preventing parenchymal necrosis. Similar immunomodulatory effects on the Treg population have been observed with bovine whey in other inflammatory models [26], but our study is among the first to detail this mechanism in the context of acute goat-whey-mediated hepatoprotection.

The superior efficacy of LGW compared to other dairy sources may be attributed to its unique biochemical matrix. Goat milk is naturally enriched with bioactive oligosaccharides that closely resemble the profile of human milk, offering potent prebiotic and anti-inflammatory properties [27,28]. Additionally, the high concentration of MCTs and specific bioactive peptides in goat whey may provide enhanced signaling for metabolic and immunological recovery compared to bovine alternatives [14,29]. While we have demonstrated the overall protective effect of the LGW matrix, future studies focusing on isolated fractions are necessary to pinpoint the exact molecular triggers within the whey.

Study Limitations and Future Perspectives

Despite the robust protective effects observed, certain limitations must be acknowledged. First, while the ConA model effectively simulates the acute phase of immune-mediated injury, it does not fully reflect the chronic, relapsing nature of human autoimmune hepatitis. However, the ConA-induced hepatitis model remains one of the most relevant mouse models for human biology, as it specifically mimics T-cell-mediated liver injury, which is a hallmark of autoimmune hepatitis and several other human liver diseases such as viral hepatitis [1,25]. Its high reproducibility and the involvement of key cytokines, such as TNF-α and IFN-γ, make it an appropriate tool for investigating fundamental immunological pathways and for the preliminary screening of new drugs and therapeutic supplements, such as LGW, aimed at treating inflammatory liver diseases [30]. Second, this study focused on a prophylactic “immunonutrition” framework; therefore, a pharmacological positive control (e.g., corticosteroids) was not included, as our primary aim was to evaluate the functional food’s preventive potential. Future research should explore chronic inflammation models and transition into clinical trials to confirm whether these immunomodulatory shifts translate to therapeutic benefits for patients with inflammatory liver diseases.

5. Conclusions

In conclusion, our data suggest that LGW provides a multi-level defense against immune-mediated liver injury by modulating innate signaling through DCs and promoting adaptive tolerance via Tregs. This positions lyophilized goat whey as a promising, safe, and effective functional food for the prevention of immune-mediated hepatic conditions.