Anti-inflammatory effects of taurocholic acid and tauroursodeoxycholic acid from rainbow trout spleen extract via NF-κB suppression

Abstract

Inflammation is an essential innate immune response, yet excessive or persistent inflammation contributes to chronic inflammatory diseases. Our previous studies demonstrated that rainbow trout spleen extracts possess potent anti-inflammatory activity, primarily concentrated in the butanol fraction (OSB), through suppression of NF-κB signaling and macrophage M1 polarization. However, the complex composition of OSB and the requirement for high effective concentrations limited its translational potential. In this study, we try to identify the key bioactive components responsible for OSB’s anti-inflammatory effects. The five major constituents enriched in OSB were further fractionated into two subfractions, among which B-TT, containing taurocholic acid (TCA) and tauroursodeoxycholic acid (TUDCA), exhibited the most pronounced anti-inflammatory activity. Mechanistically, B-TT significantly inhibited lipopolysaccharide (LPS) induced NF-κB activation in RAW264.7 macrophages by suppressing phosphorylation dependent IκBα degradation, thereby blocking nuclear translocation of the p65 subunit. This inhibition led to reduced iNOS expression, decreased pro-inflammatory cytokine transcription, suppression of M1 macrophage surface markers, and attenuation of intracellular reactive oxygen species (ROS) generation. Collectively, these findings demonstrate that B-TT effectively suppresses LPS-induced macrophage M1 polarization via inhibition of NF-κB signaling and indicate that TCA and TUDCA are major anti-inflammatory constituents present in rainbow trout spleen extracts, highlighting fish-derived visceral byproducts as sustainable bioresources for anti-inflammatory applications.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-56475-9.

Introduction

Inflammation is a fundamental innate immune response that protects the host against pathogens and tissue injury¹. However, dysregulated or persistent inflammation can progress to chronic inflammatory states, contributing to the pathogenesis of numerous diseases, including diabetes, cancer, and rheumatoid arthritis². At the molecular level, inflammatory responses are tightly regulated by intracellular signaling cascades, among which the nuclear factor kappa B (NF-κB) pathway plays a central role in controlling the transcription of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines³. Consequently, modulation of NF-κB signaling has emerged as a major therapeutic strategy for the treatment of inflammatory disorders⁴.

In our previous studies, we demonstrated that spleen crude extracts derived from rainbow trout (Oncorhynchus mykiss) exhibit potent anti-inflammatory activity in lipopolysaccharide (LPS) stimulated macrophages⁵. Through solvent fractionation, we further identified that the butanol fraction, designated OSB, was primarily responsible for this anti-inflammatory effect. OSB markedly suppressed LPS-induced activation of the NF-κB signaling pathway by inhibiting the degradation of IκBα, thereby preventing the nuclear translocation of the p65 subunit. This inhibition led to a significant downregulation of iNOS and COX-2 expression, as well as reduced production of pro-inflammatory cytokines. In addition, OSB attenuated intracellular reactive oxygen species (ROS) generation and significantly decreased the mRNA expression of M1 macrophage surface markers, including CD40 and CD86. Collectively, these findings indicated that OSB effectively suppresses M1 macrophage polarization and exerts broad anti-inflammatory effects.

Despite these promising results, several limitations remained unresolved. OSB is a complex mixture composed of multiple bioactive components, and the specific compounds responsible for its anti-inflammatory activity had not been identified. Moreover, relatively high concentrations (approximately 400 µg/mL) were required to achieve robust anti-inflammatory effects, raising concerns regarding translational applicability and therapeutic efficiency. These limitations underscored the necessity to isolate and characterize key active constituents capable of exerting potent anti-inflammatory effects at lower, more clinically relevant concentrations.

Currently, anti-inflammatory therapies used in clinical practice are broadly classified into steroidal anti-inflammatory drugs (SAIDs) and non-steroidal anti-inflammatory drugs (NSAIDs)^6,7. SAIDs exert strong anti-inflammatory effects by binding to intracellular glucocorticoid receptors and suppressing NF-κB dependent transcription of inflammatory genes⁸. However, long term steroid use is associated with severe adverse effects, including immunosuppression, receptor downregulation, reduced responsiveness, and drug resistance^9,10. NSAIDs, such as aspirin, alleviate inflammation primarily through inhibition of COX enzymes¹¹. Nevertheless, non-selective inhibition of COX-1 disrupts gastrointestinal mucosal protection and homeostasis, frequently resulting in gastrointestinal injury and ulceration¹². These drawbacks highlight the urgent need for safer and more effective anti-inflammatory agents derived from alternative sources.

In recent years, increasing attention has been directed toward natural products and endogenous metabolites with intrinsic immunomodulatory properties^13,14. Among these, bile acids and their derivatives have been reported to exert anti-inflammatory effects through modulation of NF-κB signaling, oxidative stress, and macrophage polarization¹⁵. Taurine conjugated bile acids, in particular, have been shown to suppress inflammatory signaling pathways and protect against tissue injury in various experimental models, suggesting their potential as novel anti-inflammatory agents^16–18.

In the present study, we aimed to identify the key bioactive constituents responsible for the anti-inflammatory effects of the OSB fraction derived from rainbow trout spleen. Through further fractionation and compositional analysis, we isolated two subfractions, B-THP and B-TT, and demonstrated that B-TT, enriched in taurocholic acid (TCA) and tauroursodeoxycholic acid (TUDCA), exhibited potent anti-inflammatory activity. This study not only elucidates the principal active components contributing to the anti-inflammatory effects of rainbow trout spleen extracts but also provides mechanistic insights into their mode of action. Importantly, our findings highlight the potential of fish-derived visceral byproducts, traditionally regarded as waste, as valuable bioresources for the development of sustainable and safe anti-inflammatory therapeutics.

Materials and methods

Preparation of crude extracts and fractionated samples

The experimental procedures used in this study were adopted from our previously published work⁵. Thirteen-month-old female rainbow trout were obtained from Woori Trout Farm (Chuncheon, Gangwon-do, Republic of Korea), a commercial fish farm operating under food production conditions. The rainbow trout used in this study were dissected on-site immediately after purchase. To minimize individual biological variability and ensure consistency across subsequent assays, a single large-scale preparation was performed by pooling the viscera from approximately 200 rainbow trout. From this bulk preparation, approximately 1 kg of raw spleen tissue was harvested and extracted with 70% ethanol at 60 °C for 16 h. The resulting crude extracts were lyophilized, yielding 25.0 g of total crude extract (2.5% w/w yield based on wet tissue weight), and subsequently reconstituted in 70% ethanol or diluted with culture medium for in vitro experiments. For solvent fractionation, the crude extract was suspended in 400 mL of distilled water and sequentially partitioned with n-hexane, ethyl acetate, and n-butanol to obtain solvent-soluble fractions. Briefly, 600 mL of n-hexane was added to the aqueous suspension to obtain the n-hexane-soluble fraction (OSH). The remaining aqueous phase was then extracted with 600 mL of ethyl acetate to yield the ethyl acetate-soluble fraction (OSE), followed by extraction with 600 mL of n-butanol to obtain the n-butanol-soluble fraction (OSB). The final residual aqueous phase was collected as the water-soluble fraction (OSW). Consequently, the crude extract was separated into four distinct solvent fractions. Through this sequential fractionation process, approximately 8.0 g of OSB was obtained, which corresponds to a yield of 32.0% (w/w) relative to the initial crude extract.

UHPLC-MS/MS and MPLC analysis

Ultra high performance liquid chromatography (UHPLC) analysis was performed using a Vanquish Flex-A10 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a UHPLC pump and detector. High resolution mass spectrometric analysis was conducted using an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was achieved using a Hypersil GOLD™ aQ UHPLC column (1.9 μm, 100 × 2.1 mm). For UHPLC analysis, water containing 0.1% formic acid was used as mobile phase A, and acetonitrile containing 0.1% formic acid was used as mobile phase B. The gradient elution program was set as follows: 0–0.5 min, 10% B (Curve 5); 0.5–15 min, 10–90% B (Curve 5); 15–17.5 min, 90% B (Curve 5); and 17.5–20 min, 90–10% B (Curve 1). The column temperature was maintained at 30 °C, the injection volume was 0.5 µL, and the flow rate was set to 0.3 mL/min. Medium pressure liquid chromatography (MPLC) was performed using a CombiFlash^® PF+ system (Teledyne Isco, Lincoln, NE, USA) equipped with a reversed phase silica gel column (RediSep^®, Teledyne Isco). The mobile phase consisted of methanol and water, applied with a stepwise gradient at ratios of 4:6, 6:4, 8:2, and 0:100 (v/v). Through this MPLC process, approximately 4.5 g of the B-THP sub-fraction and approximately 1.5 g of the bioactive B-TT sub-fraction were successfully obtained from the 8.0 g of loaded OSB, representing yields of 56.25% (w/w) and 18.75% (w/w) relative to the initial OSB fraction, respectively. Major components of the OSB fraction were tentatively identified by comparative analysis of UHPLC-MS/MS data using the Global Natural Products Social Molecular Networking (GNPS) web platform. Analytical standards for TCA (purity ≥ 98%, CAS No. 81-24-3) and TUDCA (purity ≥ 99.9%, CAS No. 14605-22-2) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

RAW264.7 murine macrophage cells (KCLB 40071) were obtained from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). Cells were gently detached using a cell scraper without trypsinization. Freeze dried extracts derived from rainbow trout spleens were dissolved in 70% ethanol and subsequently diluted with culture medium to the desired concentrations prior to treatment. Lipopolysaccharide (LPS, L2630, CAS No. 93572-42-0) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in triple-distilled water and diluted in DMEM to a final concentration of 1 µg/mL. OSB and its subfractions (B-THP and B-TT) were diluted in culture medium and applied at a final concentration of 100 µg/mL. Taurocholic acid sodium salt hydrate (TCA) and tauroursodeoxycholic acid sodium salt (TUDCA) (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in filtered, triple-distilled water to prepare stock solutions and further diluted in culture medium. TCA and TUDCA were used at 0–200 µM for MTT and NO assays, and at a final concentration of 50 µM for all other experiments.

Real time quantitative PCR (RT-qPCR)

RAW264.7 cells were seeded in culture dishes and incubated for 24 h, followed by treatment with fractions of the rainbow trout spleen crude extract in the presence of lipopolysaccharide (LPS; 1 µg/mL) for an additional 24 h. Total RNA was isolated using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using oligo(dT) primers, dNTPs, and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA). RT-qPCR was performed in a 20 µL reaction volume on a real-time PCR detection system using TOPreal™ qPCR 2X PreMIX (Enzynomics, Daejeon, Republic of Korea). The final concentration of each primer in the PCR reaction mixture was 50 nM. The thermal cycling conditions were as follows: an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing/extension at 60 °C for 1 min. Relative gene expression levels were calculated using the 2^−ΔΔCt method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The primer sequences and their respective accession numbers used for RT-qPCR are listed in Table 1.

Table 1.

RT-qPCR primer sequences.

Gene	The sequences of the primers	Accession numbers
iNOS	F-CCAAGCCCTCACCTACTTCC	NM_010927.4
	R-CTCTGAGGGCTGACACAAGG
IL-6	F-AGTCCTTCCTACCCCAATTTCC	NM_031168.2
	R-TAACGCACTAGGTTTGCCGA
TNF-α	F-ACCGTCAGCCGATTTGCTAT	NM_013693.3
	R-TTGGGCAGATTGACCTCAGC
IL-12p40	F-AGACCCTGCCCATTGAACTG	NM_001303244.1
	R-CAGGAGTCAGGGTACTCCCA
IL-23p19	F-CAGCAGCTCTCTCGGAATCT	NM_031252.2
	R-CAGACCTTGGCGGATCCTTT
CD40	F-GCTATGGGGCTGCTTGTTGA	NM_011611.3
	R-GGTGGCATTGGGTCTTCTCA
CD86	F-ATGGACCCCAGATGCACCA	NM_019388.3
	R-TGTGCCCAAATAGTGCTCGT
GAPDH	F-CTCATGACCACAGTCCATGC	NM_001289726.2
	R-CACATTGGGGGTAGGAACAC

Western blotting

RAW264.7 cells were seeded into 100 mm culture dishes and treated 24 h later according to the experimental conditions. Following treatment, cells were harvested using a cell scraper, and total protein was extracted using RIPA lysis buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 140 mM NaCl; 1% Triton X-100; 0.1% sodium deoxycholate; and 0.1% SDS) supplemented with a cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Protein concentrations were determined using the Bradford assay (Thermo Fisher Scientific, Waltham, MA, USA), and equal amounts of protein (20 µg per sample) were used for subsequent analyses. Equal amounts of protein were separated by SDS-PAGE and transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 30 min at room temperature and then incubated overnight at 4 °C with primary antibodies diluted in TBST containing 1% bovine serum albumin (BSA). The primary antibodies were diluted according to the manufacturer’s recommended concentrations. After washing, membranes were incubated for 2 h at room temperature with horseradish peroxidase (HRP) conjugated secondary antibodies diluted 1:10,000 in 5% skim milk. Depending on the host species of the primary antibody, either Goat anti-Rabbit IgG Poly-HRP Secondary Antibody (#32260) or Goat anti-Mouse IgG Poly-HRP Secondary Antibody (#32230) (Invitrogen, Carlsbad, CA, USA) was used. Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection kit (GE Healthcare, Chicago, IL, USA). Primary antibodies against iNOS (13120 S), IκBα (9242 S), phosphorylated p65 (p-p65; 3033 S) and CD86 (19589 S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against p65 (ab32536) and Lamin B1 (ab16048) were obtained from Abcam (Cambridge, UK). Primary antibodies against β-actin (sc-47778) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Immunofluorescence staining

RAW264.7 cells were seeded onto coverslips placed in 6-well plates and treated 24 h after seeding according to the experimental conditions. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 3 min, and blocked with 5% skim milk in PBS for 1 h at room temperature. Cells were then incubated for 2 h with primary antibodies against p65 (ab32536; Abcam, Cambridge, UK) or CD86 (19589 S; Cell Signaling Technology, Danvers, MA, USA) diluted 1:200 in blocking buffer, followed by incubation with Alexa Fluor™ 488-conjugated Goat anti-Rabbit IgG Cross-Adsorbed Secondary Antibody (1:200; A-11008, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA) diluted 1:2,000 in PBS, and fluorescence images were acquired using a confocal laser scanning microscope (Nikon, Tokyo, Japan).

Image-based quantitative analysis

For quantitative analysis of immunofluorescence images, fluorescence intensity was measured using ImageJ software (NIH, USA). Nuclear and cytoplasmic regions were defined based on DAPI staining, and the nuclear-to-cytoplasmic fluorescence ratio of p65 or mean fluorescence intensity (MFI) of CD86 was calculated. At least three independent experiments were analyzed, with a minimum of 30 cells quantified per condition. The quantified data were used for statistical analysis and graphical representation.

Reactive oxygen species (ROS) analysis

RAW264.7 cells were seeded into 6-well plates and allowed to stabilize for 24 h in a humidified incubator at 37 °C with 5% CO₂. Cells were then co-treated with OSB subfractions and lipopolysaccharide (LPS; 1 µg/mL) for 24 h according to the experimental conditions. Following treatment, cells were incubated with Hoechst 33,342 (1 µg/mL; Sigma-Aldrich, St. Louis, MO, USA) and carboxy-H₂DCFDA (10 µM) in phosphate-buffered saline (PBS) for 30 min at 37 °C in the dark. Cells were subsequently washed with PBS, and intracellular ROS levels were observed using a fluorescence microscope.

MTT assay

Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RAW264.7 cells were seeded into 96-well plates at a density of 6 × 10³ cells per well and allowed to stabilize for 24 h. Cells were then treated with TCA or TUDCA at concentrations ranging from 0 to 200 µM for an additional 24 h. After treatment, the culture medium was removed and replaced with fresh medium containing MTT solution, followed by incubation for 3 h at 37 °C. The medium was subsequently removed, and the resulting formazan crystals were dissolved in dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm with a reference wavelength of 690 nm using a microplate reader (Allsheng, Hangzhou, China). Cell viability was expressed as a percentage relative to the untreated control.

Nitric oxide assay

RAW264.7 cells were seeded into 96-well plates and incubated for 24 h. Cells were then co-treated with lipopolysaccharide (LPS; 1 µg/mL) and TCA or TUDCA at concentrations ranging from 0 to 200 µM for an additional 24 h. Following treatment, culture supernatants were collected and transferred to a new 96-well plate for nitric oxide (NO) measurement using the NO Plus Detection Kit (iNtRON Biotechnology, Seongnam, Republic of Korea), according to the manufacturer’s instructions.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was determined using Student’s t-test, and differences were considered statistically significant at p < 0.05. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01.

Results

Differential effects of OSB sub-fractions on LPS-induced iNOS expression

Our previous results indicated that the anti-inflammatory activity of the spleen crude extract is predominantly enriched in the OSB fraction, which has been previously shown to exhibit no significant cytotoxicity at the concentrations used⁵. Based on this observation, we investigate to identify the specific bioactive components within OSB responsible for this effect. The chemical composition of OSB was analyzed using UHPLC-MS/MS, followed by molecular networking and spectral matching using the GNPS web platform. UHPLC-MS/MS profiling revealed that the chemical constituents of OSB segregated into two distinct clusters according to polarity (Fig. 1B). One cluster consisted primarily of highly polar metabolites that eluted earlier, whereas the second cluster comprised less polar bile acid derivatives that eluted later. Through comparison with the standard markers, five major compounds present at high abundance were unequivocally identified as taurine, hypoxanthine, phenylalanine, taurocholic acid (TCA), and tauroursodeoxycholic acid (TUDCA), based on matched retention times and MS/MS fragmentation patterns. To functionally evaluate these clusters, OSB was further fractionated by MPLC, yielding two sub-fractions: B-THP, a relatively high-polarity fraction enriched in taurine, hypoxanthine, and phenylalanine, and B-TT, a low-polarity fraction predominantly composed of bile acid derivatives TCA and TUDCA (Fig. 1B). Semi-quantitative UHPLC-MS/MS peak area analysis further indicated that TCA and TUDCA were present in comparable proportions within the B-TT sub-fraction, with TCA representing a slightly higher relative abundance than TUDCA. To compare the anti-inflammatory efficacy of these sub-fractions, RAW264.7 macrophages were stimulated with LPS (1 µg/mL) in the presence of OSB, B-THP, or B-TT (100 µg/mL). The anti-inflammatory response was assessed by monitoring the expression of inducible nitric oxide synthase (iNOS) at both the mRNA and protein levels. RT-qPCR and Western blot analyses demonstrated that B-THP treatment did not suppress LPS-induced iNOS expression; rather, iNOS mRNA and protein levels were increased relative to the LPS only control. In contrast, B-TT treatment markedly attenuated LPS-induced iNOS expression and exhibited a stronger inhibitory effect than the unfractionated OSB (Fig. 1C, D). Therefore, these results demonstrate that the anti-inflammatory activity of OSB is primarily attributable to the B-TT sub-fraction. Given that B-TT is enriched in TCA and TUDCA, our findings suggest that bile acid derivatives are the principal components to the OSB mediated suppression of inflammatory signaling in macrophages.

Fig. 1.

Regulatory effects of OSB subfractions on iNOS expression. (A) Schematic illustration of the fractionation procedure of crude extracts from rainbow trout spleen. The anti-inflammatory activities of crude extracts derived from various rainbow trout organs were comparatively analyzed, and the spleen extract exhibited the most prominent anti-inflammatory activity. The spleen extract was further fractionated by liquid-liquid extraction, and among the four resulting fractions, the butanol fraction (OSB) was identified as the primary fraction mediating anti-inflammatory effects through modulation of the NF-κB signaling pathway. (B) The five major components exhibiting the highest signal intensities in negative ion mode were definitively identified from UHPLC-MS/MS analysis of OSB by comparison of their retention times with those of authentic standards. These components were categorized into two groups, and OSB was further fractionated into B-THP and B-TT using medium-pressure liquid chromatography (MPLC). (C, D) RAW264.7 cells were co-treated with lipopolysaccharide (LPS; 1 µg/mL) and OSB, B-THP, or B-TT (100 µg/mL) for 24 h. iNOS mRNA expression was analyzed by RT-qPCR (C), and protein expression was determined by Western blot analysis (D). Data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Regulatory effects of OSB sub-fractions on the NF-κB signaling pathway

Our previous studies demonstrated that OSB, a fraction derived from the spleen crude extract, exerts anti-inflammatory effects through modulation of the NF-κB signaling pathway⁵. Based on these findings, we comparatively evaluated the effects of OSB derived sub-fractions, B-THP and B-TT, on LPS-induced NF-κB activation. RAW264.7 macrophages were co-treated with LPS and either B-THP or B-TT, and the activation status of the NF-κB signaling cascade was analyzed. Upon LPS stimulation, IκBα undergoes rapid phosphorylation followed by proteasomal degradation, which subsequently allows phosphorylated p65 to translocate from the cytoplasm to the nucleus, where it functions as a transcription factor that promotes the expression of pro-inflammatory genes¹⁹. Treatment with the B-THP fraction failed to suppress LPS-induced NF-κB activation. In contrast, B-TT treatment effectively inhibited the phosphorylation dependent degradation of IκBα and markedly attenuated p65 phosphorylation (Fig. 2A). To further assess the effect of OSB sub-fractions on p65 nuclear translocation, cytoplasmic and nuclear fractions were isolated and analyzed by Western blotting. Consistent with the signaling data, B-THP treatment did not significantly alter the LPS-induced accumulation of p65 in the nucleus. However, B-TT treatment substantially reduced nuclear p65 levels, indicating effective inhibition of p65 nuclear translocation (Fig. 2B). These findings were further validated by immunofluorescence analysis. While B-THP did not interfere with LPS-induced p65 nuclear localization, B-TT treatment effectively retained p65 in the cytoplasm, thereby preventing its translocation to the nucleus (Fig. 2C). This observation was further supported by quantitative analysis of the p65 nuclear-to-cytoplasmic fluorescence intensity ratio, which revealed a significant reduction in nuclear p65 accumulation following B-TT treatment compared with LPS alone. Collectively, these results demonstrate that B-TT recapitulates the NF-κB inhibitory and anti-inflammatory effects previously observed with OSB. This strongly suggests that the principal anti-inflammatory bioactive components of OSB are enriched within the B-TT sub-fraction.

Fig. 2.

Regulatory effects of OSB subfractions on the NF-κB signaling pathway. (A) Protein expression levels of IκBα and phosphorylated p65 (p-p65) were analyzed by Western blot in RAW264.7 cells co-treated with lipopolysaccharide (LPS; 1 µg/mL) and either B-THP or B-TT (100 µg/mL) for 5–15 min. Total p65 was used as a loading control to normalize changes in p-p65 levels. (B) RAW264.7 cells were co-treated with LPS (1 µg/mL) and B-THP or B-TT (100 µg/mL) for 2 h. Cytosolic and nuclear fractions were subsequently isolated and analyzed by Western blot to assess the nuclear translocation of p65. Cytosolic fractions were normalized to β-actin, and nuclear fractions were normalized to lamin B1. (C) Subcellular localization of p65 was examined by immunofluorescence microscopy following co-treatment with LPS (1 µg/mL) and B-THP or B-TT (100 µg/mL) for 2 h. Nuclei were stained with DAPI (blue), and p65 was visualized using an Alexa Fluor 488-conjugated secondary antibody. Quantification of p65 nuclear translocation was performed using ImageJ software by calculating the nuclear-to-cytoplasmic fluorescence intensity ratio of p65 from at least three independent experiments (n ≥ 3). For each condition, fluorescence intensity was analyzed from randomly selected cells. Data are presented as mean ± SD. Statistical significance was determined using Student’s t-test. *p < 0.05, **p < 0.01.

Effects of OSB sub-fractions on LPS-induced M1 macrophage polarization

We next investigated whether the anti-inflammatory effect of the B-TT sub-fraction is associated with the suppression of LPS-induced M1 macrophage polarization. The mRNA expression levels of representative pro-inflammatory cytokines, including IL-6, TNF-α, IL-12p40, and IL-23p19, were analyzed by RT-qPCR. LPS stimulation markedly upregulated the transcription of these cytokines; however, this induction was significantly attenuated by B-TT treatment. These results indicate that B-TT suppresses the transcriptional activation of inflammatory cytokines, likely through inhibition of LPS-induced NF-κB signaling. In parallel, the mRNA expression levels of M1 macrophage surface markers, CD40 and CD86, were also significantly reduced following B-TT treatment (Fig. 3A). Consistent with the transcriptional data, immunofluorescence analysis revealed that LPS stimulation strongly increased CD86 protein expression, whereas B-TT treatment markedly suppressed this upregulation. This inhibitory effect was not observed with the B-THP fraction, further highlighting the distinct anti-inflammatory activity of B-TT (Fig. 3B). Quantitative analysis of CD86 fluorescence intensity further confirmed a significant reduction in CD86 expression in the B-TT treated group compared with LPS alone. To further assess the functional consequences of M1 polarization, intracellular reactive oxygen species (ROS) levels were measured using H2DCFDA. In agreement with previous observations, B-THP treatment did not significantly affect LPS-induced ROS generation. In contrast, B-TT treatment substantially reduced intracellular ROS levels in LPS stimulated macrophages (Fig. 3C). Taken together, these findings demonstrate that B-TT effectively suppresses LPS-induced M1 macrophage polarization by downregulating pro-inflammatory cytokine expression, inhibiting M1 surface marker expression, and reducing intracellular ROS production. These results further support the conclusion that the principal anti-inflammatory bioactive components of OSB are enriched in the B-TT sub-fraction rather than in B-THP.

Fig. 3.

Regulatory effects of OSB subfractions on M1 macrophage polarization. (A) The mRNA expression levels of pro-inflammatory cytokines (IL-6, TNF-α, IL-12p40, and IL-23p19) and M1 macrophage surface markers (CD40 and CD86) were analyzed in RAW264.7 cells co-treated with lipopolysaccharide (LPS; 1 µg/mL) and B-THP or B-TT (100 µg/mL) for 24 h. (B) Immunofluorescence analysis of CD86 expression was performed in RAW264.7 cells following co-treatment with LPS (1 µg/mL) and B-THP or B-TT (100 µg/mL) for 24 h. Nuclei were stained with DAPI (blue), and CD86 expression was detected using an Alexa Fluor 488-conjugated secondary antibody. CD86 expression was quantified using ImageJ software by measuring mean fluorescence intensity from immunofluorescence images. (C) Intracellular reactive oxygen species (ROS) levels were measured using H₂DCFDA after co-treatment with LPS (1 µg/mL) and B-THP or B-TT (100 µg/mL) for 24 h. Nuclei were stained with Hoechst (blue), and intracellular ROS levels were visualized as green fluorescence. Intracellular ROS levels were quantified using ImageJ software by measuring mean fluorescence intensity from immunofluorescence images. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Effects of co-treatment with TCA and TUDCA on LPS-induced iNOS expression

To determine whether the anti-inflammatory activity of the B-TT sub-fraction is attributable to taurocholic acid (TCA), tauroursodeoxycholic acid (TUDCA), or their combined action, the anti-inflammatory effects of TCA and TUDCA standards were evaluated in RAW264.7 macrophages. Both TCA and TUDCA exhibited no detectable cytotoxicity at concentrations up to 200 µg/mL and significantly suppressed LPS-induced nitric oxide (NO) production in a dose dependent manner (Fig. 4A, B). We next examined the effects of TCA and TUDCA on iNOS expression under inflammatory conditions. RAW264.7 cells were stimulated with LPS (1 µg/mL) and treated with either TCA or TUDCA (50 µM), alone or in combination. RT-qPCR analysis revealed that LPS-induced upregulation of iNOS mRNA was significantly reduced by treatment with either TCA or TUDCA. Notably, co-treatment with TCA and TUDCA produced the most pronounced inhibitory effect on iNOS transcription. Consistent with the mRNA data, Western blot analysis demonstrated that the LPS-induced increase in iNOS protein expression was also most effectively suppressed by combined TCA and TUDCA treatment (Fig. 4C, D). Collectively, these results indicate that TCA and TUDCA exert potent anti-inflammatory effects in RAW264.7 macrophages without inducing cytotoxicity and that their combined treatment confers enhanced suppression of LPS-induced iNOS expression. These findings suggest that the anti-inflammatory activity of the B-TT sub-fraction is largely mediated by the combined action of TCA and TUDCA, which represent the principal candidate bioactive components contributing to the observed anti-inflammatory effects of B-TT.

Fig. 4.

Regulatory effects of co-treatment with TCA and TUDCA on iNOS expression. (A) Cell viability was assessed by MTT assay in RAW264.7 cells treated with taurocholic acid (TCA) or tauroursodeoxycholic acid (TUDCA) at concentrations ranging from 0 to 200 µM for 24 h. (B) Nitric oxide (NO) production was measured following co-treatment with lipopolysaccharide (LPS; 1 µg/mL) and TCA or TUDCA (0–200 µM) for 24 h. (C) The mRNA expression levels of iNOS were analyzed by RT-qPCR 24 h after co-treatment with LPS (1 µg/mL) and TCA or TUDCA at 50 µM. (D) Protein expression levels of iNOS were determined by Western blot analysis 24 h after co-treatment with LPS (1 µg/mL) and TCA or TUDCA at 50 µM. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Effects of co-treatment with TCA and TUDCA on the NF-κB signaling pathway

We next compared the inhibitory effects of TCA and TUDCA, administered either individually or in combination, on the LPS-induced NF-κB signaling pathway. Western blot analysis revealed that LPS stimulation induced rapid IκBα degradation accompanied by increased phosphorylation of p65. In contrast, treatment with TCA or TUDCA attenuated IκBα degradation and reduced p65 phosphorylation, with co-treatment exerting the strongest inhibitory effect on both events (Fig. 5A). To determine whether TCA and TUDCA inhibit the nuclear translocation of p65, cytoplasmic and nuclear fractions were isolated and analyzed by Western blotting. LPS-induced accumulation of p65 in the nuclear fraction was substantially reduced by TCA or TUDCA treatment and was most effectively suppressed under co-treatment conditions (Fig. 5B). These findings were further corroborated by immunofluorescence analysis. While treatment with TCA or TUDCA alone resulted in only modest inhibition of LPS-induced p65 nuclear translocation, combined treatment with both compounds robustly prevented p65 localization to the nucleus (Fig. 5C). Taken together, these results demonstrate that TCA and TUDCA recapitulate the NF-κB inhibitory mechanism underlying the anti-inflammatory activity of the B-TT sub-fraction. Although each compound alone suppresses IκBα degradation and subsequent p65 nuclear translocation, their combined treatment elicits a markedly enhanced inhibitory effect on NF-κB signaling, thereby producing a stronger anti-inflammatory response.

Fig. 5.

Regulatory effects of co-treatment with TCA and TUDCA on the NF-κB signaling pathway. (A) Protein expression levels of IκBα and phosphorylated p65 (p-p65) were analyzed by Western blot in RAW264.7 cells co-treated with lipopolysaccharide (LPS; 1 µg/mL) and taurocholic acid (TCA) and tauroursodeoxycholic acid (TUDCA) (50 µM each) for 5–15 min. Total p65 was used as a loading control to normalize changes in p-p65 levels. (B) RAW264.7 cells were co-treated with LPS (1 µg/mL) and TCA and TUDCA (50 µM each) for 2 h. Cytosolic and nuclear fractions were subsequently isolated and analyzed by Western blot to assess the nuclear translocation of p65. Cytosolic fractions were normalized to β-actin, and nuclear fractions were normalized to lamin B1. (C) Subcellular localization of p65 was examined by immunofluorescence microscopy following co-treatment with LPS (1 µg/mL) and TCA or TUDCA (50 µM) for 2 h. Nuclei were stained with DAPI (blue), and p65 was detected using an Alexa Fluor 488-conjugated secondary antibody. Quantification of p65 nuclear translocation was performed using ImageJ software by calculating the nuclear-to-cytoplasmic fluorescence intensity ratio of p65 from at least three independent experiments (n ≥ 3). For each condition, fluorescence intensity was analyzed from randomly selected cells. Data are presented as mean ± SD. Statistical significance was determined using Student’s t-test. *p < 0.05, **p < 0.01.

Effects of co-treatment with TCA and TUDCA on LPS-induced M1 macrophage polarization

We next evaluated the effect of co-treatment with TCA and TUDCA on LPS-induced M1 macrophage polarization. RT-qPCR analysis revealed that the LPS-induced upregulation of pro-inflammatory cytokines was significantly attenuated by combined TCA and TUDCA treatment. In parallel, the mRNA expression levels of M1 macrophage specific surface markers were also markedly reduced under co-treatment conditions, indicating effective suppression of the M1 polarization program (Fig. 6A). Consistent with these transcriptional changes, immunofluorescence analysis demonstrated that LPS-induced upregulation of the M1 surface marker CD86 at the protein level was substantially diminished following co-treatment with TCA and TUDCA (Fig. 6B). Quantitative analysis of CD86 fluorescence intensity demonstrated a significant decrease in CD86 expression following TCA and TUDCA treatment compared with LPS stimulation alone. We further assessed intracellular reactive oxygen species (ROS) generation, a functional hallmark of M1 macrophage activation. LPS stimulation markedly increased intracellular ROS levels, whereas co-treatment with TCA and TUDCA significantly reduced LPS-induced ROS production (Fig. 6C). Collectively, these results demonstrate that combined treatment with TCA and TUDCA effectively suppresses LPS-induced M1 macrophage polarization by downregulating pro-inflammatory cytokine expression, reducing M1 surface marker expression, and attenuating intracellular ROS accumulation. Importantly, the observed effects closely recapitulate the anti-inflammatory regulatory mechanisms underlying the activity of the B-TT sub-fraction. These findings suggest that TCA and TUDCA represent the principal candidate bioactive constituents contributing to the anti-inflammatory efficacy of B-TT and support the hypothesis that the observed activity of B-TT is largely associated with the combined action of these bile acids.

Fig. 6.

Regulatory effects of co-treatment with TCA and TUDCA on M1 macrophage polarization. (A) The mRNA expression levels of pro-inflammatory cytokines (IL-6, TNF-α, IL-12p40, and IL-23p19) and M1 macrophage surface markers (CD40 and CD86) were analyzed in RAW264.7 cells co-treated with lipopolysaccharide (LPS; 1 µg/mL) and taurocholic acid (TCA) and tauroursodeoxycholic acid (TUDCA) (50 µM each) for 24 h. (B) Immunofluorescence analysis of CD86 expression was performed in RAW264.7 cells following co-treatment with LPS (1 µg/mL) and TCA and TUDCA (50 µM each) for 24 h. Nuclei were stained with DAPI (blue), and CD86 expression was visualized as fluorescence using an Alexa Fluor 488-conjugated secondary antibody. Quantitative analysis of CD86 immunofluorescence was conducted using ImageJ software by measuring mean fluorescence intensity. (C) Intracellular reactive oxygen species (ROS) levels were measured using H₂DCFDA after co-treatment with LPS (1 µg/mL) and TCA and TUDCA (50 µM each) for 24 h. Nuclei were stained with Hoechst (blue), and intracellular ROS levels were visualized as green fluorescence. Intracellular ROS levels were quantified using ImageJ software by measuring mean fluorescence intensity from immunofluorescence images. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.

Discussion

In the present study, we systematically extended our previous findings on the anti-inflammatory activity of rainbow trout spleen extracts by identifying the key bioactive constituents responsible for this effect. Our earlier work demonstrated that the butanol fraction (OSB) effectively suppressed LPS-induced inflammatory responses in macrophages by inhibiting NFκB signaling, attenuating oxidative stress, and suppressing M1 macrophage polarization. However, the complex nature of OSB as a multicomponent mixture and the requirement for relatively high treatment concentrations limited its translational potential. By further fractionating OSB and conducting compositional analyses, the current study successfully addressed these limitations and established a direct link between the observed anti-inflammatory effects and specific bile acid derivatives, namely taurocholic acid (TCA) and tauroursodeoxycholic acid (TUDCA) (Fig. 7).

Fig. 7.

Proposed mechanism underlying the anti-inflammatory effects of rainbow trout (Oncorhynchus mykiss) spleen derived bile acids in LPS-stimulated macrophages. OSB and its active components, taurocholic acid (TCA) and tauroursodeoxycholic acid (TUDCA), suppress LPS-induced NF-κB activation by inhibiting IκBα degradation and p65 nuclear translocation. This inhibition reduces iNOS-mediated NO production, pro-inflammatory gene expression, intracellular ROS generation, and M1 macrophage polarization in RAW264.7 cells.

The identification of TCA and TUDCA enriched B-TT as the principal anti-inflammatory fraction represents a critical advancement in the mechanistic understanding of rainbow trout spleen derived bioactivity. Consistent with our previous observations using OSB, B-TT effectively suppressed LPS-induced NF-κB activation, reduced the expression of pro-inflammatory mediators such as iNOS attenuated inflammatory cytokine production²⁰. Importantly, these effects were achieved at substantially lower concentrations compared to the parent OSB fraction, underscoring the functional relevance of isolating defined active components. This continuity between OSB and B-TT mediated effects strongly supports the notion that bile acid derivatives are key drivers of the anti-inflammatory phenotype observed in our earlier studies.

Bile acids, traditionally recognized for their roles in lipid digestion and metabolic regulation, have recently emerged as important immunomodulatory molecules^21,22. Previous studies have reported that TUDCA exerts anti-inflammatory effects by suppressing NF-κB signaling, reducing endoplasmic reticulum stress, and limiting oxidative damage in macrophages and epithelial cells^23–25. Similarly, TCA has been shown to modulate inflammatory signaling pathways and macrophage activation, although its immunological role has been less extensively characterized than that of TUDCA^16,26. However, the upstream mechanisms underlying NF-κB inhibition by TCA and TUDCA remain to be fully elucidated. Previous studies have demonstrated that bile acids can regulate inflammatory signaling through bile acid sensing receptors, including the farnesoid X receptor (FXR) and the G protein coupled receptor TGR5. Activation of FXR has been shown to suppress pro-inflammatory gene expression through inhibition of the NF-κB signaling pathway^27,28. In addition, activation of TGR5 elevates intracellular cAMP levels, which can attenuate NF-κB dependent inflammatory responses in macrophages²⁹. Furthermore, TUDCA is well known for its ability to alleviate cellular stress and oxidative damage, processes that are closely linked to NF-κB activation³⁰. Consistent with these reports, the reduction in intracellular ROS observed in our study suggests that TCA and TUDCA may suppress NF-κB signaling through a combination of receptor-mediated pathways and stress-attenuating mechanisms, ultimately limiting macrophage activation and M1 polarization.

Our findings are in agreements with these reports, particularly in terms of NF-κB inhibition and suppression of inflammatory gene expression. However, unlike most prior studies that focused on chemically synthesized or commercially sourced bile acids, our work uniquely demonstrates that naturally derived TCA and TUDCA from fish visceral tissues are biologically active and capable of modulating macrophage-driven inflammatory responses. In this study, we confirmed that TCA was present in greater abundance than TUDCA in the B-TT fraction (Fig. 1B). However, in vitro experiments showed that co-treatment of the two compounds resulted in significantly stronger anti-inflammatory effects compared to when each was treated alone (Figs. 4 and 5). These interesting observations suggest that TUDCA exerts a significant synergistic effect on the overall efficacy of B-TT, despite being present in relatively small amounts. Mechanistically, this synergy may stem from complementary upstream targets. For example, TCA and TUDCA may selectively activate different bile acid receptors (e.g., FXR and TGR5) or simultaneously regulate receptor-mediated signaling and intracellular stress responses (e.g., ER stress). Future studies are needed to elucidate the precise mechanisms of interaction between these specific bile acid derivatives.

A notable aspect of our study is the demonstrated impact of TCA and TUDCA containing fractions on macrophage polarization. While previous studies have largely emphasized bile acid mediated suppression of cytokine production, our data reveal a broader immunomodulatory role involving the downregulation of M1 macrophage surface markers, including CD40 and CD86, as well as attenuation of intracellular ROS accumulation^26,31. These findings suggest that bile acid derivatives influence not only inflammatory signaling pathways but also macrophage functional identity. This expanded mechanistic insight highlights a previously underappreciated role of bile acids in regulating innate immune cell plasticity.

The originality of the present study lies in several key aspects. First, it establishes a clear progression from a complex bioactive extract to defined molecular constituents, thereby enhancing mechanistic clarity and translational relevance. Second, it identifies fish spleen derived bile acids as active anti-inflammatory agents, expanding the current understanding of bile acid biology beyond mammalian systems. Third, the study links bile acid mediated NF-κB inhibition with macrophage polarization control, providing an integrated view of inflammatory regulation that has not been fully explored in prior research.

Beyond its scientific implications, this work holds significant industrial and environmental value. Fish visceral tissues, including spleen, are typically regarded as low-value byproducts or waste in the aquaculture and seafood processing industries^32,33. Our findings demonstrate that these materials can serve as sustainable sources of high-value bioactive compounds with therapeutic potential. The identification of TCA and TUDCA enriched fractions from rainbow trout spleen supports the feasibility of developing value-added functional ingredients, nutraceuticals, or anti-inflammatory therapeutics derived from marine byproducts. This aligns with global efforts toward sustainable bioresource utilization and circular bioeconomy strategies.

Nevertheless, several limitations of the present study should be acknowledged. Although TCA and TUDCA were detected at comparable levels within the B-TT sub-fraction, their relative proportions may vary among individual rainbow trout due to biological and physiological factors, including age, sex, diet, and metabolic state, as bile acid composition reflects systemic homeostasis. Such variability warrants further investigation. In addition, while our in vitro data demonstrate robust anti-inflammatory effects of TCA and TUDCA enriched fractions through suppression of NF-κB signaling, in vivo validation will be required to further assess their biological relevance and translational potential. Finally, although this study focused on inhibition of pro-inflammatory signaling and M1 macrophage polarization, future studies may expand these findings by examining additional immunomodulatory pathways and macrophage phenotypes.

In conclusion, the present study provides compelling evidence that TCA and TUDCA are key bioactive components responsible for the anti-inflammatory effects of rainbow trout spleen extracts. Notably, while the anti-inflammatory properties of TCA and TUDCA have been reported in other biological systems, our study uniquely identifies these bile acids from fish spleen tissue and establishes a direct experimental progression from crude extract to active fraction and defined molecules. By bridging our previous extract-based observations with molecularly defined mechanisms, this work advances both fundamental and applied aspects of anti-inflammatory research. Our findings highlight fish-derived bile acids as promising, sustainable candidates for the development of novel anti-inflammatory interventions and lay the groundwork for future translational and industrial applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

DYK performed the majority of the experiments, analyzed the data, and contributed to drafting the initial manuscript. WSC, JHP, and HHN carried out supporting experiments. SK and JC assisted with the dissection and provision of rainbow trout samples. JP, WS, and HY were responsible for the extraction, fractionation, and compositional analyses of the spleen extracts. SM provided valuable consultation and technical support and contributed to manuscript writing and funding acquisition. KCK conceived and supervised the study, designed the experiments, and wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.