Asiatic acid inhibits NF-κB signaling and ameliorates experimental acute and chronic colitis in mice

Abstract

Objective:

This study aimed to elucidate the anti-inflammatory mechanisms of asiatic acid (AA) by focusing on its modulation of the nuclear factor-κB (NF-κB) signaling pathway and to evaluate its therapeutic effects in murine models of both acute and chronic colitis.

Introduction:

AA, a naturally occurring triterpenoid compound derived from Centella asiatica, is known for its anti-inflammatory activity. However, its comprehensive effects on both acute and chronic intestinal inflammation, particularly through detailed modulation of the NF-κB pathway, have not been fully elucidated.

Methods:

Human intestinal epithelial cells COLO 205 and murine macrophage cells RAW 264.7 were pretreated with AA, followed by stimulation with tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), respectively. The mRNA expression of pro-inflammatory cytokines, including interleukin (IL)-8, TNF-α, and IL-6, was quantified using real-time RT-PCR. Western blotting was performed to assess the phosphorylation and degradation of the NF-κB inhibitor IκBα, and NF-κB DNA-binding activity was assessed via electrophoretic mobility shift assay (EMSA). In vivo, acute colitis was induced using dextran sulfate sodium (DSS) in wild-type mice, and chronic colitis was established by piroxicam administration in IL-10⁻/⁻ mice. Following AA treatment, colon length, body weight, histology (H&E) with histologic scoring, and colonic NF-κB p65 immunohistochemistry (IHC) were evaluated.

Results:

AA significantly downregulated cytokine expression in both cell lines. It inhibited IκBα phosphorylation and degradation, and EMSA demonstrated a marked reduction in NF-κB DNA-binding activity. In mice, AA attenuated body weight loss, colonic shortening, and histologic inflammation in both DSS and IL-10⁻/⁻ models. Concomitantly, colonic IHC showed reduced nuclear NF-κB p65.

Conclusions:

AA alleviates intestinal inflammation by suppressing NF-κB signaling in vitro and exhibits therapeutic efficacy in both acute and chronic colitis models, suggesting its potential as a therapeutic candidate for inflammatory bowel disease.

Introduction

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic systemic inflammatory disorders primarily affecting the gastrointestinal tract. As incurable diseases, they can significantly impair patients’ quality of life and are associated with considerable morbidity. ¹ Although the precise etiology of IBD remains elusive, it is widely accepted that the disease results from a complex interplay among genetic susceptibility, environmental factors, alterations in the gut microbiota, and immune system dysregulation. ² Various therapeutic strategies have been developed to target these multifactorial pathogenic mechanisms. ³

Among the key molecular targets, nuclear factor-κB (NF-κB) has emerged as a critical regulator of immune responses and is notably activated in intestinal inflammatory cells of individuals with IBD. NF-κB activation induces the transcription and secretion of multiple pro-inflammatory cytokines, thereby triggering and sustaining intestinal inflammation.^4,5 Therefore, modulation of the NF-κB pathway represents a promising therapeutic strategy for treating IBD. ⁶

Asiatic acid (AA) is a pentacyclic triterpene compound isolated from Centella asiatica, a plant traditionally used in herbal medicine, and is well recognized for its anti-inflammatory properties.^7–10 Several studies have explored the therapeutic potential of AA in experimental colitis models.^11,12 AA protected intestinal tissues by attenuating molecular, biochemical, and histopathological changes associated with UC development. ¹¹ AA also alleviated dextran sulfate sodium (DSS)-induced colitis in mice through inhibition of NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation. ¹² While these findings highlight AA’s therapeutic potential, prior research has primarily focused on acute colitis and targeted either general inflammatory markers or inflammasome-related pathways.

In the present study, we aimed to evaluate the effects of AA in both acute and chronic murine colitis models, including DSS-induced colitis and piroxicam-accelerated colitis in interleukin (IL)-10 knockout mice. Given that intestinal inflammation involves both epithelial disruption and immune cell activation, ¹³ we employed COLO 205 intestinal epithelial cells and RAW 264.7 macrophages to reflect these two key features of IBD. Furthermore, we investigated the regulatory role of AA in NF-κB signaling in both intestinal epithelial cells and macrophages.

Materials and methods

Cell preparation

In this study, we used the human colon epithelial cell line COLO 205 (Korean Cell Line Bank [KCLB], #10222, Seoul, Korea) and the murine macrophage cell line RAW 264.7 (KCLB, #40071, Seoul, Korea). Cell line identity was confirmed for COLO 205 by concordance with the supplier’s reference short tandem repeat (STR) profile, ¹⁴ and for RAW 264.7—for which human STR profiling is not applicable—by procurement from KCLB and characteristic morphology. COLO 205 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). RAW 264.7 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 2 mM L-glutamine, and the same concentrations of antibiotics. For experiments, cells were seeded into 6-well plates at a density of 0.5–2 × 10⁶ cells per well and incubated overnight to allow for adequate adhesion.^15–17 All assays were conducted using cells between passages 20 and 25. Cells were maintained under aseptic conditions and routinely screened for mycoplasma using a commercial detection kit; all tests were negative prior to and throughout the experimental period. Both COLO 205 and RAW 264.7 showed the expected responsiveness to pro-inflammatory stimuli, supporting pathway engagement and model suitability.^18–20 Representative prior publications from our laboratory employing both lines have been reported.^21,22

Cell viability assay

COLO 205 and RAW 264.7 cells were seeded in 96-well plates and treated with AA at 0, 10, 20, 50, and 100 μM for 48 h. Cell viability was assessed using the CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI, USA). After adding the MTS reagent, plates were incubated at 37°C for 2 h, and absorbance was measured at 490 nm. Cell viability (%) was calculated relative to vehicle-treated controls (0.1% dimethyl sulfoxide (DMSO)). Doses maintaining ⩾80% viability were considered non-cytotoxic.

Mice

Specific pathogen-free (SPF) C57BL/6 wild-type (WT) mice were purchased from Young-Bio (Seongnam, Korea) and used to establish a DSS-induced acute colitis model. IL-10 knockout (IL-10⁻/⁻) mice on a C57BL/6 background were obtained from the Biomedical Center for Animal Resource Development of Seoul National University (Seoul, Korea) and utilized to generate a chronic colitis model.^23–25 All animals were housed under controlled conditions (24 ± 2°C, 12 h light/dark cycle, 50 ± 5% humidity) with ad libitum access to food and water. Mice used in the experiments were 6–7 weeks old and weighed between 19 and 21 g.

Real-time reverse transcription polymerase chain reaction

To evaluate the mRNA expression of pro-inflammatory cytokines, including IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α), real-time reverse transcription polymerase chain reaction (RT-PCR) was performed in COLO 205 and RAW 264.7 cells. Cells were pretreated with AA (10, 20, or 50 μM; Sigma-Aldrich, St. Louis, MO, USA) for 48 h or left untreated as controls. Following pretreatment, COLO 205 cells were stimulated with 10 ng/mL of TNF-α (R&D Systems, Minneapolis, MN, USA) for 1 h, and RAW 264.7 cells were stimulated with 10 μg/mL of lipopolysaccharide (LPS) from Escherichia coli O127:B8 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. We pretreated cells with AA for 48 h and then applied a 1 h stimulation with TNF-α (COLO 205) or LPS (RAW 264.7) to capture hour-scale transcriptional responses of NF-κB target genes (IL-8, IL-6, TNF-α), consistent with published NF-κB time-course studies.^26–28 This schedule also aligns with prior AA protocols in RAW 264.7. ²⁹ Total RNA was extracted from the cells using TRIzol reagent (Gibco/BRL, Gaithersburg, MD, USA) and complementary DNA (cDNA) was synthesized using standard protocols for reverse transcription. RT-PCR was performed using SYBR Green PCR Master Mix and the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). For COLO 205 cells, the expression of IL-8 and TNF-α mRNA was analyzed, while for RAW 264.7 cells, IL-6 and TNF-α expression was assessed. β-actin served as an internal control for normalization. All primer sequences were designed using Primer Express version 2.0 (Applied Biosystems, Foster City, CA, USA), and each reaction was performed in triplicate. Relative gene expression was normalized to β-actin and expressed as fold change.^23,30 All RT-PCR primer sequences used in this study are listed in Supplemental Table 1.

Western blot

To investigate the effects of AA on the NF-κB signaling pathway, Western blot analysis was performed to examine the expression levels of inhibitor of NF-κB alpha (IκBα) and its phosphorylated form. COLO 205 and RAW 264.7 cells were either untreated or treated with AA at low (10 μM) or high (30 μM) concentrations for 24 h. After treatment, COLO 205 cells were stimulated with TNF-α (10 ng/mL) for 30 min, and RAW 264.7 cells were stimulated with LPS (10 μg/mL) for 30 min. ¹⁷ This timing was chosen to capture IκBα phosphorylation and degradation, which occur early in the NF-κB signaling cascade.^26–28 NF-κB activation was assessed by Ser32/36 phosphorylation of IκBα, a required IKK-dependent step in the canonical pathway.^26,31 We did not use subunit phosphorylation (e.g. RelA/p65) as a general marker because it is site- and stimulus-dependent and not required in all contexts.^32,33 Following stimulation, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed according to previously described protocols.^23,34 Protein concentrations were quantified using the Bradford assay. Equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (pore size: 0.2 μm). Membranes were probed with primary antibodies against phospho-IκBα and IκBα (Cell Signaling Technology, Danvers, MA, USA), as well as β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After washing, membranes were incubated with HRP-conjugated anti-rabbit and anti-mouse IgG secondary antibodies, as appropriate (Cell Signaling Technology, Danvers, MA, USA). Protein bands were detected using an enhanced chemiluminescence system. ³⁵

Electrophoretic mobility shift assay

To assess the effect of AA on NF-κB DNA-binding activity, electrophoretic mobility shift assays (EMSA) were conducted using the LightShift^® Chemiluminescent EMSA Kit (Thermo Scientific, Rockford, IL, USA).^23,25 COLO 205 cells were either left untreated or exposed to AA at low (10 μM) or high (30 μM) concentrations for 24 h, followed by stimulation with 10 ng/mL TNF-α for 30 min. Nuclear extracts were prepared and incubated with a biotin-conjugated DNA oligonucleotide probe (labeled at the 3′ end) complementary to the NF-κB consensus sequence to assess binding activity. To confirm the specificity of DNA-protein interactions, a supershift assay was performed using anti-NF-κB p50 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). The DNA-protein complexes and free probes were then subjected to polyacrylamide gel electrophoresis, transferred onto a nylon membrane, and visualized using chemiluminescence.

DSS-induced acute colitis in mice

To investigate the preventive potential of AA, an acute colitis model was established in mice using DSS (MP Biomedicals, Irvine, CA, USA).^23,25 A total of 24 mice were randomly divided into 3 groups (n = 8 per group). The control group received only filtered water throughout the study. The vehicle group was administered DMSO (Sigma-Aldrich, St. Louis, MO, USA) via oral gavage once daily from day −2 to day 5. Similarly, the AA group received AA at a dose of 30 mg/kg, dissolved in an equivalent volume of DMSO, following the same administration schedule. Acute colitis was induced by adding DSS to the drinking water of both the vehicle and AA groups starting on day 0. Body weight was recorded daily, and all mice were euthanized on day 5. On the same day, the entire colon was harvested for measurement of colon length. Colonic tissues were then stained with hematoxylin and eosin (H&E), and the severity of inflammation was assessed based on established histological scoring criteria. ³⁶

Chronic colitis in IL-10⁻/⁻ mice

This study further investigated the therapeutic efficacy of AA in IL-10-/- mice, a well-established model of chronic colitis characterized by spontaneous intestinal inflammation due to impaired anti-inflammatory function. To accelerate colitis development, piroxicam (Sigma-Aldrich, St. Louis, MO, USA) was incorporated into the chow at a concentration of 200 ppm and administered from day 0 to day 14. The mice were randomly assigned to three groups: a control group (n = 8) provided standard chow and filtered water without piroxicam (day 0 to day 28); a vehicle group (n = 8) given DMSO via oral gavage once daily from day 14 to day 28; and an AA group (n = 8) treated with AA at 30 mg/kg, dissolved in an equivalent volume of DMSO, once daily from day 14 to day 28. Body weight was recorded daily, and all mice were euthanized on day 28. Total colon length was measured, and colitis severity was evaluated using Berg’s grading system. ³⁷

Immunohistochemical analysis

Paraffin-embedded colon sections (4 µm) mounted on poly-L-lysine slides were deparaffinized and rehydrated. Antigens were unmasked by heating in Tris/EDTA buffer (pH 9.0) for 20 min. BLOXALL was used to block endogenous peroxidase (10 min), and normal serum was applied to reduce nonspecific binding (20 min, room temperature). Sections were incubated with rabbit anti–NF-κB p65 (RelA; Cell Signaling Technology, Danvers, MA, USA) for 30 min. After washing, signal development followed the VECTASTAIN Original ABC procedure according to the manufacturer’s instructions (biotinylated anti-rabbit, 30 min; ABC reagent, 30 min), then developed with DAB and counterstained with hematoxylin. Images were acquired on a bright-field microscope. Four non-overlapping high-power fields (HPFs; 400×) were evaluated per mouse, and the mean of the four fields was used as the animal-level score. For epithelial nuclei, we applied the Remmele–Stegner immunoreactive score (IRS, 0–12), calculated as intensity (0–3; none/weak/moderate/strong) × proportion of nuclear-positive cells (0–4; 0%, <10%, 10%–30%, 31%–60%, 61%–100%). ³⁸

Statistical analysis

All statistical analyses were performed using GraphPad Prism software (version 10; GraphPad Software, San Diego, CA, USA). Differences between experimental groups were assessed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons for continuous outcomes and Kruskal–Wallis with Dunn’s multiple comparisons for ordinal endpoints. A two-way repeated-measures ANOVA was employed to assess changes in body weight, with Bonferroni correction applied for multiple comparisons. Figures display mean ± standard deviation (SD). A p-value < 0.05 was considered statistically significant; significance was denoted by * for p < 0.05 and ** for p < 0.01.

Results

AA identifies a non-cytotoxic concentration range in COLO 205 and RAW 264.7 cells

In COLO 205 cells, treatment with AA at 10, 20, and 50 μM maintained ⩾80% cell viability, while 100 μM resulted in a marked decrease to approximately 30%. RAW 264.7 cells showed high viability (⩾95%) across all tested concentrations. These data indicate that 10–50 μM is a non-cytotoxic range suitable for further experiments. Full results are provided in Supplemental Figure 1.

AA suppressed pro-inflammatory cytokine gene expression in COLO 205 and RAW 264.7 cells

To assess the anti-inflammatory effects of AA, we analyzed the mRNA expression levels of pro-inflammatory cytokines using real-time RT-PCR. In COLO 205 cells, AA pretreatment significantly suppressed TNF-α-induced IL-8 and TNF-α mRNA expression in a dose-dependent manner (Figure 1(a) and (b)). Similarly, in RAW 264.7 cells, AA pretreatment significantly reduced LPS-induced IL-6 and TNF-α mRNA expression (Figure 1(c) and (d)).

Figure 1.

Asiatic acid (AA) downregulates pro-inflammatory cytokine mRNA expression in COLO 205 and RAW 264.7 cells.

AA inhibited NF-κB signaling in COLO 205 and RAW 264.7 cells

We performed Western blot analyses to assess whether AA suppresses the NF-κB pathway by evaluating both phosphorylated IκBα and IκBα levels. In COLO 205 cells pretreated with AA and subsequently stimulated with TNF-α, phosphorylated IκBα levels were decreased, whereas IκBα levels were preserved compared to stimulated vehicle controls. A similar pattern was observed in RAW 264.7 cells pretreated with AA and then stimulated with LPS (Figure 2).

Figure 2.

AA inhibits IκBα phosphorylation and degradation in stimulated COLO 205 and RAW 264.7 cells.

AA attenuated the DNA-binding activity of NF-κB in COLO 205 cells

To determine whether AA affects the DNA-binding activity of NF-κB, EMSA was performed. AA pretreatment led to a dose-dependent decrease in nuclear NF-κB binding activity following TNF-α stimulation in COLO 205 cells (Figure 3).

Figure 3.

AA suppresses NF-κB DNA-binding activity in TNF-α-stimulated COLO 205 cells.

AA prevented DSS-induced acute colitis in mice

Mice treated with AA exhibited significantly less body weight loss compared to the vehicle group (Figure 4(a)). Furthermore, colon shortening was significantly attenuated in the AA-treated group (Figure 4(b) and (c)). Histological analysis revealed that colonic inflammation was markedly reduced following AA administration, with significantly lower histological scores than those observed in the vehicle group (Figure 4(d) and (e)). Consistent with these findings, NF-κB p65 IHC showed reduced nuclear staining with a significantly lower epithelial nuclear p65 IRS in AA-treated mice compared with vehicle (Figure 6(a) and (b)).

Figure 4.

Protective effects of AA in dextran sulfate sodium (DSS)-induced acute colitis in mice. (a) Body weight changes during the experimental period. Mice treated with AA exhibited significantly less weight loss than those in the vehicle group. Data are presented as mean ± SEM. (b) Representative images of excised colons on day 5. Colons from AA-treated mice were visibly longer than those from vehicle-treated mice. (c) Colon length measurements. AA treatment significantly attenuated DSS-induced colonic shortening. Data are presented as mean ± SD. (d) Representative hematoxylin and eosin (H&E)-stained colon sections (×100 magnification; scale bar = 100 μm). Compared to the vehicle group, AA-treated tissues showed milder crypt damage and reduced inflammatory cell infiltration. (e) Histological inflammation scores. AA administration significantly reduced mucosal injury, as indicated by lower histological scores. Data are presented as mean ± SD.

AA ameliorated chronic colitis in IL-10⁻/⁻ mice

In IL-10⁻/⁻ mice, AA treatment showed a trend toward reduced body weight loss compared to the vehicle group, although the difference was not statistically significant (Figure 5(a)). However, colon length was significantly preserved in the AA-treated group compared to the vehicle group (Figure 5(b) and (c)). H&E-stained colon sections further demonstrated that AA administration effectively reduced inflammation and improved histological scores, reflecting its therapeutic effect in chronic colitis (Figure 5(d) and (e)). Similarly, NF-κB p65 IHC showed reduced nuclear staining, yielding a significantly lower epithelial nuclear p65 IRS in the AA group compared with vehicle (Figure 6(c) and (d)).

Figure 5.

Therapeutic effects of AA in the interleukin-10 knockout (IL-10⁻/⁻) mouse model of chronic colitis. (a) Changes in body weight. AA-treated mice showed a trend toward reduced weight loss, although the difference was not statistically significant. Data are presented as mean ± SEM. (b) Gross images of colons collected at necropsy. Colons from AA-treated mice were visibly longer than those from vehicle-treated mice. (c) Colon length analysis. AA significantly attenuated colonic shortening in IL-10⁻/⁻ mice. Data are presented as mean ± SD. (d) Representative H&E-stained colon sections (×100 magnification; scale bar = 100 μm). AA treatment maintained epithelial integrity and reduced histological features of severe injury, including ulceration and submucosal inflammatory infiltration. (e) Histological scores. AA significantly decreased the severity of chronic inflammation compared to the vehicle group. Data are presented as mean ± SD.

Figure 6.

NF-κB p65 immunohistochemistry (IHC) in DSS-induced acute colitis and IL-10⁻/⁻ chronic colitis. (a) Representative IHC images of colon sections from the DSS model (×400 magnification; scale bar = 50 μm). AA-treated mice showed visibly reduced nuclear p65 staining compared with the vehicle group (antibody diluted 1:400). (b) Immunoreactivity score (IRS) for nuclear NF-κB p65 in the DSS model. AA significantly lowered nuclear p65 immunoreactivity compared with the vehicle group. Data are shown as mean ± SD. (c) Representative IHC images from the IL-10⁻/⁻ model (×400 magnification; scale bar = 50 μm). AA treatment decreased nuclear p65 positivity relative to the vehicle group (antibody diluted 1:400). (d) IRS for nuclear NF-κB p65 in the IL-10⁻/⁻ model. AA significantly decreased the immunoreactivity compared to the vehicle group. Data are shown as mean ± SD.

Discussion

In this study, we investigated the anti-inflammatory potential of AA using both in vitro and in vivo models of acute and chronic intestinal inflammation. AA effectively reduced inflammatory markers in various experimental settings. Treatment with AA led to a dose-dependent suppression of key pro-inflammatory cytokines—IL-8, IL-6, and TNF-α—in COLO 205 intestinal epithelial cells and RAW 264.7 macrophages, suggesting that AA regulates cytokine production at the transcriptional level. To elucidate the underlying mechanism, we focused on the NF-κB signaling pathway, a central regulator of inflammatory responses. In COLO 205, we used TNF-α rather than LPS to activate NF-κB because epithelial cell lines, including COLO 205, often exhibit limited or variable responses to LPS owing to regulated expression and trafficking of Toll-like receptor 4 (TLR4) and its co-receptor myeloid differentiation factor-2 (MD-2).^39,40 RAW 264.7 cells served as a TLR4-dependent macrophage model and were stimulated with LPS. ⁴¹ We measured TNF mRNA after TNF-α stimulation to capture potential autocrine TNF feedback, which can sustain or amplify NF-κB activity.^42,43 The increase in IL-8 with TNF-α is consistent with the functional NF-κB binding site in the IL-8 promoter; while mitogen-activated protein kinase (MAPK; JNK/p38)–activator protein-1 (AP-1) can contribute, NF-κB typically predominates in epithelial IL-8 regulation.^44,45 This is consistent with our prior report in COLO 205, in which TNF-α robustly induced NF-κB–dependent IL-8 and pathway modulation attenuated the response. ¹⁹ In qualitative Western blots, AA decreased phosphorylated IκBα while maintaining IκBα, a pattern consistent with IκBα stabilization and reduced NF-κB signaling. This mechanism was further supported by EMSA results, which demonstrated a dose-dependent reduction in NF-κB DNA-binding activity. In vivo, AA alleviated DSS-induced acute colitis and improved outcomes in IL-10⁻/⁻ mice. Histologically, p65 IHC showed reduced nuclear positivity in AA-treated mice in both models, consistent with the in vitro findings. To our knowledge, this study is among the first to comprehensively demonstrate that AA mitigates inflammation in both acute and chronic colitis models through modulation of the NF-κB pathway.

AA is a naturally occurring triterpenoid compound derived from Centella asiatica, a medicinal herb traditionally used in Eastern medicine. ⁹ It has been studied for its regulatory effects on pro-inflammatory cytokine expression and its ability to inhibit the progression of immune-mediated inflammatory diseases. ⁴⁶ Several prior studies have reported that AA suppresses the NF-κB signaling pathway in various disease models.^29,47–49 For instance, AA was shown to inhibit proliferation and promote apoptosis in fibroblast-like synoviocytes—cells critically involved in rheumatoid arthritis—by downregulating NF-κB and activating the nuclear factor erythroid 2–related factor 2–heme oxygenase-1 (Nrf2–HO-1) pathway. ⁴⁹ In a mouse model of fulminant hepatic failure, AA inhibited both NF-κB and MAPK signaling. ⁴⁷ Additionally, AA has been reported to suppress NF-κB and nuclear factor of activated T cells 1 (NFATc1) activity during osteoclast differentiation and bone resorption. ⁴⁸ In macrophages stimulated with LPS, AA reduced IκBα phosphorylation and reactive oxygen species (ROS) generation, partly through the suppression of the Notch signaling pathway. ²⁹

Although a few studies have investigated the effects of AA in experimental colitis, these have primarily focused on acute experimental colitis models, with limited attention given to chronic inflammatory models.^11,12 AA administration reduced inflammatory tissue damage, cytokine production, and oxidative stress in the colon, likely through modulation of the inflammasome or antioxidant pathways. In addition, AA ameliorated acetic acid-induced colonic injury by suppressing biochemical and histopathological markers of disease, including NF-κB p65 expression. ¹¹ However, these studies were primarily limited to models of acute inflammation or simplified in vitro systems involving single cell types.

In contrast, we designed our study to overcome these limitations by using both an acute DSS model and a chronic model based on IL-10⁻/⁻ mice. Acute colitis in the DSS model results from chemical injury to the epithelial barrier, which rapidly leads to neutrophil infiltration and strong innate immune activation. ⁵⁰ This model is particularly useful for studying early epithelial damage and short-term inflammatory responses. ⁵¹ Conversely, IL-10⁻/⁻ mice develop a slow-onset, persistent form of colitis due to the absence of IL-10, a pivotal anti-inflammatory cytokine.^52,53 This chronic pathology is marked by sustained activation of adaptive immune pathways, continuous lymphocyte accumulation, and elevated cytokine release, resembling the long-standing mucosal injury seen in CD. ⁵⁴ Using both models enabled us to capture different stages and mechanisms of intestinal inflammation. To investigate AA’s mechanism of action more thoroughly, we analyzed its effects in two cell types central to intestinal inflammation: epithelial cells and macrophages. ⁵⁵ We tracked NF-κB signaling from early events—such as IκBα phosphorylation and degradation—to downstream DNA-binding activity by EMSA. This multi-layered approach revealed a clear link between molecular changes and tissue-level improvement in colitis. Together, these assays provide a more comprehensive mechanistic profile of AA than prior reports. To better evaluate AA’s effects under these different conditions, we also tailored histological evaluation methods to each model. For the DSS-induced model, we used the scoring approach described by Dieleman et al., ³⁶ which assesses crypt structure, epithelial integrity, and neutrophil presence. For the IL-10⁻/⁻ model, we applied Berg’s grading system, ³⁷ emphasizing goblet cell depletion, thickening of the mucosa, and infiltration by mononuclear cells. These tailored criteria enabled a more accurate assessment of inflammation severity and tissue recovery in each model, thereby increasing the potential clinical applicability of our findings. Taken together, our findings highlight the relevance of AA not only in acute inflammation, but also in chronic inflammatory contexts, supporting its broader potential for IBD treatment. In both DSS-induced acute colitis and IL-10⁻/⁻ chronic colitis, AA treatment reduced nuclear p65 staining by IHC. These tissue-level findings are consistent with the suppression of NF-κB signaling observed in vitro. This concordance between molecular data and therapeutic outcomes supports the notion that NF-κB pathway inhibition plays a role in the anti-inflammatory activity of AA.

Nonetheless, the in vivo component of our study has certain limitations. Specifically, AA was administered over a relatively short period and within a narrow dosing range. Although favorable outcomes were observed, it remains uncertain whether these effects can be maintained with prolonged treatment; potential long-term toxicity was not evaluated. In vitro, we did not conduct a same-batch, head-to-head comparison of 24-h and 48-h AA pre-exposure. The timings were pre-specified to match the temporal characteristics of the measured endpoints and to align with prior reports; a systematic evaluation relating pre-exposure duration to early signaling events and hour-scale transcriptional responses is warranted. Therefore, further research is needed to optimize dosing strategies and evaluate the safety and efficacy of extended AA administration.

Conclusion

In conclusion, AA significantly alleviated inflammation in both acute and chronic experimental models of colitis, likely through suppression of the NF-κB signaling pathway and the downstream expression of pro-inflammatory cytokines. Altogether, these results position AA as a promising candidate for further development as an immunomodulatory agent in IBD therapy.

Supplemental Material

sj-docx-1-iji-10.1177_03946320251398285 – Supplemental material for Asiatic acid inhibits NF-κB signaling and ameliorates experimental acute and chronic colitis in mice

Supplemental material, sj-docx-1-iji-10.1177_03946320251398285 for Asiatic acid inhibits NF-κB signaling and ameliorates experimental acute and chronic colitis in mice by Seona Park, Hyun Jung Lee, Seong-Joon Koh, Jong Pil Im and Joo Sung Kim in International Journal of Immunopathology and Pharmacology