Cedrol ameliorates ulcerative colitis via myeloid differentiation factor 2-mediated inflammation suppression, with barrier restoration and microbiota modulation

Abstract

BACKGROUND

Ulcerative colitis (UC) is a chronic and treatment-resistant disorder requiring potent therapeutics that are effective and safe. Cedrol (CE) is a bioactive natural product present in many traditional Chinese medicines. It is known for its suppression of inflammation and mitigation of oxidative stress. Its therapeutic efficacy and mechanistic underpinnings in UC remain uncharacterized.

AIM

To investigate the therapeutic potential and mechanisms of CE in UC.

METHODS

The anti-inflammatory activity and intestinal barrier-repairing effects of CE were assessed in a dextran sulfate sodium-induced murine colitis model. Network pharmacology was employed to predict potential targets and pathways. Then molecular docking and dynamics simulations were utilized to confirm a stable interaction between CE and the toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex. The anti-inflammatory mechanisms were further verified using in vitro assays. Additionally, the gut microbiota composition was analyzed via 16S rRNA gene sequencing.

RESULTS

CE significantly alleviated colitis symptoms, mitigated histopathological damage, and suppressed inflammation. Moreover, CE restored intestinal barrier integrity by enhancing mucus secretion and upregulating tight junction proteins (zonula occludens 1, occludin, claudin-1). Mechanistically, CE stably bound to MD2, inhibiting lipopolysaccharide-induced TLR4 signaling in RAW264.7 cells. This led to suppression of the downstream mitogen-activated protein kinase and nuclear factor kappa B signaling pathways, downregulating the expression of tumor necrosis factor-alpha, interleukin-1β, and interleukin-6. Gut microbiota analysis revealed that CE reversed dextran sulfate sodium-induced dysbiosis with significant enrichment of butyrogenic Christensenella minuta.

CONCLUSION

CE acted on MD2 to suppress proinflammatory cascades, promoting mucosal barrier reconstitution and microbiota remodeling and supporting its therapeutic use in UC.

Key Words: Cedrol; Ulcerative colitis; Toll-like receptor 4; Myeloid differentiation factor 2; Signaling pathways; Gut microbiota

Core Tip: Therapeutics to treat ulcerative colitis (UC) must be effective and safe. The natural compound cedrol (CE) possesses anti-inflammatory and antioxidant properties. However, its therapeutic efficacy and mechanisms in UC remain unclear. We demonstrated that CE ameliorated dextran sulfate sodium-induced colitis in mice through multifaceted mechanisms: Attenuating inflammation; Promoting intestinal barrier repair; And restoring gut microbial homeostasis. Mechanistically, CE exerted its anti-inflammatory effects by functionally inhibiting the toll-like receptor 4/myeloid differentiation factor 2 complex, thereby inhibiting the activation of proinflammatory signaling pathways. These findings highlighted the potential of CE as a promising therapeutic agent for UC.

INTRODUCTION

Ulcerative colitis (UC) is a chronic, relapsing idiopathic inflammatory condition primarily affecting the colorectum. It is marked by abdominal discomfort, chronic diarrhea, and bloody mucoid stools, leading to significant weight loss and diminishing overall well-being[1,2]. The precise etiology and pathogenesis of UC are elusive although multifactorial origins involving genetic predisposition, environmental factors (diet/Lifestyle), intestinal barrier dysfunction, gut microbiota dysbiosis, and aberrant immune activation are implicated[1,3]. Notably, the incidence of UC has surged globally over recent decades, particularly in Asia. UC is classified by the World Health Organization as a contemporary refractory condition and is often termed “green cancer” due to its persistent, treatment-resistant nature[4]. Current mainstream therapies (aminosalicylates, corticosteroids, immunomodulators, biologics, small molecules) yield a clinical remission rate ≤ 60% with frequent adverse effects during prolonged use[1,2,5]. Consequently, developing novel UC therapeutics with enhanced efficacy and long-term safety profiles is imperative.

Natural compounds are a promising source for drug discovery. Phytochemicals including terpenoids, alkaloids, and flavonoids have demonstrated potential for managing UC by functioning through diverse mechanisms such as modulation of gut microbial ecosystems and metabolomes, preservation of intestinal epithelial barrier integrity, and immunoregulation[6,7].

Cedrol (CE, C₁₅H₂₆O; Figure 1A), a natural sesquiterpene alcohol, is widely distributed in nature. It shows notable enrichment in coniferous species (e.g., Cedrus atlantica, Juniperus virginiana) and is found in various traditional Chinese medicinal herbs (e.g., Ginseng, Portulaca oleracea, Angelica sinensis, Ligusticum chuanxiong, Magnolia officinalis, Ganoderma lucidum). It has multifunctional bioactivities including anti-inflammatory, antioxidant, analgesic, anxiolytic, and antitumor properties[8-11]. Recent research has focused predominantly on the effects of CE in the treatment of arthritis and oncology. A singular in vitro study using Caco-2 cells suggested that CE may restore inflammation-compromised intestinal barriers via dual mechanisms: An adenosine triphosphate-dependent proliferation stimulation and an amino acid-mediated tight junction (TJ) protein expression[12]. Nevertheless, the therapeutic potential and mechanistic basis of CE in UC remain unexplored.

Figure 1 Cedrol ameliorated dextran sulfate sodium-induced colitis symptoms. A: Chemical structure of cedrol; B: Experimental scheme diagram; C: Body weight changes during the experimental period; D: Representative colons from each group; E: Quantitative analysis of colon length; F: Disease activity index scores. Data are expressed as mean ± SD (n = 6). ^bP < 0.01. ^cP < 0.001. CE: Cedrol; DSS: Dextran sulfate sodium; i.g.: Gavage; DAI: Disease activity index.

This investigation intended to employ an integrated methodology combining in vivo/in vitro models, network pharmacology, computational simulations (docking/dynamics), and 16S rRNA gene sequencing to evaluate the efficacy and mechanisms of CE, with a focus on inflammatory pathway modulation, intestinal barrier preservation, and microbiome remodeling. It also attempted to explore potential direct pharmacological targets of CE, aiming to provide innovative strategic perspectives for UC intervention.

MATERIALS AND METHODS

Materials

CE (purity ≥ 98%, C154086) and olive oil (S30503) were sourced from Aladdin Biochemical Technology and Yuanye Bio-Technology (Shanghai, China), respectively. Dextran sulfate sodium (DSS) (molecular weight 40 kDa, DCJ1606A) and lipopolysaccharide (LPS) (L8880) were procured from Seebio Bio-Technology (Shanghai, China) and Solarbio Science and Technology (Beijing, China).

Animal models and treatments

Female C57BL/6 mice (8-week-old, 18-22 g) were procured from Shanxi Provincial People’s Hospital Experimental Animal Center [No. SYXK(Jin)2024-0002] and acclimated for 7 days under specific pathogen-free conditions maintained at 22 ± 1 °C with 55% ± 5% relative humidity and a 12-hour light/dark cycle. Animals were randomly assigned to four experimental groups (n = 6): (1) The control group (olive oil 10 mL/kg/day by gavage, days 0-9); (2) The CE group (CE suspension in olive oil 10 mg/kg/day by gavage, days 0-9)[13,14]; (3) The DSS group (olive oil 10 mL/kg/day by gavage, days 0-9 + 2.5% DSS in deionized water ad libitum, days 3-9); and (4) The DSS + CE group (CE suspension in olive oil 10 mg/kg/day by gavage, days 0-9 + 2.5% DSS in deionized water ad libitum, days 3-9) (Figure 1B). The CE suspension was prepared fresh daily in olive oil (final concentration 1 mg/mL).

The disease activity index (DAI) was calculated using standardized criteria (Table 1) derived from daily evaluations of body weight, stool consistency, and rectal bleeding[15,16]. On day 10 cervical dislocation euthanasia preceded colon excision and length measurement. A 1 cm distal segment underwent 4% paraformaldehyde (Servicebio, Wuhan, Hubei Province, China) fixation, and residual tissue was stored at -80 °C. Experimental protocols received approval from Shanxi Provincial People’s Hospital Ethics Committee [approval No. (2024) 569].

Table 1 Disease activity index criteria.

Score	Weight loss (%)	Stool condition	Bleeding
0	None	Normal, well-formed	Negative
1	< 5	Soft but formed	Occult blood (+)
2	5-10	Soft	Occult blood (++)
3	10-20	Loose	Occult blood (+++)
4	> 20	Watery diarrhea	Naked bloody stool

Hematoxylin and eosin and alcian blue periodic acid-Schiff staining

Distal colon tissues were fixed overnight in 4% paraformaldehyde, followed by paraffin embedding and sectioning into 3 μm slices. Tissue sections were stained with hematoxylin and eosin (Servicebio) and alcian blue periodic acid-Schiff (Servicebio) following standard protocols, then examined via optical microscopy (CKX53; Olympus, Tokyo, Japan) with digital imaging. Histopathological damage was assessed using the criteria outlined in Table 2[17], with the overall score (0-13) representing the sum of individual scores for inflammation extent, crypt damage, ulceration, and edema to ensure a standardized evaluation.

Table 2 Histopathological scoring criteria.

Score	Inflammation extent	Crypt damage	Ulceration	Edema
0	None	Intact crypt architecture	No ulcer	Absent
1	Scattered submucosal cells	Some crypt damage	Small focal ulcers	Present
2	Focal submucosal infiltrates	Increased crypt spacing, goblet cell loss, crypt shortening	Frequent small ulcers
3	Focal submucosal, lamina propria infiltrates	Large crypt-deficient areas amid normal crypts	Extensive surface epithelial loss
4	Extensive submucosal, lamina propria, perivascular infiltrates	Crypt absence
5	Transmural inflammation

Immunohistochemistry

Following dewaxing, 3 μm paraffin-embedded tissue sections were subjected to antigen retrieval in preheated Tris-ethylenediaminetetraacetic acid buffer (potential of hydrogen = 8.0) at 95 °C for 30 minutes, endogenous peroxidase blocking with 3% hydrogen peroxide (Annjet, Shandong Province, China) (room temperature, 15 minutes in the dark), and nonspecific binding blockade using 3% bovine serum albumin (30 minutes). Primary antibodies (rabbit anti-zonula occludens 1/occludin and mouse anti-claudin-1, 1:500) were incubated with sections overnight at 4 °C. Subsequently, sections were incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:200) for 50 minutes. Diaminobenzidine chromogen was freshly prepared for staining, followed by hematoxylin counterstaining (3 minutes) for nuclear visualization. All reagents were sourced from Servicebio unless otherwise noted.

16S rRNA gene sequencing

Colon content samples were snap-frozen in liquid nitrogen and stored at -80 °C. Four colon content samples were then randomly selected per group and subjected to genomic DNA extraction using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol for 16S rRNA gene sequencing. DNA was assessed by 1% agarose gel electrophoresis, followed by quantification employing a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The V3-V4 hypervariable regions of bacterial 16S rRNA genes were amplified via polymerase chain reaction (PCR) and prepared for sequencing using Illumina protocols (San Diego, CA, United States). Finally, paired-end sequencing (2 × 250 bp) was carried out on the Illumina NovaSeq 6000 platform (Shanghai Wei Huan Biological Technology Co. Ltd, Shanghai, China).

After preprocessing the raw sequence data, we employed DADA2 for comprehensive analysis to produce high-resolution amplicon sequence variants (ASVs). These ASVs were subsequently cross-referenced against the Silva-138-9 reference database to assign accurate taxonomic classifications. Alpha and beta diversity analyses were conducted in QIIME2[18]. Significant differences in microbial community structure between groups were further determined with the Kruskal-Wallis test and the linear discriminant analysis effect size method.

Network pharmacology

Potential CE targets were identified using SwissTargetPrediction, PharmMapper, and SuperPred databases with gene nomenclature standardization via UniProt. Disease targets of UC were identified via the DisGeNET, GeneCards, and OMIM databases. Common targets were intersected using Venny 2.1.0 and analyzed for protein-protein interactions through the STRING database. The resulting network was constructed and visualized in Cytoscape 3.7.2. Functional enrichment assessments for Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed using the DAVID database and visualized via the WeiShengXin Cloud Platform.

Molecular docking

The three-dimensional structure of CE was acquired from PubChem (CID: 65575) and prepared using Chem³D 2020 for molecular docking. The crystal structure of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) complex (PDB ID: 3FXI) was obtained from the Protein Data Bank. Molecular docking was performed with AutoDock Vina 1.5.7 software. The resulting docking poses were analyzed and visualized using PyMOL 2.6.0.

Molecular dynamics simulation

For the ligand (CE) the general amber force field was applied; for the protein (MD2) the AMBER14SB force field coupled with the TIP3P (transferable interatomic potential with three points model) water model was used. Subsequently, files of MD2 and CE were merged to construct the simulation system of the complex, and molecular dynamic simulations were conducted using the Gromacs 2022. Simulations were run under periodic boundary conditions at 298 K and 1 bar. Hydrogen-bond-related covalent bonds were constrained via LINCS with a 2 fs time step. Electrostatic interactions were computed via Particle Mesh Ewald with a 1.2 nm cutoff. Non-bonded interactions were truncated at 10 Å and updated every ten steps. The system underwent 100 ps of number of particles, volume, and temperature and number of particles, pressure, and temperature equilibration before a 100 ns molecular dynamics simulation. Conformations were saved every 10 ps. Trajectory analysis was performed using VMD and PyMOL.

Cell culture and treatment

The RAW264.7 macrophage cell line (Chinese Academy of Sciences, Shanghai, China) was cultured in Dulbecco’s modified Eagle’s medium (Gibco, China; Thermo Fisher Scientific) with 10% fetal bovine serum (Gibco, Australia; Thermo Fisher Scientific) at 37 °C in a humidified incubator with 5% carbon dioxide (HF100; Heal Force Group, Shanghai, China). For experimental treatments cells were plated into 6-well culture dishes (Wuxi NEST Biotechnology, Jiangsu Province, China) and propagated until achieving > 80% confluence. Pretreatment was performed by exposing cells to 20 μmol/L CE for 1 hour, followed by stimulation with 1 μg/mL LPS for 5 hours[13,19]. Finally, cells were collected for subsequent experiments.

Cell viability assay

Cells were plated into 96-well culture dishes (Wuxi NEST Biotechnology) at a density of 1 × 10³ cells/well to 1 × 10⁴ cells/well and cultured overnight. Following exposure to serial CE concentrations for 24 hours, 10 μL cell counting kit-8 reagent (MedChemExpress, Shanghai, China) was added per well and incubated for 30 minutes. Absorbance at 450 nm was measured with a microplate reader (Epoch; BioTek Instruments, Agilent Technologies, Winooski, VT, United States), and cell viability was determined.

Real-time quantitative PCR

Total RNA was extracted with RNAiso Plus (Takara, Kyoto, Japan) and quantified. Then reverse transcribed into complementary DNA with the PrimeScrip^TM RT reagent Kit (Takara). Real-time quantitative PCR amplification was conducted on a real-time PCR system (CFX96; Bio-Rad, Hercules, CA, United States) employing 2x M5 HiPer SYBR Green quantitative PCR SuperMix (Mei5 Biotechnology, Beijing, China). Relative messenger RNA (mRNA) expression levels were calculated via the 2^-ΔΔCT method. Primers were custom designed and synthesized commercially by Sangon Biotech (Shanghai, China) with sequences detailed in Table 3.

Table 3 Primer sequence.

Gene	Forward	Reverse
TNF-α	5’-CCCCAAAGGGATGAGAAGTTC-3’	5’-CCTCCACTTGGTGGTTTGCT-3’
IL-1β	5’-GTTCCCATTAGACAACTGCACTACAG-3’	5’-GTCGTTGCTTGGTTCTCCTTGTA-3’
IL-6	5’-CCAGAAACCGCTATGAAGTTCC-3’	5’-GTTGGGAGTGGTATCCTCTGTGA-3’
β-actin	5’-GTCAGGTCATCACTATCGGCAAT-3’	5’-AGAGGTCTTTACGGATGTCAACGT-3’

IL: Interleukin; TNF: Tumor necrosis factor.

Western blot

Protein lysates were extracted using radio immunoprecipitation assay buffer supplemented with 1% phenyl methyl sulfonyl fluoride and 1% phosphatase inhibitor (Solarbio). Protein quantification was performed by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Samples (30 μg/Lane) underwent 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and transferred to a polyvinylidene difluoride membrane (Millipore, Dublin, Ireland). After blocking in 5% skim milk, membranes were incubated overnight at 4 °C with primary antibodies targeting: Phospho-extracellular regulated protein kinase (ERK) 1/2 (Thr202/Tyr204); ERK1/2; Phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185); JNK; Phospho-p38 (Thr180/Tyr182); P38, phospho-nuclear factor kappa B (NF-κB) p65 (Ser536); NF-κB p65 (Cell Signaling Technology, Daners, MA, United States); Cyclooxygenase-2 (COX-2); Inducible nitric oxide synthase (iNOS); Glyceraldehyde-3-phosphate dehydrogenase (Proteintech, Wuhan, Hubei Province, China). Secondary antibodies (ABclonal, Woburn, MA, United States) were then applied for 1 hour. The proteins were then subjected to enhanced chemiluminescence (Boster Biological Technology, Wuhan, Hubei Province, China) and visualized with the ChemiDoc X-Ray Spectrometer + Image Lab system (Bio-Rad). The results were analyzed by Image J software (La Jolla, CA, United States).

Statistical analysis

GraphPad Prism 9.5 was employed for analyses (results presented as mean ± SD). The t-test, one-way analysis of variance, or non-parametric test was applied as appropriate with significance defined as P < 0.05.

RESULTS

CE ameliorated DSS-induced colitis symptoms in mice

To assess the protective potential of CE in UC, a murine colitis model was induced by administering 2.5% DSS in their drinking water for 7 days. Figure 1B presents the experimental scheme. The DSS group lost a significant amount of weight compared with the control group and the CE group. Notably, weight reduction in the DSS + CE group was considerably attenuated compared with the DSS group (Figure 1C). The colon length was markedly reduced in the DSS group compared with the control and CE groups. However, CE treatment substantially mitigated DSS-induced colon shortening (Figure 1D and E). Similarly, DAI scores increased significantly in the DSS group while declining markedly in the DSS + CE group (Figure 1F), indicating attenuation of colitis severity following CE administration.

CE attenuated colonic injury/inflammation in DSS-induced colitis

Histopathological damage was further assessed using hematoxylin and eosin staining. The DSS group displayed inflammatory cell infiltration, mucosal damage, crypt atrophy, and edema in colonic sections (Figure 2A). In contrast the DSS + CE group showed marked attenuation of the aforementioned pathological features. Additionally, the histopathological score was significantly elevated in the DSS group while it was reduced in the DSS + CE group (Figure 2B). These observations demonstrated that CE attenuated colonic injury in colitis. Following this, the anti-inflammatory capacity of CE was assessed. Proinflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6 were markedly increased in the colonic tissues of the DSS group. CE administration effectively suppressed this DSS-induced upregulation (Figure 2C-E).

Figure 2 Cedrol attenuated histopathological damage and inflammatory cytokine expression in dextran sulfate sodium-induced colitis. A: Typical hematoxylin and eosin colon sections (× 100); B: Histopathological scores; C-E: Transcript levels of tumor necrosis factor-alpha (C), interleukin (IL)-1β (D), and IL-6 (E) messenger RNA in colonic tissues were measured by real-time quantitative polymerase chain reaction. Data are presented as mean ± SD (n = 6). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. CE: Cedrol; DSS: Dextran sulfate sodium; TNF: Tumor necrosis factor; IL: Interleukin; mRNA: Messenger RNA.

CE restored intestinal barrier function in DSS-induced colitis

To investigate the ameliorative effects of CE on gut barrier integrity in DSS-induced colitis, goblet cell mucin production was determined via alcian blue periodic acid-Schiff staining. TJ protein (zonula occludens 1, occludin, claudin-1) expression was examined through immunohistochemistry. DSS exposure significantly reduced mucus secretion and downregulated TJ protein expression compared with the control group (Figure 3). CE treatment reversed these alterations, increasing both mucus production and TJ protein expression. These results demonstrated that CE ameliorated intestinal barrier dysfunction in DSS-induced colitis.

Figure 3 Cedrol re-established intestinal barrier integrity in dextran sulfate sodium-induced colitis. A: Characteristic alcian blue periodic acid-Schiff staining (× 100) and histogram of stained regions; B-D: Representative immunohistochemistry images (× 100) and statistical analysis of the average optical density for zonula occludens 1 (B), occludin (C), and claudin 1 (D). Data are expressed as mean ± SD (n = 6). ^aP < 0.05. ^bP < 0.01. ^dP < 0.0001. CE: Cedrol; DSS: Dextran sulfate sodium; AB-PAS: Alcian blue periodic acid-Schiff staining; AOD: Average optical density; ZO-1: Zonula occludens 1.

Network pharmacology-based prediction of CE mechanisms in UC treatment

To elucidate the potential mechanisms of CE against UC, the network pharmacology analysis identified 323 CE-related targets and 2164 UC-associated targets, yielding 138 overlapping therapeutic targets (Figure 4A). Protein-protein interaction analysis of these targets revealed a network comprising 138 nodes and 1527 edges (Figure 4B) with subsequent screening identifying 28 core targets including ALB, STAT3, EGFR, SRC, CASP3, HSP90AA1, NFKB1, PPARG, ESR1, and TLR4 (Figure 4C). Functional enrichment analysis identified the following: Biological processes (109 terms) primarily encompassing negative regulation of apoptosis, protein phosphorylation, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling activation, and signal transduction (Figure 4D); Cellular components (21 terms) predominantly involving receptor complexes, cytosol, and cytoplasm (Figure 4E); Molecular functions (54 terms) principally associated with histone H3-Y41/H2A-X142-targeted and tyrosine protein kinase activities (Figure 4F). KEGG-based analysis identified 123 significantly enriched pathways (top 20 shown in Figure 4G) with PI3K-AKT, mitogen-activated protein kinase (MAPK), and FoxO signaling pathways demonstrating particular relevance to UC pathogenesis.

Figure 4 Network-based pharmacological investigation. A: Venn diagram identifying 138 overlapping targets between cedrol and ulcerative colitis; B: Comprehensive protein-protein interaction network of overlapping targets; C: Core target subnetwork (28 high-connectivity nodes); D-F: Bubble plots for gene ontology-biological processes, gene ontology-cellular components, and gene ontology-molecular functions; G: Kyoto Encyclopedia of Genes and Genomes pathway enrichment bubble plot (top 20 pathways by significance). CE: Cedrol; UC: Ulcerative colitis; ERK: Extracellular regulated protein kinase; COVID-19: Coronavirus disease 2019; PD-L1: Programmed cell death ligand 1; PD-1: Programmed cell death 1; HIF: Hypoxia inducible factor; AGE: Advanced glycation end products; RAGE: Receptor for advanced glycation end products; MAPK: Mitogen-activated protein kinase; PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B.

CE exhibited direct binding to the TLR4/MD2 complex

Numerous preclinical investigations have demonstrated that TLR4-mediated signaling pathways are central to the pathophysiology of UC, positioning this receptor as a promising therapeutic target[20-22]. TLR4 assembles with MD2 on the cell surface to form a functional complex that recognizes pathogen-associated ligands like LPS to initiate downstream inflammatory signaling. Based on the prediction from network pharmacology, we first utilized molecular docking to explore the potential interaction between CE and the TLR4/MD2 complex. The docking analysis revealed CE binding within MD2 through Van der Waals interactions with CYS133, VAL24, and VAL48 in addition to hydrophobic contacts (alkyl/π-alkyl) with ILE153, ILE52, ILE32, ILE46, LEU61, and PHE151 (Figure 5A-C). The calculated binding affinity of -6.7 kcal/mol suggested favorable binding.

Figure 5 Computational docking and molecular dynamics simulations. A-C: Docking results of cedrol and the toll-like receptor 4/myeloid differentiation factor 2 complex; D-H: Root mean square deviation (D), radius of gyration (E), root mean square fluctuation (F), centroid evolution (G), and buried solvent accessible surface area analysis (H) during molecular dynamics simulations. RMSD: Root mean square deviation; Rg: Radius of gyration; RMSF: Root mean square fluctuation; SASA: Solvent accessible surface area.

Molecular dynamics simulations confirmed complex stability via convergent root mean square deviation values indicating structural equilibrium, consistent radius of gyration reflecting structural compactness, residue-specific fluctuation profiles from root mean square fluctuation analysis, stable ligand-pocket distances in centroid evolution, and persistent molecular interfaces demonstrated by equilibrated buried solvent accessible surface area (Figure 5D-H). These computational analyses substantiated direct and stable binding between CE and MD2.

CE suppressed inflammation via inhibition of TLR4/MD2-mediated MAPK and NF-kB signaling

When TLR4 forms a complex with MD2 (its coreceptor), the complex recognizes LPS and initiates downstream activation of the MAPK and NF-κB signaling pathways, culminating in a proinflammatory cascade[23]. To demonstrate this in our model, we utilized western blot analysis to measure phosphorylated and total ERK1/2, JNK, and p38 levels in mouse colon tissues. The phosphorylation ratios of ERK1/2, JNK, and p38 were significantly increased in the DSS group. CE treatment significantly reverted the phosphorylation status toward baseline (Figure 6A-D).

Figure 6 Cedrol inhibited toll-like receptor 4-mediated mitogen-activated protein kinase and nuclear factor kappa B signaling in vivo and in vitro. A-D: Phosphorylated and total protein abundance of mitogen-activated protein kinase signaling molecules [extracellular regulated protein kinase (ERK)1/2, c-Jun N-terminal kinase (JNK), p38] in colonic tissues of mice (n = 6); E: Cellular viability was quantified with the cell counting kit-8 method (n = 3); F-H: Proinflammatory cytokine expression in RAW264.7 macrophages across experimental groups (n = 3); I-K: Inducible nitric oxide synthase and cyclooxygenase-2 protein expression in RAW264.7 cells across groups (n = 3); L-P: Phosphorylated and total protein expression of ERK1/2, JNK, p38 and nuclear factor kappa B p65 in RAW264.7 macrophages (n = 3). Data are expressed as mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. CE: Cedrol; DSS: Dextran sulfate sodium; ERK: Extracellular regulated protein kinase; p-ERK: Phospho-extracellular regulated protein kinase; JNK: C-Jun N-terminal kinase; p-JNK: Phospho-c-Jun N-terminal kinase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TNF: Tumor necrosis factor; IL: Interleukin; mRNA: Messenger RNA; LPS: Lipopolysaccharide; iNOS: Inducible nitric oxide synthase; COX-2: Cyclooxygenase-2.

To further explore the potential role of CE in TLR4/MD2-mediated inflammatory signaling, we conducted in vitro experiments in RAW264.7 cells. The cell counting kit-8 assay confirmed that CE (0-30 μM) exerted no cytotoxicity on RAW264.7 cells (Figure 6E). Based on prior studies[13,19], 20 μM of CE was selected for subsequent experiments. LPS (1 μg/mL, 5 hours; a canonical TLR4 activator) sharply elevated TNF-α, IL-1β, and IL-6 mRNA levels and upregulated iNOS and COX-2 protein expression in RAW264.7 cells. Notably, pretreatment with CE (20 μM) substantially attenuated these inflammatory markers (Figure 6F-K). CE pretreatment markedly suppressed the LPS-induced phosphorylation of MAPK family members (ERK1/2, JNK, p38) and the RelA subunit (NF-κB p65) (Figure 6L-P).

Collectively, these findings demonstrate that CE’s anti-inflammatory effects are mediated through suppression of TLR4/MD2-dependent MAPK and NF-κB pathway activation. Importantly, quantification of TLR4 protein expression revealed no significant differences across experimental groups (Supplementary Figure 1), indicating that CE’s mechanism involves functional inhibition of the TLR4/MD2 complex rather than reduced receptor expression.

CE mitigated gut microbiota imbalance induced by DSS

Cumulative evidence suggests that there is a robust correlation linking intestinal microbiome dysbiosis to UC pathogenesis[24]. To evaluate the modulatory impact of CE on DSS-mediated gut microbiota alterations in murine models, we performed 16S rRNA gene sequencing of intestinal contents. Analyses revealed distinct ASV profile shifts in the DSS and DSS + CE groups vs controls (Figure 7A). α-diversity metrics (Shannon and Simpson indices) revealed significantly lower microbial richness and diversity in the DSS group compared with the control group. Conversely, the DSS + CE group exhibited a significant restoration of microbial diversity (Figure 7B and C). β-diversity analysis (principal coordinates analysis and non-metric multidimensional scaling) clearly discriminated microbial communities among the control group, the DSS group, and the DSS + CE group (Figure 7D and E). Notably, the DSS + CE group showed closer proximity to the control group compared with the DSS group, indicating that CE improves DSS-induced dysbiosis by restoring microbial composition.

Figure 7 Cedrol ameliorated dextran sulfate sodium-induced gut microbiota dysbiosis. A: Venn diagram of amplicon sequence variants for each group; B and C: Alpha diversity analysis (Shannon and Simpson indices); D and E: Beta diversity was displayed via principal coordinates analysis and non-metric multidimensional scaling based on weighted UniFrac algorithm; F: Phylum-level abundance distribution; G: Genus-level relative abundance; H: Differences in bacterial abundance were analyzed at the genus level across various groups (P < 0.05); I: Linear discriminate analysis effect size-derived linear discriminant analysis scores; J: Kyoto Encyclopedia of Genes and Genomes functional prediction heatmap. Data are expressed as mean ± SD (n = 4). CE: Cedrol; DSS: Dextran sulfate sodium; PCoA: Principal co-ordinates analysis; NMDS: Non-metric multidimensional scaling; LDA: Linear discriminate analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes.

Phylum-level analysis revealed a marked reduction in Bacteroidota and Bacillota and elevation of Pseudomonadota in the DSS group. CE administration partially reversed these perturbations (Figure 7F). Genus-level analysis further indicated elevated Escherichia-Shigella, and Akkermansia and reduced Muribaculaceae in the DSS group. CE treatment partially counteracted these shifts (Figure 7G). Specifically, CE significantly increased beneficial genera (Muribaculaceae, Muribaculum, Alistipes, Alloprevotella) while suppressing taxa (Akkermansia, Bacteroides, Escherichia-Shigella) that were increased in the DSS group (P < 0.05) (Figure 7H). Linear discriminant analysis (> 2) identified Christensenella minuta as a key discriminative species in the DSS + CE group (Figure 7I). Finally, KEGG functional prediction suggested the potential involvement of CE in key metabolic pathways (pantothenate/CoA, peptidoglycan, branched-chain amino acid, lysine, and aminoacyl-tRNA biosynthesis) and cellular processes (ribosome and mismatch repair) (Figure 7J).

DISCUSSION

UC is a chronic, recurrent, and cancer-prone inflammatory bowel disease. Current treatment strategies are unsatisfactory, and there is an urgent need to develop safer and more effective therapies. Natural compounds are valuable sources for drug discovery due to their diverse chemical structures and potential therapeutic benefits[1,2,5]. CE is a natural compound that exists in various medicinal herbs and possesses anti-inflammatory and antioxidant properties[8-11]. However, its efficacy and mechanisms in UC have not been thoroughly investigated. This study examined the therapeutic effects and mechanistic basis of CE in experimental models of UC to provide novel insights into future intervention strategies.

We utilized the extensively validated DSS-induced colitis murine model to faithfully recapitulate the hallmark clinical manifestations of UC including body weight decline, hematochezia, higher DAI values, shortened colon length, and histopathological findings (e.g., crypt loss, neutrophil infiltration). Notably, CE administration significantly ameliorated disease phenotypes and concomitantly attenuated histopathological damage and downregulated the production of key proinflammatory cytokines in colonic tissues. This finding was consistent with the anti-inflammatory properties of CE on arthritis[8,9,19].

CE also enhanced the expression of TJ proteins and stimulated mucus production by goblet cells, leading to restored gut barrier function. Unlike corticosteroids, which often exacerbate mucosal fragility and increase relapse rates despite their anti-inflammatory efficacy[25], CE exhibited superior therapeutic potential by concurrently suppressing inflammatory responses and restoring intestinal barrier integrity, resulting in a comprehensive and safe clinical profile. Analogous mechanisms have been documented in other natural products and bioactive constituents of traditional Chinese medicine[26], such as berberine[27,28] and curcumin[29].

To build upon these findings, network pharmacology analysis predicted that the therapeutic efficacy of CE in UC may be mediated through the PI3K-AKT, MAPK, and FoxO signaling pathways while simultaneously identifying 28 core molecular targets including pivotal regulators such as STAT3, NFKB1, and TLR4. The MAPK signaling cascade is a well-established inflammatory pathway implicated in UC pathogenesis. Extensive prior research confirmed that pharmacological inhibition of this cascade effectively ameliorated UC inflammatory responses[30,31]. Our investigation confirmed that CE intervention significantly suppressed phosphorylation of critical MAPK signaling molecules such as ERK1/2, JNK, and p38 within the colonic tissues of the DSS-induced colitis murine model. This observation aligned with established mechanisms documented in a prior CE study investigating rheumatoid arthritis[9]. Collectively, these findings demonstrated that CE mediates its therapeutic efficacy against UC by inhibiting activation of the MAPK signaling pathway.

TLR4 is a critical pattern recognition receptor residing on the cell membrane. It has the capacity to trigger downstream signaling primarily through the MAPK and NF-κB pathways. This signaling cascade potently amplifies the inflammatory response, ultimately leading to damage within the mucosal lining. Consequently, this receptor serves as a critical mediator in the initiation and progression of UC[20,32]. Targeting the TLR4 pathway is a promising therapeutic strategy for UC. Preclinical studies indicate that many plant-derived compounds (phytochemicals) effectively modulate TLR4 signaling. This activity has been observed in diverse experimental systems including animal models of colitis and in vitro cell culture, highlighting the potential to ameliorate intestinal inflammation[33-35].

MD2 is a secreted glycoprotein that associates with the extracellular domain of TLR4. This coreceptor directly binds bacterial LPS and is essential for facilitating the dimerization of TLR4, leading to the activation of downstream inflammatory signaling pathways[36]. Building upon the network pharmacology analysis in this study that identified TLR4 as a core therapeutic target, we subsequently employed molecular docking and molecular dynamics simulations to investigate its potential interaction with CE. Computational analyses revealed that CE exhibited high-affinity binding (docking energy: -6.7 kcal/mol) to MD2. Furthermore, extended molecular dynamics simulations demonstrated stable binding as evidenced by root mean square deviation, radius of gyration, root mean square fluctuation, centroid evolution, and buried solvent accessible surface area. To complement these computational findings, subsequent cell-based experiments demonstrated that CE markedly inhibited LPS-triggered activation of the MAPK and NF-κB pathways. Critically, we confirmed that CE achieved this suppression without altering total TLR4 protein levels, indicating functional inhibition rather than receptor downregulation. The collective evidence supports the hypothesis that CE exerts its anti-inflammatory activity by directly binding to MD2 and subsequently suppressing activation of the downstream MAPK and NF-κB signaling pathways of TLR4.

To investigate the influence of CE on gut microbiota homeostasis, we conducted a detailed assessment of the intestinal microbiota composition. The results demonstrated that the DSS group exhibited a pronounced decline in α-diversity indices and disruptions of the microbial community architecture relative to controls. Specifically, we detected a decline in the abundance of Bacteroidota and Bacillota accompanied by an expansion of the potentially pathogenic phylum Pseudomonadota, particularly Escherichia-Shigella. These dysbiotic changes were consistent with established findings in colitis models[37,38]. CE administration effectively countered these deleterious shifts. Treatment with CE restored gut microbiota α-diversity and richness and promoted a compositional shift towards a more beneficial profile. We observed selective enrichment of putatively beneficial taxa, including Muribaculaceae, Muribaculum, Alistipes, and Alloprevotella, while simultaneously suppressing the proliferation of taxa such as Akkermansia, Bacteroides, and Escherichia-Shigella that were elevated in the pathological state induced by DSS. A particularly intriguing finding emerged from the linear discriminant analysis that highlighted Christensenella minuta as a key discriminant feature significantly associated with the CE treatment group. Christensenella minuta DSM 22607 produces substantial amounts of acetic acid and fair quantities of butyric acid. These microbially derived short-chain fatty acids are known to dampen inflammation via suppression of the NF-κB pathway and to enhance mucosal healing in a mouse colitis model[39]. Consequently, Christensenella minuta is a viable candidate for microbiome-targeted therapeutic strategies in inflammatory bowel disease[40].

A key finding of this study is that CE concurrently targets multiple interconnected components of UC pathophysiology. We propose that CE’s therapeutic efficacy functions through an integrated mechanism wherein its anti-inflammatory action initiates a self-reinforcing cycle that enhances both intestinal barrier integrity and microbial homeostasis. The suppression of TLR4/MD2-mediated inflammation by CE directly reduces mucosal damage while creating a more favorable microenvironment for commensal bacteria. Simultaneously, the restoration of intestinal barrier function prevents bacterial translocation, thereby further mitigating systemic immune activation. The observed microbiota remodeling particularly the enrichment of short-chain fatty acid-producing bacteria such as Christensenella minuta likely contributes to this cycle through metabolites that both suppress NF-kB signaling and enhance epithelial barrier function, establishing a positive feedback loop. This multi-level intervention, simultaneously curbing inflammation, restoring the mucosal barrier, and rebalancing beneficial microbial communities, explains CE’s marked efficacy against colitis and represents a comprehensive therapeutic strategy superior to single-target approaches. Although correlation analyses between inflammatory markers, barrier proteins, and specific bacterial taxa could further clarify these relationships, the current sample size for microbiota analysis (n = 4 per group) may limit the statistical reliability of such correlations. Future studies with larger cohorts will help in characterizing these relationships quantitatively and in validating the proposed interaction network.

Our study provided the first evidence that CE exerts notable therapeutic effects against DSS-induced colitis through a coordinated multi-mechanistic action, including anti-inflammatory activity via functional suppression of the TLR4/MD2-MAPK/NF-κB axis, restoration of intestinal barrier integrity, and amelioration of gut microbiota dysbiosis. Beyond the current findings, several important questions remain for future investigation. While our data strongly suggest TLR4/MD2 as a functional target, definitive confirmation will require more direct interventions, such as TLR4-neutralizing antibodies, TLR4-knockout models, or TLR4-overexpression systems. Furthermore, given the central role of immune dysregulation in UC, the impact of CE on specific immune cell populations represents a critical avenue for future research. Subsequent studies should systematically evaluate the compound’s influence on the functional polarization of macrophages (e.g., M1/M2), the differentiation of T helper (Th) cell subsets (e.g., Th1, Th2, Th17, and regulatory T cell), and the activity of dendritic cells. Elucidating these immunomodulatory effects will be essential to efforts that will fully uncover the mechanism by which CE restores immune homeostasis in the colonic microenvironment.

These robust preclinical outcomes position CE as a promising translational candidate for UC. However, it is important to acknowledge that the current investigation employed a single dosage without a comprehensive dose-response assessment. Future studies should include detailed pharmacokinetic and pharmacodynamic analyses, chronic toxicity evaluation, and validation in clinically relevant animal models to confirm the efficacy and safety profile of CE prior to advancing to clinical translation.

CONCLUSION

CE ameliorated DSS-induced colitis through three complementary mechanisms: (1) Attenuating inflammation via TLR4/MD2-mediated suppression of MAPK and NF-κB signaling; (2) Restoring intestinal barrier function; and (3) Modulating the gut microbiota (Figure 8). These multifaceted actions demonstrated that CE is a promising natural therapeutic candidate for UC.

Figure 8 Cedrol treated dextran sulfate sodium-induced colitis by inhibiting inflammation, restoring the intestinal barrier, and rebalancing gut microbiota. TNF: Tumor necrosis factor; IL: Interleukin; DSS: Dextran sulfate sodium; LPS: Lipopolysaccharide; MD2: Myeloid differentiation factor 2; TLR4: Toll-like receptor 4; MAPK: Mitogen-activated protein kinase; ERK: Extracellular regulated protein kinase; JNK: C-Jun N-terminal kinase; NF-κB: Nuclear factor kappa B; iNOS: Inducible nitric oxide synthase; COX-2: Cyclooxygenase-2.