AKBA was isolated from the resin seeping (Olibanum) from the bark of Boswellia carterii Birdw. The olibanum medicinal materials were from Grand Bazaar in Urumqi City, Xinjiang, China, and were identified as olibanum from Boswellia carteri Birdw by Prof. Tengfei Ji. Purified AKBA was a colorless and crystalline compound, the molecular formula was C₃₂H₄₈O₅ and the formula weight was 512.4, purity was greater than 98%, the HR-ESI-MS spectrum (Fig. S1), ¹H NMR (Fig. S2) and ¹³C NMR (Fig. S3) spectrum were provided by Prof. Tengfei Ji. AKBA was dissolved in Dimethyl sulfoxide (DMSO, MP Biomedicals Co., Ltd., 196055, Solon, OH, USA) at 100 mM as a stock solution, and stored at −20°C for use in cells. The final concentration of DMSO in cell culture was no more than 0.1%. For the animal experiment, AKBA was suspended with 0.5% carboxymethylcellulose (CMC-Na, Solarbio Life Science Co., Ltd., C8620, Beijing, China) at 9 mg/mL as stock solution.

Human colorectal carcinoma HCT116, SW620 cell lines, RAW 264.7 cells (mouse leukemia cells of monocyte-macrophage) were obtained from the Procell (Pricella Life Science & Technology Co., Ltd., Wuhan, China). Cells were cultured in RPMI-1640 medium (Pricella Life Science & Technology, PM150110) containing 10% fetal bovine serum (FBS, v/v, Pricella Life Science & Technology, 164210-50) and 1% penicillin/streptomycin (Solarbio, P1400). Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂ in a CO₂ incubator (Hera cell Vios 160I, Thero Scientific, Munich, Germany). Cells within 10 passages and tested negative for mycoplasma contamination (Myco-Lumi^TM Detection kit for mycoplasma by luminescence method, Beyotime Biotechnology Co., Ltd., C0298S, Shanghai, China) were used for the experiments.

Cell proliferation was checked by the CCK-8 assay after AKBA treatment. Cells were seeded in 96-well plates at 3 × 10³ cells/well (Beaver Biomedical Engineering Co., Ltd., Suzhou, China) and treated with 0–100 µM AKBA for 24, 48, and 72 h. The CCK-8 assay was done according to user manual.

Cell-Light EdU Apollo 567 In Vitro kit (RIBOBIO Co. Ltd., C10310-1, Guangzhou, China) was used to check DNA synthesis activity in HCT116 and SW620 cells treated with 0, 20, 30, and 40 µM AKBA for 24 h. Briefly, 50 μM 5-ethynyl-2^′-deoxyuridine (EdU) solution was added and incubated for 2 h, followed by fixation with 4% paraformaldehyde and permeabilization by 0.5% Triton X-100. Apollo stain reaction solution was used to stain cells. Cells were counter-stained by Hoechst 33342. EdU-positive cells were imaged and quantified. Data were obtained in triplicate.

The plate cloning assay was done to assess the growth ability in 2D dimension of HCT116 and SW620 cells treated with 0, 20, 30, and 40 μM AKBA. Cells were seeded at 500 cells/well in 6-well plates and incubated for 24 h before AKBA treatment. After 14 days, cells were fixed with 4% paraformaldehyde (Solarbio, P1110) and stained with 0.1% crystal violet solution (Solarbio, G1063). The experiment was performed in triplicate.

Cells (2 × 10⁶) were seeded in 6 cm dishes, treated with 20, 30, and 40 μM AKBA for 24 h. Cells were harvested, fixed in 70% ethanol at −4°C for 12 h, then stained with RNase A (20 µg/mL) and Propidium iodide PI (50 μg/mL). Fluorescence was analyzed using a C6 flow cytometer at 488 nm, with data processed using FlowJo 10.8.1 software.

Cells (2 × 10⁶) were planted into 6 cm dishes and treated with 20, 30, and 40 μM AKBA for 24 h. The detailed experimental methods were described previously [21].

To explore the anti-tumor mechanism of AKBA, differentially expressed genes (DEGs) were detected via mRNA transcriptome sequencing in HCT116 and SW620 cells that had been exposed 30 µM AKBA for 48 h. The detailed methods and procedures were described previously [21]. DEGs were screened using the following criteria |log₂(Fold Change)| ≥ 3 and adjusted p value ≤ 0.05.

Cells were plated at a density of 3 × 10⁵ cells per well and allowed to incubate for 24 h. They were then stimulated with 1 µg/mL LPS (Solarbio, L8880) for 1 h and subsequently treated with 20, 30, and 40 µM AKBA for 24 h. The RNA extraction, reverse transcription, PCR, and quantitative methods were described previously [22]. The primer sequences for important cytokines (IFN-γ, IL-1β, IL-6, TNF-α, and IL-10) are listed in Table S1.

RAW 264.7 cells were seeded at 3 × 10⁵ cells/well in 6-well plates, incubated for 24 h, stimulated with 1 µg/mL LPS for 1 h, and treated with 20, 30, and 40 µM AKBA for 24 and 48 h. Supernatants were collected, centrifuged, and analyzed using ELISA kits to measure cytokine concentrations IL-1β (the mouse IL-1β ELISA kit, Biocreative, Shenzhen, China), IL-6 (4A biotech, Suzhou, China) and TNF-α (4A Biotech, Suzhou, China).

Cells were lysed with RIPA buffer (Beijing applygen Co., Ltd., C1053, Beijing, China) containing protease (Applygen, P1265) and phosphatase inhibitors (Applygen, P1260). Protein concentration was determined using a BCA kit (Beyotime, P0009, Shanghai, China). A total of 20–50 µg of protein was separated using 10% SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in TBS-T and incubated with primary antibodies overnight at 4°C. Following washing, the membrane was treated with secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were detected using a BeyoECL Plus kit (Beyotime, P0018M) and imaged with a chemiluminescence system (Shanghai Tanon Technology Co., Ltd., 5200, Shanghai, China). Details of the primary antibodies can be found in Table S2. BMS345541 (MCE, HY-10519) was utilized as an IKK inhibitor.

BALB/c mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice, approximately seven weeks old and weighing between 18 to 20 g, were used in this study. The breeding conditions of the mice were described previously [10], and the ethical considerations are detailed in the Ethics Approval section. The number of animal experimental ethical inspections is No. 00005266.

The body weight of BALB/c male mice was 18–20 g and acclimated for 3 days before the experiment then the 30 mice were randomized into the following groups, normal, LPS (5 mg/kg) model, dexamethasone (DXM, 5 mg/kg, Solarbio, D6950) as positive control group, and AKBA groups (30 and 90 mg/kg). Each group was continuously gavaged administration for 6 d. The control group and model group were given equal volumes of 0.05% CMC-Na (Solarbio, IS9000), 0.1 mL/10 g. Weight changes of mice were recorded daily. On day 7th, mice received LPS (5 mg/kg) to prepare the acute inflammation model except for the normal control group, and the volume of intraperitoneal injection was 0.1 mL/10 g. After 6 h, blood and spleen were collected.

The AOM/DSS-induced colitis-associated CRC model is currently the most widely used model for the chemical induction of CRC [23]. 50 male BALB/c mice were adaptively fed for 5 days. Azoxymethane (AOM, Sigma Aldrich, A5486, Milwaukee, WI, USA) was administered to 40 mice by subcutaneous injection. From the second week, mice were given water with 2% Dextran sulfate sodium (DSS, molecular weight 36,000–50,000, MP Biochemicals, 0210151680, Santa Ana, CA, USA) every two weeks for 3 times. Mice were then divided into four groups (n = 10): AOM/DSS model, AOM/DSS + AKBA (30 mg/kg), AOM/DSS + AKBA (90 mg/kg) groups and 5-Fluorouracil (5-Fu, 20 mg/kg, Solarbio, IF0170) groups. For the control group and AOM/DSS model group, mice received sterile distilled water drinking. For the AOM/DSS + AKBA groups, mice were received the corresponding dose of AKBA by intragastric administration for 7 weeks. The 5-Fu group received intraperitoneal 5-Fu injections weekly. The mice’s weight was measured every week, and mouse activity status, fecal traits, as well as hematochezia were monitored and measured daily, with mice being sacrificed directly on the last day without drug administration. At the end of the experiment, all the mice were euthanized and the tumor number of colon, as well as the tumor volume (mm³, major diameter × minor diameter²/2) were recorded. The colon tissues were subsequently collected for further study.

Colon samples from mice were fixed in 4% paraformaldehyde, embedded in paraffin wax, and cut into 4 μm transverse sections, then stained with H & E as described in previous research [24]. Images were taken with a light microscope (Ti-U Nikon eclipse, Tokyo, Japan).

Inflammation factors were significantly associated with colon cancer [25]. The mice were anesthetized with sodium pentobarbital, and blood was collected from the fundus venous plexus, centrifuged at 1000 rpm using a centrifuge (Allegra X-22R, Beckman coulter, Brea, CA, USA) for 10 min to obtain the serum. spleens were homogenized in PBS (1:10) and centrifuged at 2000 rpm for 10 min. Supernatant protein concentration was measured by BCA assay, and the samples were tested by V-PLEX Proinflammatory Panel 1 Mouse Kit (Univ Life Science and Technology Co., Ltd., No. K15048D-1, Shanghai, China) using MSD hypersensitive factor electrochemical luminescence analyzer (Meso scale disc., SQ120, Rockville, MD, USA).

The colonic tissue was fixed with 4% formaldehyde (Servicebio Life Science and Technology Co., Ltd., G1101—500 mL, Wuhan, China) embedded in paraffin, and sectioned. Slides were deparaffinized, rehydrated, and washed in PBS. Antigen retrieval was done with citrate buffer (Solarbio, G2700) at 100°C for 10 min slides were treated with 3% H₂O₂ to block endogenous peroxidase and blocked with 5% BSA (Solarbio, A8020) for 30 min. Primary anti-Ki67 antibody (1:500, Abcam, ab15580) was applied overnight, followed by 1 h incubation with biotinylated Goat Anti-Rabbit IgG (1:200, Cell Signaling Technology, 35401). Staining was completed with diaminobenzidine (DAB Substrate kit, Solarbio, SA1010), and hematoxylin (Solarbio, G1120). A microscope (Ti-U Nikon Eclipse, Tokyo, Japan) was used to take photographs of stained sections.

The profiling and analysis of gut microbiota in fecal samples from CRC mice, both treated and untreated with AKBA, were conducted using metagenomic sequencing and analysis methods as previously described [26,27]. The alpha and beta diversity were calculated in Qiimeplatform (http://qiime.org/scripts/assign_taxonomy.html (accessed on 10 January 2025) and RDP Classifier (version 2.11, http://sourceforge.net/projects/rdp-classifier/ (accessed on 10 January 2025). Community differences were analyzed using permutational multivariate analyses of variance (PERMANOVA) with 1000 iterations employing the Arrhenius z distance [28].

Data are expressed as means ± SEM (the standard error of the mean), obtained from three separate experiments. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was utilized for creating graphs and conducting statistical analyses. For comparison between the two groups, unpaired Student’s t-tests were used. For multiple comparisons, one-way ANOVA followed by Bonferroni post-hoc tests was conducted. A p-value of less than 0.05 was considered statistically significant.

To check if AKBA has an inhibitory effect on the growth of CRC, CCK-8 assay was carried out. The Boswellia carterii Birdw, resin seeping from the bark, and the chemical structure of AKBA was depicted in Fig. 1A. Our results revealed that AKBA significantly reduced the growth of HCT116 and SW620 cells in a time- and dose-dependent manner. The IC₅₀ values for AKBA in HCT116 cells at 24, 48, and 72 h were 41.86, 20.2, and 15.02 μM; and the values for SW620 were 74.2, 57.3, and 39.8 μM at 24, 48, and 72 h, respectively (Fig. 1B).

Therefore, AKBA at concentrations of 20, 30, and 40 μM were used to further investigate its anti-CRC effects and the underlying mechanisms. The morphology with HCT116 and SW620 cells was changed after treated by AKBA for 24 and 48 h (Fig. 1C). To check the inhibitory effects of AKBA on cell proliferation, an EdU-DNA synthesis assay was also done. After 24 h of treatment with AKBA, a dose-dependent decrease in the proliferation of HCT116 and SW620 cells was observed (Fig. 1D). Furthermore, the rate of clone formation of HCT 116 and SW620 were significantly decreased as concentrations of AKBA was increased (Fig. 1E).

To check whether AKBA affects the cell cycle and apoptosis of HCT116 and SW620 cells, Flow cytometry was used. Results indicated that AKBA treatment arrested the cell cycle at the G2/M phase (Fig. 1F) and increased apoptosis rates in a dose-dependent manner (Fig. 1G). Taken together, these findings suggest that AKBA exerts a growth-inhibitory effect on CRC cells.

To further explore the anti-tumor mechanism of AKBA, an RNA-seq assay was carried out. Compared to control cells, |log₂(Fold Change)| ≥ 3 and adjust p value ≤ 0.05 (Fig. 2A), a total of 2483 differential expression genes altered in both HCT116 and SW620 cells (Fig. 2B). Gene ontology enrichment analyses showed that the enriched biological processes involved cell cycle checkpoint, DNA replication, protein targeting to ER, sister chromatid segregation and G1/S phase transition. The identified cellular components were associated with a structural constituent of ribosome, glucosyltransferase activity, single-stranded DNA-dependent ATPase activity, protein phosphatase 1 binding, and histone kinase activity. Molecular functions included cytosolic ribosome, condensed chromosome, replication fork, centrosome, nuclear chromosome part, and condensed nuclear chromosome (Fig. 2C). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed significant changes in several biological processes including cellular senescence, cell cycle, DNA replication, NF-κB pathway, p53 signaling pathway, apoptosis and IL-17 pathway (Fig. 2D). A complete list of top 20 KEGG pathways related to cell growth, inflammation and tumors were listed in Table S3. The identified enriched KEGG pathways, including p53 signaling pathway, IL-17 signaling pathway, MAPK signaling pathway and NF-κB pathway, have been implicated in inflammation and tumor progression of CRC. Notably, the NF-κB signaling pathway is closely associated with both tumor development and inflammatory processes [29]. Additionally, cytokines are also associated with the NF-κB signaling pathway, thereby contributing to inflammation and cancer. Hut genes of the NF-κB pathway and IL-17 pathway were illustrated in Fig. 2E. All in all, these results indicated that the mechanism of AKBA was closely associated with the NF-κB pathway.

It was well known that alteration in the expression of various inflammatory factors was related to the NF-κB pathway during colon cancer. To confirm the effects of AKBA on inflammatory factors and the NF-κB pathway, ELISA analysis was carried out to check the concentrations of cytokines in the cell supernatant, as well as Western blot was checked the total and phosphorylated (p) levels of NF-κB, NF-κB, IκB, and IKK in HCT116 and SW620 cells treated by AKBA. Results from ELISA analysis showed that AKBA significantly reduced the levels of TNF-α and IL-1β compared to the untreated control group at 48 h (Fig. 3A). Furthermore, results from Western blot showed that the protein expression of NF-κB, p-NF-κB and IKK were obviously decreased whereas the expression of IκB was increased after treatment of AKBA (Fig. 3B). This means that AKBA can inhibit the activation of the NF-κB pathway. To further confirm that the NF-κB pathway was involved in the effect of AKBA on the CRC. BMS345541 (an IKK inhibitor) was also used to treat HCT116 and SW620 cells. It was showed in Fig. 3C that BMS345541 reduced the expression of IKK, NF-κB, and p-NF-κB whereas expression of IκB was increased in cells treated by BMS345541, which is similar to AKBA. Collectively, these findings suggest that AKBA exerts its inhibitory effects on CRC by regulating NF-κB pathway.

To further investigate the effects of AKBA on the inflammatory factors both in cellular and animal models of inflammation, we measured the levels of inflammatory factors in RAW 264.7 cells, as well as in the serum and spleen of the mice. In the LPS-induced RAW 264.7 cell model, LPS treatment significantly increased the mRNA levels of IL-6, IL-1β, IFN-γ, TNF-α and IL-10. AKBA treatment, however, reduced the mRNA levels of IL-6, IL-1β, IFN-γ, and TNF-α, while increasing the level of IL-10 compared to the LPS-treated group (Fig. 4A). In addition, results from the ELISA assay showed that AKBA decreased the levels of NO, IL-1β, IL-6 and TNF-α in LPS-induced RAW264.7 cells (Fig. 4B). Next, we used LPS-induced inflammation mice model to further confirm the effects of AKBA on cytokines. A schematic diagram of mice treatment is illustrated in Fig. 4C. The body weight of the DXM group decreased following DXM administration, with significant weight loss observed on days 3,4,5, and 6 compared to the normal group. In contrast, AKBA had no effect on the body weight of the mice (Fig. 4D). The treatment of LPS led to a notable increase of inflammatory factors, such as IL-1β, IFN-γ, IL-12p70 and TNF-α both in serum and spleen compared to the control group. After treatment of AKBA, the levels of IL-1β, IFN-γ, IL-12p70 and TNF-α were decreased both in serum and spleen (Fig. 4E,F). AKBA also decreased the level of IL-5 in serum (Fig. S4A), as well as IL-2 and IL-6 in the spleen (Fig. S4B), which indicated that AKBA alleviated systemic inflammation. These findings indicate that AKBA possesses significant anti-inflammatory properties, as shown in both in vitro and in vivo models.

Previous study had indicated that the NF-κB pathway is significantly activated in DSS-induced inflammatory bowel disease [12]. To check whether AKBA suppressed the tumorigenicity of CRC in vivo. A CRC model induced by AOM/DSS in BalB/c mice was established, and the mice were treated with AKBA at doses of 50 and 100 mg/kg daily for a duration of 7 weeks (Fig. 5A). During the 7 weeks’ administration of AKBA, no abnormal behavior or obvious weight loss was observed (Fig. 5B). The representative images of colon tumors from normal control, vehicle and AKBA treatment group were shown in Fig. 5C. Oral administration of AKBA significantly reduced the number (Fig. 5D) and the volume of tumors (Fig. 5E). HE staining of colon tissue showed that AKBA significantly alleviated the pathological changes of colon cancer tissue in mice (Fig. 5F). Immunohistochemistry of intestinal tissue showed that the expression of Ki67 was reduced in the in AKBA-treated group compared to the vehicle group (Fig. 5G). The index of the thymus, spleen, and colon wasn’t significantly changed after treatment of AKBA compared to the normal group (Fig. S5A–C). Together, all these results showed that AKBA suppressed the tumorigenicity and ameliorated AOM/DSS-induced colonic pathological changes in mice.

The role of microbial communities in human pathophysiology, especially in oncogenesis, has become a focal point of contemporary biomedical research. Gastrointestinal cancers demonstrate particularly significant interactions with enteric microbiota, a relationship attributed to their anatomical adjacency within the digestive ecosystem. This unique biological positioning has rendered gut microbiome-oncology correlations a priority subject in current translational research, with numerous studies elucidating microbial contributions to carcinogenesis, tumor progression, and therapeutic responses [30]. To determine whether AKBA influences the gut microbiota in mice with CRC, the intestinal flora of the mice was analyzed by the 16S rDNA pyrosequencing. After the amplification of 338F_806R, a total of 930653472 (bp) bases were detected, optimized for 649635838 OTUs, with an average length of 420.22. A total of 575 species were identified. When compared with the control group, significant differences were observed in the Shanno index among the control, model, and AKBA groups (Fig. 6A and Table S4). Additionally, differences among the groups were also significant in the PCoA analysis (Fig. 6B). The 92 differential species were shared among the 3 groups (Fig. 6C). There was no significant difference in dominant species at the phylum level (Fig. 6D). At the family level, the dominant species included Muribaculaceae, Lachnospiraceae, Marinifilaceae, and Rikenellaceae, Bacteroidaceae (Fig. 6E). The top 20 species with the most pronounced changes at both the phylum and species level were shown in Fig. 6F,G. LEfSe multi-level species difference discriminant analysis (which includes various levels including phyla, class, genus, family, order and species) was conducted to examine the hierarchical differences among species. The LDA value was used to measure the differential effect of species, suggesting that the species may play an important role in the process of environmental change. This analysis can also be used as one of the ways to find the biomarker between the control group and the treatment groups (Fig. 6H,I). The intestinal microflora consists of directly carcinogenic microflora, bacterial toxins (e.g., colibactin, BFT), passenger bacteria (opportunistic microorganisms), Fungi (Penicillium spp., Aspergillus spp., and Fusarium spp.). Changes in the microbiota can lead to dysbiosis and the development of CRC [31]. Compared with model group, Clostridium sp. KNHs209, Bacteroides ovatus [32], Bacteroides sp. CAG:20, Parabacteroides merdae, Paraprevotella clara, Rikenella microfusus, Bacteroides sp. CAG:702 were increased. Whereas Barnesiella viscericola [33], Bacteroides oleiciplenus, Coprobacter secundus [34], Bacteroides sp. CAG:633, Bacteroides sp. CAG:462, Bacteroides salyersiae, Bacteroides salanitronis were decreased after treatment of AKBA (Fig. 6J). All these results showed that AKBA can restore the intestinal microbiota by increasing probiotics and reducing harmful bacteria.

Based on the above findings, we have proposed a mechanistic scheme, shown in Fig. 7, demonstrating that AKBA exerts an anti-cancer effect on the colon by modulating inflammation and the gut microbiota. AKBA inhibits the production of pro-inflammatory cytokines, such as IL-1β and TNF-α in macrophages, thus reducing systemic inflammation. AKBA also inhibits tumor cells by reducing the NF-κB signaling pathway, which reduces TNF-α production and prevents further inflammation. In addition, AKBA improves microbial diversity and increases beneficial gut bacteria that further support anti-inflammatory and anti-tumor activities. Taken together, these effects help AKBA to suppress the progression of colon cancer.