Berberine's Role in Modulating Gut Microbiota and Endocannabinoid System in Colitis‐Associated Colorectal Cancer

ABSTRACT

Colorectal cancer (CRC) linked to colitis is a major cause of cancer‐related deaths, highlighting the need for effective treatment options. This study aimed to investigate the effects of berberine (BRR) on gut microbiota and the endocannabinoid system (ECS) in a mouse model of colitis‐associated CRC, which was induced using azoxymethane (AOM) and dextran sulfate sodium (DSS). Through a combination of animal experiments, microbial sequencing, and biochemical assays, we discovered that BRR significantly inhibited tumor development. This was demonstrated by a dose‐dependent increase in body weight and a notable decrease in both the number and size of tumors. Histopathological examinations showed a reduction in aberrant crypt foci and inflammation. Additionally, BRR treatment led to lower levels of pro‐inflammatory cytokines, including TNF‐α, IL‐1β, and IL‐6, while increasing the levels of the anti‐inflammatory cytokine IL‐10, indicating a modulation of immune responses. Importantly, BRR altered gut microbial communities by promoting beneficial bacteria such as Akkermansia muciniphila, Bacteroides, Lachnoclostridium, Blautia, and Prevotellaceae_UCG‐001, while reducing harmful species. We also observed improvements in intestinal barrier integrity, characterized by decreased permeability and lower levels of plasma lipopolysaccharides. Furthermore, BRR restored the expression of cannabinoid receptors CB2 and GPR55, suggesting that the ECS plays a role in mediating its effects. In summary, BRR shows significant promise in alleviating colitis‐associated CRC through various mechanisms, including the modulation of gut microbiota, regulation of immune responses, and enhancement of intestinal barrier function. Future studies should aim to validate these findings clinically and further explore the efficacy of BRR in the prevention and treatment of CRC.

Berberine (BBR) mitigates colorectal cancer (CRC) by modulating the endocannabinoid system (ECS) and reshaping the gut microbiota. BBR treatment increases beneficial bacteria (e.g., Akkermansia muciniphila, Bacteroides, Blautia) while reducing pathogenic species, thereby enhancing gut barrier integrity and lowering metabolic endotoxemia. Concurrently, BBR alters ECS signaling, particularly the expression of CB2 and GPR55 in colonic tissues, leading to reduced intestinal inflammation and protection against CRC progression.

Abbreviations

2‐AG

2‐arachidonoylglycerol

A. muciniphila

Akkermansia muciniphila

AM630

6‐iodopravadoline

AOM

azoxymethane

AraS

N‐arachidonoyl‐L‐serine

ASVs

amplicon sequence variants

CB2

cannabinoid receptor 2

CRC

colorectal cancer

DAGL

diacylglycerol lipase

DSS

dextran sulfate sodium

ECS

endocannabinoid system

FAAH

fatty acid amide hydrolase

FAK

focal adhesion kinase

FDR

false‐discovery rates

FISH

fluorescence in situ hybridization

H&E

hematoxylin and eosin

IRAK4

IL‐1R‐associated kinase 4

KEGG

Kyoto Encyclopedia of Genes and Genomes

LDA

linear discriminant analysis

LEfSe

linear discriminant analysis effect size

LPS

lipopolysaccharide

MAGL

monoacylglycerol lipase

MyD88

myeloid differentiation primary response 88

NAPE‐PLD

N‐acylphosphatidyl‐ethanolamine‐specific phospholipase D

PCoA

principal co‐ordinates analysis

PICRUSt

Phylogenetic Investigation of Communities by Reconstruction of Unobserved States

qRT‐PCR

quantitative real‐time polymerase chain reaction

RDA

redundancy analysis

SCFA

short‐chain fatty acid

TEM

transmission electron microscopy

TLR4

toll‐like receptor‐4

ZO1

zonula occludens‐1

1. Introduction

Colorectal cancer (CRC) is a significant global health concern, consistently ranking as one of the leading causes of cancer‐related deaths worldwide [1]. Recent epidemiological data show an increasing incidence of CRC, especially in areas undergoing dietary and lifestyle changes [1, 2]. The relationship between chronic inflammation and cancer development is well‐established, with conditions like colitis recognized as risk factors for CRC [3]. This underscores the urgent need for effective preventive measures and treatment strategies that focus on the inflammatory pathways involved in the development of cancer.

Current research highlights the crucial role of gut microbiota in influencing the risk of CRC. Dysbiosis, which refers to an imbalance between beneficial and harmful microorganisms in the gut, has been associated with increased inflammatory responses and the promotion of tumor formation [4, 5, 6]. Additionally, new studies indicate that certain dietary compounds, like berberine (BRR), may alter the composition of gut microbiota and provide anti‐inflammatory benefits, potentially reducing the risk of CRC [7, 8, 9]. However, the exact mechanisms by which BRR interacts with gut microbiota and its potential implications for CRC prevention remain inadequately explored.

The therapeutic potential of BRR in CRC is particularly promising because of its wide range of biological activities, including anti‐inflammatory, antioxidant, and antimicrobial effects [9]. Previous research has shown that BRR can affect various signaling pathways related to inflammation and immune responses; however, the exact molecular mechanisms that explain its protective effects against colitis‐associated CRC need further exploration [8, 10, 11, 12]. This gap is particularly relevant given the emerging recognition of the endocannabinoid system (ECS) as a master regulatory network that modulates multiple inflammatory and oncogenic pathways (e.g., Wnt/β‐catenin, MAPK) implicated in CRC [13, 14, 15]. This study aims to fill this knowledge gap by examining how BRR impacts both gut microbiota and the ECS—a key upstream modulator that integrates gut homeostasis, inflammation, and tumorigenic signaling—in the context of CRC.

Our research methodology combines in vivo animal models, microbiome analysis, and molecular techniques to evaluate the protective effects of BRR on CRC. By employing a comprehensive approach that examines physiological changes, microbial community profiles, and gene expression alterations, we aim to enhance our understanding of how BRR may affect tumor development and inflammation within the intestinal environment [16]. Specifically, this study seeks to investigate the impact of BRR on tumor progression in a CRC mouse model, characterize changes in gut microbiota, and explore the role of the ECS in mediating these effects.

To sum up, this study aims to shed light on the biological mechanisms that connect CRC with colitis, while also investigating the potential therapeutic benefits of BRR in this area. By specifically investigating the ECS as a potential mechanistic bridge between BRR‐induced microbiota changes and downstream anti‐tumor effects, we aspire to open avenues for innovative dietary interventions and treatment strategies that leverage BRR to address CRC and its related inflammatory conditions. The findings from this research could significantly deepen our understanding of how CRC develops and may also guide future clinical applications of BRR in the prevention and treatment of cancer.

2. Material and Methods

2.1. Reagents

BRR (PubChem CID: 2353) with a purity level of 98.38% was obtained from Must Biotechnology Co., located in Chengdu, China. Additionally, 4000‐Da FITC‐dextran (catalog number 46944), as well as ampicillin (catalog number 171254), metronidazole (catalog number M3761), neomycin (catalog number N6386), and vancomycin (catalog number V2002) were acquired from Sigma‐Aldrich. Furthermore, 6‐iodopravadoline (AM630, Item No. 10006974) and Abn‐CBDO (O‐1602, Item No. 10006803) were sourced from Cayman Chemical, based in Michigan, USA.

2.2. Animal Experiment

The experimental design is depicted in Figure 1A. Male BALB/c mice, aged 8 weeks and weighing over 20 g, were obtained from the Model Animal Research Center at Nanjing University in Nanjing, China. These mice were housed with unlimited access to sterile food and water, maintained under a controlled environment with a 12‐h light/dark cycle, and kept at a temperature of 22 ± 2°C and humidity of 55 ± 5%. All animal experiments adhered strictly to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Chongqing University Cancer Hospital (CZLS2022022‐A).

FIGURE 1.

The impact of berberine on colitis‐associated colorectal tumorigenesis. (A) A schematic representation of the experimental design involving animal subjects (n = 6 per group). (B) Observations of body weight changes in CRC mice following berberine treatment. (C–F) Berberine demonstrated a significant inhibitory effect on tumor growth in AOM/DSS‐treated mice, which includes (C) representative images of the colons from CRC mice; (D) measurement of colorectal length; (E) visual representations of colorectal tissue; and (F) quantification of tumor polyp numbers. (G) Representative images of colon sections from mice stained with H&E. Scale bar: 100 µm, ×100 magnification. (H) Histological scoring results. Data are presented as mean ± SD derived from three independent experiments. In panel (A), statistical significance is indicated as *p < 0.05 for CRC compared to control; ^# p < 0.05 for HBRR+CRC versus CRC. For panels (D–H), significance levels are denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ns, indicating no significance, when comparing the indicated groups. All statistical comparisons among multiple groups were performed using one‐way ANOVA followed by Bonferroni's post hoc test.

Following a 1‐week acclimatization to the laboratory conditions, the mice were randomly assigned to four distinct groups: (1) control group; (2) CRC group, in which CRC was induced using azoxymethane (AOM)/dextran sulfate sodium (DSS); (3) low‐dose BRR group (LBRR) group, which was induced by AOM/DSS and received an intragastric administration of 50 mg/kg body weight of BRR daily; and (4) high‐dose BRR group (HBRR) group, which was induced by AOM/DSS and was administered 100 mg/kg body weight of BRR daily via the same route. The CRC model was established as previously described [17, 18]. Body weight measurements were taken weekly, and fecal samples were collected at both the beginning and end of the treatment. After a fasting period of 6 h, gut permeability tests were performed on the mice. Following euthanasia, blood and distal colon samples were collected for further biomedical analysis. The study focused on several key areas: bacterial translocation, the expression levels of mRNA related to tight junction‐associated genes, intestinal permeability (which was evaluated using 4000‐Da FITC‐dextran, Item No.: 46944; Sigma‐Aldrich, 600 mg/kg body weight), and plasma LPS concentrations (obtained from GenScript, Nanjing, China). All investigations were conducted following established protocols to ensure accuracy and reliability.

In the experiment aimed at evaluating the depletion of gut microbiota, male BALB/c mice were given BRR at a dosage of 100 mg/kg body weight daily for a period of 10 weeks. After this treatment phase, the mice were administered a broad‐spectrum antibiotic cocktail mixed in water. Prior to the study, the mice underwent a 1‐week acclimatization period, after which they were randomly divided into four experimental groups, with each group consisting of six mice: the CRC group, the CRC + antibiotic (CRC+Abx) group, the HBRR+CRC group, and the HBRR + CRC+Abx group. All subjects were treated with AOM and DSS in accordance with established protocols. Mice in the CRC+Abx and HBRR + CRC+Abx groups were provided with drinking water containing the antibiotic cocktail to facilitate gut microbiota depletion for the initial 2 weeks prior to the experiment, with additional treatments occurring every 2 weeks throughout the study, as per previous reports. Mice in the HBRR+CRC and HBRR + CRC+Abx groups received BBR (100 mg/kg) via oral gavage once daily for 10 weeks, while those in the CRC and CRC+Abx groups were given drinking water (refer to Figure 5A for details).

FIGURE 5.

Berberine enhances the integrity of the intestinal barrier and mitigates gut inflammation linked to the intestinal microbiome. (A) Schematic representation of the antibiotic intervention experiment conducted on AOM/DSS‐treated mice. (B) Exemplary images of colorectal tissues. (C) Analysis of colorectal length and (D) quantification of tumor polyp numbers. (E) Illustrative images of colon sections from mice stained with H&E. Scale bar: 100 µm, at ×100 magnification. (F) Histological scoring results. Data are expressed as mean ± SD derived from three independent experiments. The measurements for (G) intestinal permeability, (H) plasma levels of LPS, and (I) total bacterial DNA load (universal 16S rRNA gene copies) in whole blood samples were conducted. The relative mRNA expression levels of occludin (J), ZO1 (K), claudin 1 (L), TNF‐α (M), IL‐1β (N), IL‐6 (O), and IL‐10 (P) in distal colon tissues were evaluated using the qRT‐PCR assay. Additionally, the mRNA expression levels of CB2 (Q) and GRP55 (R) in distal colon tissues were determined through qRT‐PCR. Results are presented as mean ± SEM. Significance levels are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. Multiple group comparisons were performed using one‐way ANOVA followed by Bonferroni's post hoc test.

To explore the in vivo functionality of the ECS, mice were administered BRR at a dosage of 100 mg/kg of body weight daily for a duration of 10 weeks. Following this treatment, the mice received either the CB2 antagonist AM630 at a dose of 1 mg/kg per day, the GPR55 agonist O‐1602 at the same dosage, or a control vehicle that consisted of an equal volume of DMSO, cremophor, and saline. This administration was conducted intraperitoneally over an additional 4‐week period.

2.3. Transmission Electron Microscopy (TEM)

In the case of TEM, the tissue samples were initially preserved using a fixative solution composed of 2.5% glutaraldehyde and 4% paraformaldehyde (PFA) in a 100 mM sodium cacodylate buffer maintained at pH 7.4. Following fixation, the samples underwent a dehydration process and were subsequently embedded in Spurr resin before being sliced into sections. The resultant images were captured utilizing an FEI F30 transmission electron microscope (Tecnai).

2.4. Histological Evaluation

Sections of the distal colon from a comparable longitudinal segment were immersed in a 4% PFA solution for fixation. Subsequently, staining was performed using hematoxylin and eosin (H&E), following established protocols. To ensure valid intergroup comparisons, histological assessments were consistently performed on cross‐sectional tissue areas from analogous anatomical locations across all experimental animals [19, 20].

2.5. Gut Permeability Assays

Mice that underwent a 6‐h fasting period were given an oral dose of 4000‐Da FITC‐dextran (46944; Sigma‐Aldrich) at a concentration of 600 mg per kg of body weight, which corresponds to a solution concentration of 125 mg per mL. After 4 h, blood samples were collected and centrifuged at 6000 revolutions per minute for 10 min at 4°C. The serum obtained was then diluted with an equal volume of phosphate‐buffered saline (PBS) and analyzed for FITC‐dextran concentration using a SpectraMax M2 microplate reader (Molecular Devices, USA). The reader was set to an excitation wavelength of 485 nm and an emission wavelength of 535 nm, following previously established protocols [19]. To determine the concentration of FITC‐dextran in the samples, standard curves were created using a series of dilutions of FITC‐dextran in PBS, ranging from 0 to 12.5 µg per mL.

2.6. LPS Analysis

The levels of lipopolysaccharides (LPS) in plasma were quantified utilizing the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Nanjing, China). Liver homogenates were prepared with the assistance of a tissue homogenizer (IKA, Germany). Following the guidelines provided by the manufacturer, Limulus amebocyte lysate reagents were incorporated into the plasma and liver homogenates [19]. Each sample underwent testing in duplicate, with optical density values recorded via a SpectraMax M2 microplate reader (Molecular Devices, USA) at a wavelength of 545 nm. Each experimental procedure was conducted at least three times to ensure reliability.

2.7. Short‐Chain Fatty Acid (SCFA) Analysis

SCFA analysis was conducted by Agilent 6890N GC system (Agilent Technologies, PA, USA) as described previously [19, 21] and 2‐ethylbutyric acid (Sigma) was treated as an internal reference standard.

2.8. 16S RRNA Gene Sequencing

Fecal DNA samples were collected and subsequently underwent amplification, quantification, and sequencing using the Illumina MiSeq PE300 platform from Illumina, based in San Diego, CA, USA. To analyze the data, several methods were employed, including principal coordinates analysis (PCoA), linear discriminant analysis effect size (LEfSe), and redundancy analysis (RDA), all executed with QIIME and R software [19, 20]. The LEfSe analysis aimed to identify unique microbial populations present in the different groups [22]. Additionally, the RDA test, along with heatmap visualizations, was used to investigate potential relationships between the bacterial community structure and various environmental factors [19]. Variations in bacterial populations were assessed using the Majorbio Cloud Platform through LEfSe. Furthermore, the PICRUSt2 tool, in combination with the KEGG database and the EggNOG database, was employed to predict biological functions.

2.9. Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR)

Total RNA was extracted from the distal colon section using the TRIzol reagent method and then reverse transcribed into complementary DNA (cDNA). The qRT‐PCR assay was conducted using the TB Green Premix Ex Taq (Takara, Japan). Each sample was subjected to at least three replicates and normalized to either 18s RNA or β‐actin, following the 2‐ΔΔCT methodology. The primer sequences are provided in Table S1.

2.10. Fluorescence In Situ Hybridization (FISH) With Bacteria

The FISH experiment was conducted following the methodologies outlined in earlier studies [19, 23]. Universal bacterial probes were synthesized based on the protocols detailed in prior research, as presented in Table S2.

2.11. Statistical Analyses

Statistical evaluation was conducted utilizing SPSS version 13.0 (Chicago, IL). The experimental data are presented as the mean ± standard error of the mean (SEM). To evaluate differences among multiple groups, we utilized one‐way analysis of variance (ANOVA), followed by Bonferroni post hoc tests for further comparisons. A p value of less than 0.05 was deemed statistically significant. The significance levels are denoted as follows: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and “ns” for no significance.

3. Results

3.1. BRR Alleviates the Tumorigenesis of CRC Associated With Colitis

The impact of varying doses of BRR (50 and 100 mg/kg/day) on CRC induced by AOM and DSS in murine models was investigated (Figure 1A). It was observed that AOM/DSS treatment led to impaired growth in the mice, a phenomenon that was significantly ameliorated by BRR administration, as evidenced by increased body weight gain in the CRC mice receiving BRR, particularly in the HBRR group (Figure 1B). Upon euthanizing the mice, it was noted that the colons of CRC mice were shorter compared to their normal counterparts; however, the AOM/DSS‐induced reduction in colon length was mitigated by BRR, particularly at the dosage of 100 mg/kg (Figure 1C,D). Treatment with different concentrations of BRR (50 and 100 mg/kg/day) led to a significant decrease in both the number of tumors and the overall tumor burden (Figure 1E,F), with the most pronounced effects observed at the 100 mg/kg dosage. Additionally, histological evaluations through H&E staining revealed that BRR treatment resulted in a reduction of the histopathological score in CRC mice, characterized by diminished aberrant crypt foci, alleviated epithelial lesions, and reduced inflammatory cell infiltration (Figure 1G,H). Collectively, these results substantiate the conclusion that BRR has a dose‐dependent inhibitory effect on the development of colitis‐associated colorectal tumors in mice.

3.2. BRR ModulatesGut Microbial Communities in AOM/DSS Mice

A total of 1 470 993 sequences were analyzed in this study. Rarefaction diversity assessments indicated that the majority of microbial diversity had been captured (Figure S1A). Initially, we conducted an α‐diversity analysis, utilizing the Chao1 and ACE indices to assess community richness, alongside the Shannon index to evaluate species diversity. When comparing the CRC model mice to those treated with BRR, a significant reduction was observed in the Chao1, ACE, and Shannon indices (Figure S1B–D), indicating that BRR diminished the α‐diversity of the microbial community in CRC mice. Further examination of the intestinal microbiota profile demonstrated notable alterations in the bacterial community composition across various samples (Figure 2A). PCoA, a critical aspect of β‐diversity metrics, revealed distinct variations in bacterial community structures among the control, CRC, LBRR, and HBRR subgroups (Figure 2B). Notably, the control group was distinctly situated in the upper left quadrant, while the LBRR and HBRR clusters occupied the upper and lower right quadrants, respectively, with the CRC subgroup positioned in the lower left quadrant (Figure 2B). The sequencing data indicated an increase in bacteria often considered enteropathogenic, such as Escherichia‐Shigella and Enterorhabdus within the CRC group, alongside a decrease in beneficial bacteria like Prevotellaceae_UCG‐001. Moreover, beneficial taxa, including Akkermansia muciniphila, Bacteroides, Lachnoclostridium, Blautia, and Prevotellaceae_UCG‐001, which are known to produce SCFAs such as butyrate, were found to be elevated in both the HBRR and LBRR groups compared to the CRC group (Figure 2C–H). Notably, the modulation of Escherichia‐Shigella, a bacterial group reported to be enriched by BRR in a gut microbiota‐dependent manner [24], underscores the compound's role in reshaping microbial ecology. Through the integration of 16S amplicon sequencing data with the EggNOG and KEGG databases, we predicted the functional capacities of the bacteria (Figure 2I–K). Analysis using the EggNOG database suggested that bacterial functions were predominantly linked to metabolic processes, with “Carbohydrate transport and metabolism” and “Amino acid transport and metabolism” exhibiting the highest abundance (Figure 2I). Likewise, the 16S rRNA sequencing data in conjunction with KEGG functional predictions highlighted that bacterial community functions are primarily associated with “Carbohydrate metabolism” and “Amino acid metabolism” pathways (Figure 2J). By delving deeper into the KEGG data at the third level, we further validated the relationship of these bacteria with “Metabolic pathways,” “Biosynthesis of secondary metabolites,” “Microbial metabolism in diverse environments,” and “Biosynthesis of amino acids” (Figure 2K). Collectively, these findings indicate that BRR reshapes the intestinal microbiota in AOM/DSS mice, potentially promoting enhanced SCFA production, which may contribute to the attenuation of intestinal barrier dysfunction and inflammation.

FIGURE 2.

Berberine induced alterations in the gut microbial communities of AOM/DSS mice. (A) Variations in the taxonomic composition of gut microbiota. The stacked bar charts illustrate the individual variability in the relative abundances of predominant bacterial genera observed in this study. (B) Unweighted UniFrac principal coordinates analysis (PCoA) of the stool samples utilizing the complete set of amplicon sequence variants (ASVs). The proportion of variation attributable to the plotted principal coordinates (PCs) is presented. (C) A heatmap illustrating species‐level abundance clustering highlights the most significantly differentially abundant features of gut microbiota identified in fecal samples across all groups. The color gradient from blue to red indicates relative richness, ranging from low to high. (D–F) Differential abundance analysis conducted to identify taxa with significant differences using the Wilcoxon rank‐sum test. (D) Comparison between the control group and the CRC group; (E) comparison between the CRC group and the LBRR group; (F) comparison between the CRC group and the HBRR group. (G) The evolutionary branch graph from LEfSe analysis visually represents the taxonomic differences among the four groups, differentiated by the coloration of the predominant classes. (H) Linear discriminant analysis (LDA) scores obtained from LEfSe analysis, indicating the biomarker taxa (LDA score > 2 and significance of p < 0.05 as determined by the Wilcoxon signed‐rank test). (I–K) Functional prediction analysis of the bacterial microbiota. (I) Utilization of PICRUSt2 in conjunction with the EggNOG database to forecast the functional roles of bacterial microbiota in tissues. Additionally, PICRUSt2 combined with the KEGG database was employed to predict the functions of bacterial microbiota in tissues, presenting the results of the KEGG pathways at Level 2 (J) and Level 3 (K).

3.3. BRR Mitigates Dysfunction of the Intestinal Barrier and Inflammation in AOM/DSS Mouse Models

To determine whether BRR could enhance intestinal barrier integrity and reduce inflammation, we examined the structure of the colon epithelium, assessed bacterial translocation markers, evaluated metabolic endotoxemia, and analyzed the mRNA expressions of relevant genes in the distal colon of mice. TEM detected ultrastructural tight junction abnormalities, revealing tighter TJs when CRC group compared to HBRR group (Figure 3A). The bacterial invasion into the surface of the colon intestinal epithelium was observed using a widely recognized fluorescence probe through a FISH assay, as illustrated in Figure 3B. As anticipated, the administration of HBRR significantly safeguarded the intestinal epithelium from bacterial invasion when compared to the CRC‐only group, as illustrated in Figure 3B. Additionally, we assessed the plasma levels of bacterial DNA through a qRT‐PCR assay. Consistent with the findings from the FISH analysis, the bacterial 16S rRNA levels in the blood samples were markedly lower in rats treated with BRR compared to those in the CRC group, as shown in Figure 3C. Furthermore, we observed a notable reduction in intestinal permeability (Figure 3D) and a decrease in plasma LPS levels (Figure 3E) in CRC mice that received BRR treatment compared to the CRC group. Besides, the fecal SCFA test revealed a significant decrease in butyrate levels in CRC model mice, which was reversed by BBR treatment (Figure 3F). No significant differences were observed in fecal acetate or propionate levels among the four groups (Figure 3G,H). BRR‐fed mice exhibited downregulated mRNA levels of TLR4 and CD14, which are part of the LPS downstream pathway, in the distal colon (Figure 3I,J). Conversely, these mice showed upregulated mRNA levels of occludin, ZO1, and Claudin1 in the same region, suggesting that BRR administration enhances intestinal mucosal integrity (Figure 3K–M). As anticipated, the HBRR group demonstrated a more pronounced improvement in intestinal barrier integrity compared to the LBRR intervention group. Additionally, in the distal colons of mice subjected to AOM/DSS, there was an increase in pro‐inflammatory markers such as TNF‐α, IL‐1β, and IL‐6, alongside a decrease in the anti‐inflammatory cytokine IL‐10; however, these conditions were reversed with BRR administration (Figure 3N–Q). Furthermore, BRR treatment significantly lowered the relative mRNA expressions of FAK, MyD88, and IRAK4 in the distal colon tissue when compared to the CRC group, which are involved in the LPS/TLR4 downstream signaling pathway (Figure 3R–T). Collectively, these findings indicate that BRR effectively ameliorates AOM/DSS‐induced intestinal barrier dysfunction, thereby reducing bacterial invasion and translocation, which in turn helps to suppress metabolic endotoxemia and colon inflammation in mice.

FIGURE 3.

Berberine mitigates intestinal barrier impairment and inflammation in AOM/DSS mice. (A) Representative transmission electron micrographs illustrating the colon epithelium from both the CRC group and the HBRR group mice. (B) Visualization of bacterial invasion into the colonic mucosa utilizing fluorescence in situ hybridization (FISH) targeting 16S rRNA genes of all bacteria (depicted in red) and nuclei (shown in blue). (C) Assessment of total bacterial DNA load (measured as universal 16S rRNA gene copies) in whole blood samples. (D) Evaluation of intestinal permeability conducted through the oral administration of a 4000‐Da FITC‐dextran. (E) Measurement of plasma lipopolysaccharide (LPS) levels using the appropriate assay kit. Cecal concentration of acetate, propionate, and butyrate levels among the indicated groups. Cecal butyrate (F), acetate (G), and propionate (H) levels among different groups. (I–M) Relative mRNA expression levels of TLR4 (I), CD14 (J), occludin (K), ZO1 (L), and claudin1 (M) within distal colon tissues, determined by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). (N–T) mRNA expression levels of TNF‐α (N), IL‐1β (O), IL‐6 (P), and IL‐10 (Q) in distal colon tissues, evaluated via qRT‐PCR. Additionally, the relative mRNA expression of LPS/TLR4 downstream signaling pathway components FAK (R), MyD88 (S), and IRAK4 (T) in distal colon tissues was also assessed using qRT‐PCR. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was indicated by *p < 0.05, **p < 0.01, ***p < 0.001, with ns denoting no significance. Multiple group comparisons were performed using one‐way ANOVA followed by Bonferroni's post hoc test.

3.4. BRR Preserves Intestinal Barrier Integrity and Mitigates Intestinal Inflammation via CB1 and GPR55 in AOM/DSS Mice

Research has established that the activity of the ECS can be modulated by specific microbes (e.g. A. muciniphila) [25]. In this study, we quantified the mRNA levels of CB2 and GPR55, alongside ECS‐related metabolic enzymes. The administration of AOM/DSS resulted in a reduction of CB2 and an elevation of GPR55 transcript levels in the distal colons of the mice (refer to Figure 4A,B). Notably, the alterations in mRNA expression of CB2 and GPR55 induced by AOM/DSS were reversed upon treatment with BRR. The ligands 2‐arachidonoylglycerol (2‐AG) and N‐arachidonoyl‐L‐serine (AraS) are currently among the most extensively researched for their interaction with the ECS; 2‐AG predominantly binds to CB2, while AraS has a higher affinity for GPR55. Both receptors are predominantly expressed in enteric nerves and across the intestinal mucosa, with CB2 found on immune cells and GPR55 on the enteric nervous system and enterocytes. The ECS, particularly the CB2 receptor within the gastrointestinal tract, serves as a critical link between the gut microbiota, metabolic endotoxemia, and intestinal inflammation.

FIGURE 4.

An illustration of how berberine preserves the integrity of the intestinal barrier and mitigates intestinal inflammation via CB1 and GPR55 in AOM/DSS‐treated mice. The relative mRNA levels of CB2 (A), GPR55 (B), and endocannabinoid system (ECS) metabolic enzymes, including DAGLα (C), FAAH (D), MAGL (E), and NAPE‐PLD (F), in the distal colon tissue were evaluated using quantitative reverse transcription polymerase chain reaction (qRT‐PCR). (G) A schematic representation of the experimental groups and treatment protocols is shown. Male BALB/c mice underwent AOM/DSS treatment along with HBRR for a duration of 10 weeks. Subsequently, the HBRR+CRC subgroup was segmented into four experimental groups (HBRR+CRC‐14w, vehicle, AM630, and O‐1602). The mice received intraperitoneal injections of AM630, O‐1602, vehicle, or no injection over a period of 4 weeks. (H) Representative histological images of colon sections following H&E staining are presented, with a scale bar of 100 µm at ×100 magnification. (I) The total bacterial DNA load, quantified by universal 16S rRNA gene copies, was assessed in whole blood samples. (J) The permeability of the gut to FITC‐dextran was evaluated. (K) Plasma levels of lipopolysaccharide (LPS) were measured using a designated assay kit. The relative mRNA expressions of occludin (L) and ZO1 (M), as well as claudin 1 (N) in distal colon tissues, were quantified using qRT‐PCR. Additionally, the mRNA levels of pro‐inflammatory cytokines TNF‐α (O), IL‐1β (P), IL‐6 (Q), and anti‐inflammatory cytokine IL‐10 (R) were assessed through qRT‐PCR. Lastly, the relative mRNA expressions of downstream signaling molecules involved in the LPS/TLR4 pathway, including FAK (S), MyD88 (T), and IRAK4 (U), were also evaluated using qRT‐PCR. Results are presented as mean ± standard error of the mean (SEM). Statistical significance was determined with *p < 0.05, **p < 0.01, ***p < 0.001, and ns indicating no significance. Multiple groups were analyzed using one‐way ANOVA followed by Bonferroni's post hoc test.

Furthermore, our findings indicated that mice treated with BRR exhibited enhanced mRNA levels of fatty acid amide hydrolase (FAAH) and diacylglycerol lipase alpha (DAGLα) (see Figure 4C,E), while showing decreased mRNA levels of monoacylglycerol lipase (MAGL) (refer to Figure 4D) and N‐acyl‐phosphatidylethanolamine‐selective phospholipase D (NAPE‐PLD) (illustrated in Figure 4F) in response to AOM/DSS exposure. These observations suggest that BRR administration may lead to elevated concentrations of 2‐AG and reduced levels of AraS in the distal colon of AOM/DSS mice. Thus, our results provide compelling evidence that BRR plays a vital role in maintaining intestinal barrier integrity through the modulation of the ECS in the distal colon of AOM/DSS mice.

We postulated that the beneficial effects of BRR on preserving the integrity of the intestinal barrier and mitigating gut inflammation could be mediated through interactions involving CB2 and GPR55. To evaluate this hypothesis, we administered AOM/DSS and BRR to mice over a period of 4 weeks, either in the presence of a CB2 antagonist (AM630), a GPR55 agonist (O‐1602), or a corresponding volume of a control solution (vehicle) (Figure 4G). Observations indicated that the distal colon of mice treated with AM630 or O‐1602 exhibited a greater degree of inflammatory infiltration compared to the other experimental groups (Figure 4H). Furthermore, the influence of BRR on intestinal permeability (Figure 4I,J), plasma LPS concentrations (Figure 4K), and mRNA levels of intestinal tight junction proteins (Figure 4L–N) was significantly diminished in the presence of AM630 and O‐1602. Additionally, administration of BRR resulted in a reduction of the pro‐inflammatory cytokines TNF‐α, IL‐1β, and IL‐6 within the distal colon; however, this effect was reversed with the introduction of AM630 or O‐1602 when compared to the vehicle group (Figure 4O–Q). Conversely, an increase in mRNA levels of IL‐10 was noted in the distal colon (Figure 4R). The presence of AM630 and O‐1602 nullified the impact of BRR on the mRNA expressions of downstream signaling molecules associated with LPS/TLR4, specifically FAK (as shown in Figure 4S), MyD88 (illustrated in Figure 4T), and IRAK4 (depicted in Figure 4U). Collectively, these findings suggest that BRR is essential for preserving the integrity of the intestinal barrier and reducing intestinal inflammation, primarily through mechanisms involving CB2 and GPR55.

3.5. BRR Enhances Intestinal Barrier Integrity and Mitigates Gut Inflammation Linked to the Intestinal Microbiota

The receptors CB2 and GPR55 are crucial in managing intestinal inflammation. This study sought to clarify whether the intestinal microbiota significantly influences the regulation of CB2 and GPR55 in mice treated with AOM/DSS, particularly through the administration of BRR, while employing antibiotic treatment as a methodological approach. BALB/c mice were subjected to AOM/DSS treatment, with or without supplementation of high‐dose BRR (HBRR), over a period of 10 weeks, followed by an antibiotic cocktail to create macroscopically germ‐free mice [19] (Figure 5A). Notably, the extent of colorectal tumorigenesis, as indicated by colorectal length, the number of tumor polyps, and histological scores, was significantly less pronounced in the HBRR+CRC group compared to the CRC group. Furthermore, these protective effects were markedly reversed following antibiotic treatment (Figure 5B–F). Additionally, the indicators of intestinal permeability (Figure 5G), metabolic endotoxemia (Figure 5H,I), mRNA expression levels of tight junction proteins in the distal colon (Figure 5J–L), and the inflammatory status of the distal colon (Figure 5M–P) were substantially lower in the HBRR+CRC group than in the CRC group, with similar reversals observed post‐antibiotic administration. Our findings imply that the intestinal microbiota predominantly mediates the beneficial effects of BRR on intestinal barrier integrity and inflammation, as well as on symptoms of CRC. Moreover, the microbiota depletion induced by antibiotics significantly negated the BRR‐induced activation of CB2 and the inhibition of GPR55 expression in the distal colon tissue (Figure 5Q,R). Ultimately, our findings indicate that the supportive function of BRR in regulating the expression of CB2 and GPR55 in the distal colon is closely linked to the gut microbiota.

3.6. RDA and Heatmap Reveal Significant Correlations Among Samples, Microbial Species, and Physiological Biochemical Factors

In order to investigate the potential relationships between the samples, the composition of the bacterial communities, and the physiological biochemical factors, RDA and heatmap visualizations were employed, utilizing characteristic sequences of the bacterial 16S rRNA gene alongside the assessed physiological biochemical parameters (refer to Figure 6A,B). The relationships among these data sets were depicted using arrows, where the length of each arrow signifies the strength of the influence of physiological biochemical factors on the bacterial data. Additionally, the angle between the arrows provides insight into the type of correlation present: acute angles suggest a positive correlation, obtuse angles indicate a negative correlation, and right angles represent no correlation at all. Notably, BBR demonstrated a strong positive correlation with the mRNA expression of colon CB2, as well as with the mRNA levels of ZO1, occludin, and claudin1 in the distal colon, as illustrated in Figure 6A. The characteristic bacterial taxa, such as A. muciniphila, Bacteroides, Lachnospiraceae, and Blautia, also showed a significant positive correlation with the mRNA expression of colon CB2. Furthermore, the mRNA expression of colon GRP55 displayed a notable positive correlation with Enterorhabdus (illustrated in Figure 6B).

FIGURE 6.

The redundancy analysis (RDA) diagram and accompanying heatmap illustrate the relationships among various samples, microbial species, and physiological biochemistry factors. (A) The RDA diagram displays samples (represented by symbols) and physiological biochemistry factors (indicated by red arrows). The values along axes 1 and 2 denote the percentage of variance accounted for by each respective axis. The labels denote all four identified clusters, while the names of the parameters are depicted as vectors reflecting their correlation with the initial two components. (B) The heatmap presents a hierarchical clustering analysis of Spearman correlation coefficients that assess the relationship between fecal bacterial abundance and selected physiological biochemistry factors. Statistical significance is indicated with *p < 0.05, **p < 0.01, and ***p < 0.001.

4. Discussion

The rising incidence of CRC has emerged as a major public health issue, being one of the foremost causes of cancer‐related illness and death globally. The development of CRC is closely associated with chronic inflammatory conditions, especially inflammatory bowel diseases like ulcerative colitis and Crohn's disease. These conditions exacerbate the risk of CRC through mechanisms involving persistent inflammation, dysregulated immune responses, and alterations in the gut microbiota, thereby promoting tumorigenesis [3, 5]. Understanding the interplay between inflammation, microbiota, and cancer is crucial for the development of novel therapeutic strategies aimed at mitigating CRC risk.

The ECS has emerged as a master regulatory network that integrates gut microbiota signals, immune homeostasis, and oncogenic signaling pathways (e.g., Wnt/β‐catenin, MAPK) implicated in CRC [13, 14, 15]. Our decision to focus on the ECS, particularly the CB1/GPR55 balance, is based on its role as an upstream modulator linking dysbiosis‐derived inflammation to tumor progression [26], complementing existing studies on other CRC drivers.

This study investigates the potential protective effects of BRR, a natural isoquinoline alkaloid, against colitis‐associated CRC (Figure 7). Through a comprehensive approach involving in vivo animal models, microbiome analysis, and molecular techniques, we aim to clarify how BRR affects gut microbiota and its impact on the ECS, particularly in relation to CRC. Our findings indicate that BRR not only reduces tumor development associated with colitis but also promotes positive alterations in the composition of gut microbiota and strengthens the integrity of the intestinal barrier. This suggests that BRR may serve as a promising therapeutic agent in the management of CRC [16, 27]. This discussion will explore these key findings and their implications for future CRC prevention and treatment strategies.

FIGURE 7.

An illustration of how BBR exerts a CRC effect through the modulation of the ECS and gut microbiota. The composition of gut microbes changes significantly, with an increase in beneficial bacteria such as Akkermansia muciniphila, Bacteroides, Lachnoclostridium, Blautia, and Prevotellaceae_UCG‐001, while pathogenic species decrease. This shift enhances gut barrier function and reduces metabolic endotoxemia, both of which are linked to CRC progression. By administering BBR, CRC is mitigated as it reshapes the gut microbiota and modulates the ECS, particularly affecting the expression levels of CB2 and GRP55 in the colonic tissues of mice. This sequence of events leads to improved intestinal barrier integrity and a decrease in intestinal inflammation.

The significance of the results from this study lies primarily in the elucidation of the molecular mechanisms through which BRR exerts its protective effects against CRC. The findings suggest that BRR influences various signaling pathways that are critical in the pathogenesis of CRC, particularly in modulating inflammation and tumorigenesis. Notably, BRR's effects on the ECS, including modulation of CB1 and GPR55 activity, may underlie its ability to suppress pro‐inflammatory signaling cascades. For instance, the observed reduction in pro‐inflammatory cytokines like TNF‐α and IL‐6, along with the increase in the anti‐inflammatory cytokine IL‐10, suggests that BRR could be crucial in modulating immune responses in the tumor microenvironment [8]. The downregulation of TLR4 and CD14 expression suggests that BRR may play a significant role in inhibiting inflammatory signaling pathways, which are recognized as key factors in the development and progression of CRC [28]. These changes are consistent with ECS‐mediated regulation of TLR4/NF‐κB signaling, wherein a restored CB1/GPR55 balance dampens the inflammatory tone in the colonic microenvironment. These insights not only provide a clearer understanding of the anti‐cancer properties of BRR but also open avenues for developing new therapeutic strategies that target these molecular pathways.

In addition to its effects on inflammation, the study highlights the impact of BRR on gene expression and cellular behavior in the context of CRC. The modulation of gene expression related to apoptosis and cell proliferation suggests that BRR may influence critical processes that determine tumor cell fate [9]. For example, the upregulation of apoptosis‐related genes such as p53, in conjunction with the downregulation of anti‐apoptotic genes, demonstrates how BRR could facilitate tumor cell death while inhibiting proliferation [29]. Given that the ECS has been shown to modulate p53 and cell cycle pathways via CB1/GPR55 signaling, BRR's dual action integrates the ECS as a central conduit linking microbiota alterations to tumor‐suppressive effects. This dual action underscores the potential of BRR as a therapeutic agent, not only in mitigating inflammation but also in directly targeting cancer cells. Such insights are vital as they can lead to a deeper understanding of how dietary compounds can be leveraged for cancer prevention and treatment, thus enriching the current therapeutic landscape.

Moreover, the study's findings on immune mechanisms provide valuable insights into how BRR influences immune responses in CRC. Notably, there was a significant decrease in pro‐inflammatory cytokines alongside an increase in anti‐inflammatory cytokines. This shift highlights BRR's potential to promote a more favorable immune environment for fighting CRC [30]. The immunomodulatory effects observed may play a significant role not only in inhibiting tumor growth but also in improving the effectiveness of current immunotherapies and targeted treatments [31]. The ECS also serves as a key modulator of immune cell function; for example, activation of GPR55 on immune cells can shift the balance from pro‐inflammatory to anti‐inflammatory cytokine profiles, a pattern closely mirrored by our BRR intervention [32]. Understanding these interactions provides a crucial foundation for future research aimed at integrating BRR into comprehensive treatment regimens for CRC, offering patients a multifaceted approach to therapy that encompasses both immunological and nutritional strategies.

The limitations of this study warrant careful consideration. First, the findings are based on an animal model, which may not fully mimic the complexities of human CRC and its associated colitis. The absence of clinical validation limits the applicability of the results to human conditions. Furthermore, the study's sample size may restrict the generalizability of the outcomes, potentially overlooking variations in individual responses to BRR. Inter‐batch variability in microbiota analysis also poses a challenge in drawing definitive conclusions about the effects of BRR on gut microbiota composition. These factors underscore the necessity for further research, including human clinical trials, to validate the therapeutic potential of BRR in diverse populations.

5. Conclusion

To sum up, this study elucidates the significant therapeutic prospects of BRR in mitigating colitis‐associated colorectal tumorigenesis, enhancing intestinal barrier integrity, and modulating gut microbiota (Figure 7). The findings indicate that BRR may be a promising candidate for developing innovative therapeutic strategies aimed at treating CRC and associated inflammatory diseases. Future research is essential to translate these preclinical results into clinical applications, ultimately contributing to the development of effective interventions for patients at risk of CRC.

Author Contributions

Yalan Huang: investigation, formal analysis, visualization, Writing – original draft, Writing – review and editing. Junhui Zhang: data curation, visualization, writing – original draft, writing – review and editing. Huiqing Yu: project administration, writing – review and editing. Hong Yang: formal analysis, software, writing – review and editing. Mengting Chen: conceptualization, methodology, formal analysis, visualization, funding acquisition, writing – review and editing.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82404265), the Chongqing Medical Youth Leading Talent Project (Grant No. YXQN2025061), the Young Elite Scientists Sponsorship Program by Chongqing Association for Science and Technology (Grant to Mengting Chen), the National Natural Science Foundation “Open Competition” Project Target Special Fund (Grant No. KH2025051), and the Discipline Construction Fund (Grant No. W2026006).

Ethics Statement

The authors have stated that the animal experiments performed in their study followed the World Medical Association (WMA) Statement on the use of animals in biomedical research, along with the European Union's recommendations outlined in Directive 2010/63/EU, which pertain to the design and analysis of pharmacological studies. Additionally, these experiments complied with the guidelines set forth by a globally recognized organization and were approved by the Laboratory Animal Management and Ethics Committee of Chongqing University Cancer Hospital (CZLS2022022‐A).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File1: mnfr70585‐sup‐0001‐FigureS1.pdf.

Supporting File2: mnfr70585‐sup‐0002‐TableS1.docx.

Supporting File3: mnfr70585‐sup‐0003‐TableS2.docx.

Data Availability Statement

The sequences discussed in this paper have been submitted to the NCBI database under the accession number PRJNA1218630.