Our results showed that the induction of DSS caused typical UC symptoms in cats, such as diarrhea and hematochezia (Fig. 1B), and a marked shift in disease activity index (DAI) score (Fig. 1D) and body weight (Fig. S1). Additionally, the colon length of the cat was significantly shortened in the DSS-induced group (Fig. 1C). Hematoxylin and eosin (HE) staining of the colon showed that DSS treatment caused severe enteric mucosal injury (Fig. 1E). However, all characteristic features were prevented by oral BBR supplementation. These results indicate that BBR can alleviate the overall symptoms of DSS-induced colitis in cats and alleviate colonic injury.

Species accumulation curves were shown in order to judge whether the sample numbers were sufficient (Fig. S2). Next, we analyzed the differences in intestinal flora at the family and genus levels (Fig. 2A and B). At the family level, Prevotellaceae, Lachnospiraceae, Selenomonadaceae, Veillonellaceae, and Lactobacillacrae were significant in the control group, and in the DSS-induced group, there was an increase in the richness of Bacteroidaceae and Fusobacteriaceae. After treatment with BBR, there was an increase in beneficial bacteria, such as Lactobacillacrae and Prevotellaceae and a reduction in the richness of Bacteroidaceae. At the genus level, the DSS-induced group showed an increase in the richness of Bacteroides and Fusobacterium, and after treatment with BBR, there was an increase in beneficial bacteria, such as Lactobacillus and Prevotella and a reduction in the richness of Bacteroides. The results showed that the intestinal flora of DSS-induced colitis is changed by BBR treatment.

β-Diversity analysis is mainly used to analyze differences between the groups. Using a principal-component analysis (PCA) and a principal coordinates analysis (PCoA), differences in species composition abundance were compared by analyzing the projected distances on the axes among the samples. PCA (Fig. 3A) and PCoA analyses (Fig. 3B) were used to evaluate the similarities and differences between the three groups. PCA indicated that intestinal flora diversity was higher in the DSS-induced group than in the control group and was reduced after treatment with BBR. PCoA indicated that the gut microbiota composition of the BBR group was different from the DSS group in terms of axis PCo-1. These results suggest that the administration of BBR maintains the intestinal flora diversity.

In this study, we performed 16S rRNA gene high-throughput sequencing to reveal the impact of BBR on the gut microbiota. The Venn diagram is used as a community analysis to investigate which species are common and unique among different groups. It can be seen that the difference in operational taxonomic unit (OTU) between the DSS+BBR group and the DSS group at the phylum level is not apparent, mainly with Firmicutes, Bacteroidetes, and Proteobacteria; at the genus level, Prevotella shows the most evident change, followed by Bacteroides and Clostridium. There is a difference in relative abundance between the DSS+BBR group and the DSS group in the Firmicutes, Fusobacteria, and Proteobacteria. At the genus level, significant differences can be seen in Prevotella, Bacteroides, Blautia, Cellulosilyticum, and Fusobacterium in both the DSS+BBR and DSS groups (Fig. 4A and B).

Heat maps were used to analyze species composition so as to display the trend of species abundance distribution among samples and further compare species composition differences (Fig. 5A). The Collonsella aerofaciens, Bifidobacterium adoiescentis, Megasphaera eisdenii, Bacteroides plebeius, and Lactobacillus ruminis were significant in the control group. The DSS-induced group showed an increase in Bacteroides fragilis and a reduction in Lactobacillus ruminis. After the treatment with BBR, there was an increase in the beneficial bacteria, such as Lactobacillus ruminis. A random forest analysis was used to classify the microbial community samples (Fig. 5B). The DSS-induced group showed an increase in harmful bacteria, such as Bacteroides fragilis, and a reduction in the beneficial bacteria, such as Lactobacillus ruminis. However, the balance between harmful and beneficial bacteria was restored after treatment with BBR.

The analysis of fecal flora metabolic pathways in cats allowed for predicting the relevant functions of their flora and analyzing the effect of BBR on the role of feline intestinal flora. The abundance of secondary functional pathways for all samples is shown in Fig. 6A, which shows that the highest possible of metabolism-related tracks was observed and that human disease and genetic information processing were followed. Fig. 6B shows multiple MetaCyc secondary functional pathways for all samples, which demonstrates the highest quantity of ways related to biosynthesis and degradation/utilization/assimilation. These results indicate that metabolic pathway function and species differences are improved after treatment with BBR.

Next, we assessed the effects of BBR administration on DSS-induced proinflammatory responses. The DSS-induced group had significantly increased serum IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) levels compared with the control group. Conversely, BBR supplementation remarkably reversed this tendency (Fig. 7A). Exploring the underlying mechanism by which BBR modulates DSS-induced colitis, we found that the DSS-induced group showed significant increase in mRNA level expression of inflammatory factors. The results showed that the JAK2/STAT3 signaling pathways were associated with inflammation, such as IL-6, TNF-α, IL-1β, IL-18, NLRP3, Caspase-1, JAK2, and STAT3 (Fig. 7B). As seen in Fig. 7C, the protein expressions of JAK2, STAT3, and other related proteins in the pathway were increased in the DSS group compared to the control group. The protein expressions in the BBR group were decreased compared to those in the DSS group. Still, their expressions were higher than in the control group. The above results demonstrate that BBR can reduce inflammation through the JAK2/STAT3 signaling pathway.

Our study showed that the Ca²⁺ concentration (Fig. 8A) was changed with the intestinal smooth muscle damage. DSS-induced models showed increases in Ca²⁺ concentration, while the results were the opposite in the BBR group. Intestinal motility is positively associated with MLC phosphorylation in the smooth muscle. The relative activities of MLCK regulate smooth muscle MLC phosphorylation. The present study results indicated a significant increase in MLC phosphorylation in the colonic smooth muscle of the DSS-induced cats (Fig. 8B and C). However, these effects were significantly reversed after the BBR administration. The increased MLCK mRNA expression levels also changed considerably (Fig. 8D). It is thus concluded that BBR has a substantial impact on colonic smooth muscle contraction.

Our study investigated the therapeutic effects of BBR on UC based on intestinal flora and tissue distribution analysis. BBR plays a vital role in modulating intestinal flora and intestinal smoothing mechanisms by improving UC symptoms because of its antiinflammatory effects in pathological conditions. Notably, BBR tends to have unique effects that remain consistent when used in clinical practice. BBR has a long tradition as a botanical medicine for treating gastrointestinal disorders, and these data are expected to provide necessary information for the targeted application of BBR in UC treatment.

Nine healthy adult shorthair cats (weight, 2.0 to 2.3 kg) were randomly allocated into three groups: control group (CON), DSS-induced colitis (DSS) group, and berberine treatment (DSS+BBR) group. All experimental studies were conducted in accordance with the humanitarian spirit and in accordance with the standards of the Institutional Animal Care and Use Committee of Northeast Agricultural University (grant number NEAUEC20220340). The cats in the DSS group and DSS+BBR group were given 5% DSS in water for 10 days. Between days 10 and 17, the DSS+BBR group was administered BBR (80 mg/kg/day), and the weight of each animal was recorded. On the day 18 of the experiment, the cats were anesthetized by intraperitoneal injection with pentobarbital sodium (35 mg/kg) before samples of tissues and blood were collected. Colon samples were harvested and stored at −80°C for further experiments. The animals were housed under standard conditions and received humane care complying with institutional guidelines. The experimental design used to test the effects of BBR on DSS-induced cats is shown in Fig. 1A.

DAI is a comprehensive score that has been used to evaluate the severity of DSS-induced colitis in animal models, including general health condition, weight loss, stool consistency, and degree of fecal bleeding (30). The scoring is as follows: score 0 indicates no weight loss, average stool properties, negative occult blood test; score 1 indicates a weight loss of 1 to 5%, soft stool, and negative occult blood test; score 2 indicates a weight loss of 5 to 10%, pale stool, and positive occult blood test; score 3 indicates a weight loss of 10 to 15%, diarrhea, and positive occult blood positive test; and 4 points indicates a weight loss of >15%, diarrhea, and bloody stools. From days 10 to 17, the general health condition, weight loss, stool consistency, and degree of fecal bleeding of the three groups was recorded.

Fecal samples were collected from the three groups, and the fecal microbiomes were examined using 16S rRNA gene sequencing carried out by the Shanghai Personalbio Biotechnology Corp. The total genomic DNA was extracted according to the manufacturer’s protocol. The DNA concentration and purity were tested using Nanodrop and 1% agarose gel electrophoresis. Pfu high fidelity DNA polymerase was used for PCR amplification, and the number of amplification cycles was strictly controlled. The amplified products were purified and recovered by magnetic beads. The recovered effects were amplified by PCR and quantified by fluorescent light. The fluorescent reagent was Quant-iT PicoGreen dsDNA assay kit, and MiSeq was used to perform high-throughput sequencing. Analysis was performed using QIIME2 software. The α-diversity in each sample was assessed based on the distribution of the amplicon sequence variants (ASV)/OTU in the different models. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted using tools available in the KEGG database. The OTUs reached a 97% nucleotide similarity level.

Total RNA was extracted from colon tissue and reversing transcription were performed. The cDNA was synthesized using standard procedures. The primers used to amplify related genes are designed by the software Primer 5.0. The mRNA expression of colonic TNF-α, IL-6, IL-10, IL-1β, IL-18, NLRP3, Caspase-1, JAK2, STAT3, MLC, MLCK, and calmodulin (CaM) was quantified by applying Light Cycler technology and SYBR green PCR core reagent kits. The relative changes in the gene expression were analyzed according to the 2^−ΔΔCT method. Primers used for quantitative PCR (qPCR) in Table 1.

Cat colon tissues were homogenized in ice-cold phosphate-buffered saline (PBS) and centrifuged at 13,000 × g at 4°C for 20 min. The supernatant was collected to analyze the concentrations of secretory immunoglobulin. IL-6, IL-1β, and TNF-α in the serum were measured with the corresponding enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bio, Inc., China) and measured using a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm. All steps were completed according to the manufacturer’s instructions.

Tissue homogenization was done as described above (53). Calcium ions in the sample were combined with Methyl Thyme Aroma Blue (MTA) in an alkaline solution to form a blue complex. We measured the calcium content of the samples according to the calcium standard curve. The expression of Ca²⁺ in the colon tissues was measured with the corresponding kits (Nanjing Jiancheng Bio, Inc., China, C004-2-1). All steps were completed according to the manufacturer’s instructions.

The colon tissue (0.1 g) was ground with liquid nitrogen for Western blot analysis. The protein samples were subjected to SDS-polyacrylamide gel electrophoresis with a concentration of 12%,10%, and 8% separation gels. Then, the proteins were transferred to a polyvinylidene fluoride membrane at 250 mA in a Tris-glycine buffer. Subsequently, the membranes were blocked with 5% fat-dry milk at 37°C for 2 h in the shaker, incubated overnight with diluted primary antibodies against the cat, including NLRP3, ASC, Caspase-1, JAK2, STAT3, smooth muscle contraction-associated speck-like protein containing MLC, p-MLC, CaM, and MLCK at 4°C. The membrane was incubated with secondary antibodies for 2 h (horseradish peroxidase-conjugated goat antirabbit IgG, 1:1,000, CST, 7074). The protein bands were detected and quantified using gel imaging system software (TransGen Biotech Co., Beijing, China). β-Actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal references. Antibodies information for Western Blot in Table 2.

Statistical analyses were performed using GraphPad Prism 8.0.1 software (New York, NY, USA). The data are expressed as means ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey post hoc analysis was applied when evaluating differences between three groups. P values < 0.05 were considered statistically significant.

The data sets supporting the conclusions of this article are available in the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra) under BioProject accession number PRJNA873140.