Berberine maintains intestinal homeostasis through modulating TLR4/ NF-κB / MTORC pathway and autophagy in cats

doi:10.21203/rs.3.rs-1537283/v1

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Berberine maintains intestinal homeostasis through modulating TLR4/ NF-κB / MTORC pathway and autophagy in cats

https://doi.org/10.21203/rs.3.rs-1537283/v1

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Background

Inflammatory bowel disease (IBD), a disease that seriously harms human and animal health, has attracted many researchers' attention because of its complexity and difficulty in treatment. Most research has involved rats and dogs, and very little was cats. We should know that gut microbiota varies significantly from animal to animal. Traditional Chinese Medicine and its monomer component have many advantages compared with antibiotics used in pet clinics. Numerous studies have shown berberine (berberine hydrochloride， BBr) therapeutic value for IBD; however, the specific mechanism remains to consider.

Results

We assessed gut pathology and analyzed fecal bacterial composition using Histological staining and 16S rRNA sequence. DSS administration destroyed intestinal mucosal structure and changed the diversity of intestinal flora relative to control. RT-PCR and western blot confirmed specific molecular mechanisms that trigger acute inflammation and intestinal mucosal barrier function disruption after DSS treatment. And autophagy inhibition is typical pathogenesis of IBD. Interestingly, berberine ameliorates inflammation during the development of the intestinal by modulating the TLR4(Toll-like receptors 4)/NF-κB signaling pathway and activating autophagy. Berberine significantly reduces tumor necrosis factor α(TNF-α), interleukin (IL)-6, and IL-1β expression in cats' serum. Enhancing the antioxidant effect of IBD cats is one of the protective mechanisms of berberine. We demonstrated that berberine repairs intestinal barrier function by activating the mTOR(mammalian target of rapamycin) complex, which inhibits autophagy.

Conclusions

Berberine can restore intestinal microbiota homeostasis and regulate the TLR4/ NF-Κb pathway, thereby controlling inflammatory responses. In addition, we propose a novel mechanism of berberine therapy for IBD, namely, berberine therapy can simultaneously activate MTORC and autophagy to restore intestinal mucosal barrier function in cats, which should be further studied to shed light on berberine to IBD.

Inflammatory bowel disease(IBD)

gut microbiota

intestinal barrier function

berberine

TLR4/NF-κB signaling pathway

autophagy

mTOR complex(MTORC)

Cats are the most popular pets nowadays[1]. The change of living environment led to the change of diet from high protein to high carbohydrate[2]; it is one of the essential factors that predispose domestic cats to gastrointestinal diseases[3]. The fact that the gut is known as the "second brain" speaks volumes about the importance of a healthy gut. However, treating gastrointestinal diseases has always been a challenge for humans and pets.

IBD is a common gastrointestinal disorder in cats and is a chronic immune-mediated disease affecting the gastrointestinal tract; so far, it is incurable [4]. The number of IBD patients increases year by year due to wrong living habits [5]. Many studies have investigated the therapeutic agents of intestinal flora on IBD[6, 7]. Furthermore, intestinal barrier dysfunction was an essential mechanism of IBD, which has attracted more and more researchers' attention[8].

Antibiotics using in IBD, but whether they affect the body for better or worse remains highly controversial[9]. To that end, researchers are turning to natural medicines that are safe and effective medicines. BBr is the main ingredient in many commonly used Traditional Chinese Medicines (e.g., the genus Berberis). Studies have shown that BBr displayed numerous pharmacological activities, including anti-inflammatory as a mechanism of action for its therapeutic use in treating IBD [5, 10, 11]. However, the interplay between BBr and IBD is far more complex than it has to articulate.

We hypothesized that BBr would be safe and effective for IBD prevention. Here, we established IBD models in American shorthair treated with 5%DSS for seven days to compare the clinical and histological changes with or without administration and further explore the therapeutic mechanism of BBr. In the following, we present data that demonstrates BBr restored intestinal barrier function, maintained intestinal microbiota homeostasis, regulated the expression of TLR4/NF-κB, and activated both MTORC and autophagy. Thus, it suggests that the therapeutic mechanism of BBr is remodeling intestinal flora, restoring intestinal barrier function, alleviating inflammation through modulation of the TLR4/NF-κB pathway, and preventing intestinal mucosal over-repair by activation of MTORC and autophagy.

Animal

The present study used twelve healthy adults American Shorthairs, including six females (all intact) and six males (all intact). They were vaccinated with the triple vaccine and were not in the vaccination period at the experiment. In vitro deworming was performed once a month, and in vivo deworming was performed every three months to eliminate the effects of viruses and parasites.

Drug administration

All cat adaptability raised a week with 12 h of light/12 h of the dark cycle, 25 ℃, and free access to food and water. Cats allocate to three groups: control (CON or A, n = 3), vehicle (DSS or B, n = 3), and BBr-treated vehicle group (n = 6) (BBr, Fig. 1A). To verify the effect of BBr dose on curative effect, six cats in the BBr group divide into two groups: the low-dose group (C or L-BBr, 40 mg/kg) and the high-dose group (D or H-BBr, 80 mg/kg). 5% DSS (dextran sulphate sodium, w/v, DSS, 36–50 kDa, MP Biomedical, Solon, OH) was added to the drinking water over seven days to induce experimental IBD and then replaced with normal drinking water for an additional seven days (Fig. 1A). BBr (Sigma-Aldrich, St. Louis, MO) was administered orally at 40mg/kg and 80 mg/kg daily to the BBr group for 14 days (Fig. 1A). Cats have euthanized on Day 14 for the following analysis.

Fecal collection

Feces have collected from each cat within 5 min of 2ml glycerin injected into the rectum with a 2mm diameter rectal administration tube after drug administration (day 14) and frozen at − 80°C until further analysis.

Clinical monitoring and determination of the histological score

During the process of IBD, experienced personnel determined the cat IBD activity index score (DAI) as previously reported[12] to assess the severity of inflammation in colitis, including bodyweight loss, stool consistency, and fecal blood (Additional file 6: Table S1).

On Day 14, Cats were euthanized and measured the length of the colon; about 1 cm of colon tissue was removed and fixed in 10% formalin to stain with hematoxylin and eosin (H&E). The extent of tissue damage was observed under Leica laser microdissection systems (DM6B, Heidelberg, Germany) and assessed in Additional file 1(Figure S1)[13]. Performed all injury assessments by observers unaware of the treatment and expressed the final score as an average.

Oxidative stress measurement

The experiment examined only three groups with significantly different antioxidant levels: the CON, DSS, and H-BBR groups. Normal saline was mixed in 0.1 g of colon tissue from each group, ground on ice to homogenate, and taken the supernatant after centrifugation, determined the protein concentration with a BCA protein determination kit (Mei5 Biotechnology Co., Ltd, Beijing, China). The contents of CAT, T-AOC, GSH, MOD, and SOD in colon homogenate were detected using a CAT, T-AOC, GSH, SOD, and MDA assay kit (Njjcbio, Nanjing, China) following the manufacturer's instructions.

16S rRNA sequence analysis

We used a 16S rRNA sequence to analyze the effects of BBr on the communities of intestinal flora and extracted total DNA from 0.2g of feces in each group. SEQHEALTH (Wuhan, China) conducted the sequencing work. Nanodrop was used to quantify DNA and detected the extracted DNA quality by 1.2% agarose gel electrophoresis. We used the V3 + V4 region of the 16S rRNA gene, the target for PCR amplification. The amplified products were recovered and quantified by fluorescence. The sequencing library should be prepared using the TruSeq Nano DNA LT Library Prep Kit(Illumina, San Diego, California) and performing high-quality sequencing. For this experiment's amplicon sequencing bioinformatics workflow, as follows:

The raw sequence is divided into library and sample according to index and Barcode information and then removed the Barcode sequence. Sequence denoising or OTU clustering was performed according to the QIIME2 DADA2 analysis or Searched software analysis process. Presented the specific composition of each sample (group) at the taxonomic level of different species to understand the overall situation. According to the distribution of ASV/OTU in different samples, each sample evaluated the Alpha diversity level and reflected the appropriate sequencing depth through the light curve. We calculated each sample's distance matrix at the ASV/OTU level. At the level of taxonomic composition, we further measured the diversity of species abundance composition among different samples (groups) through various unsupervised sorting, clustering, and modeling methods, combined with corresponding statistical test methods, and tried to find marker species.

Enzyme-linked immunosorbent assay (ELISA)

The forelimb saphenous vein blood was collected on the 14th day of treatment and centrifuged at 15000 RPM /min for 15 min to separate the serum. The serum freezing at -80℃ for later use. According to the manufacturer's instructions, the concentrations of IL-6, IL-1β, and TNF-α in the serum sample detecting by the ELISA kit(Cell Signaling Technology, Boston, USA). Draw a standard curve to calculate the content of cytokines in the sample.

RNA extraction and Quantitative PCR

RT-PCR was used to verify gene-level changes in the CON, DSS, and H-BBr groups. Extracted total RNA from colon tissues using RNeasy Plus Kit (Invitrogen, New York, USA). Following the manufacturer's instructions, reverse transcription performing by using the BioRT Master HisSensi cDNA First-Strand Synthesis kit (Shanghai Roche, China). PCR2720 thermal cycler (Applied Biosystems, USA) and ABI7500 PCR system perform RT-qPCR. The fluorescence reagent used in this experiment was SYBR Green I, and the cycling conditions refer to Additional file 2(Figure S2). The primers used for PCR amplification which listed in Additional file 7(Table S2).

Western blot

We detected the protein levels of the CON group, DSS group, and H-BBr group by using Western blot. Extracted tissue protein on ice, and the protein concentration was determined with a BCA protein determination kit and diluted to an equal concentration (2–4µ g). Protein was separated by 6%, 8%, 10%, and 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The membrane transfer time is proportional to the protein size. After sealing with 5% nonfat milk for two hours at room temperature, the membrane was incubated overnight with cat primary antibody (Additional file 8: Table S3) at 4℃ and acquired with peroxidase-conjugated secondary antibody goat anti-rabbit IgG C. (1:2,000, Cell Signaling Technology) for two hours at room temperature. Finally, it detected the signal with X-ray films (TransGen Biotech Co., Beijing, China).

Statistical

Using GraphPad Prism 7.00 software for experimental data, Student's T-tests and one-way analysis of variance (ANOVA) analyze differences between groups. We processed the images by using Adobe Photoshop 2020 and Imagej_V1.8.0. SPSS 13.0 and statistics were analyzed by means ± standard deviation(x ̅±s). P < 0.05 was considered statistically significant.

BBr alters the experimental features in DSS-induced IBD

All cats developed significant IBD symptoms following DSS uptake, as evidenced by a severe DAI score (Fig. 1B and C) and colon shortening (Fig. 1F and G). However, BBr reverses these clinical characteristics (Fig. 1A-G). The histological examination demonstrated that BBr showed a significant protective effect on colon damage and inflammation, with complete morphology of epithelial cells and abundant goblet cells, complete repair of the glandular structure, but still a small amount of inflammatory cell infiltration (Fig. 1D and E). Consistently, the expression of serum inflammatory factors was upregulated in DSS-treated, while IL-6, IL-1β, and TNF-α were down-regulated after BBr treatment (Additional file 3: Figure S3). Moreover, DSS exposure disrupts the antioxidant system in the gut [14]. Compared with the CON group, the content of MDA increased in the DSS group while it decreased in the H-BBr group (Additional file 4༚Figure S4). Meanwhile, the H-BBr group had an increased antioxidant effect, as evidenced by increased T-AOC, GSH, CAT, and SOD values (Additional file 4༚Figure S4). Notably, the greater the dose of BBr, the less pronounced the DSS-induced IBD features.

BBr modulates DSS-Induced gut microbiota dysbiosis

To observe the effects of BBr on the gut microbiota, we evaluated the composition and diversity of intestinal microbiota using 16S rRNA sequence analysis. It can be seen in Fig. 2A that the species accumulation curve of cat feces collected in this experiment tends to be smooth, which proves that the sequencing depth is adequate. To comprehensively assess the alpha diversity of the microbial community, the Chao1and Observed Species index is used in this process to characterize the richness. The Shannon and Simpson index characterized diversity, and Faith's PD index characterized evolution-based diversity. Pielou's Evenness index characterized evenness, and Good's Coverage index characterized coverage. Compared with the CON group, the diversity of the DSS group increased, but its richness and evolutionary diversity decreased (Fig. 2B). As a carnivore, the diversity of intestinal flora of cats is much smaller than that of omnivores. The smaller the diversity of intestinal flora, the worse its stability. After DSS application, many harmful bacteria and opportunistic bacteria colonized, resulting in increased species diversity of the DSS group, but after BBr treatment, species diversity decreased (Fig. 2B). Estimated differences in gut microbiota composition between groups by employing the principal coordinates analysis (PCoA) and nonmetric multidimensional scaling (NMDS). In the coordinate graph, species composition and structure differences are proportional to the distance between the two samples. The results showed little difference between the CON and H-BBr groups, while the composition and structure of intestinal flora in the DSS group were different from the other groups(Fig. 2C and D). Taken together, BBr can partially alleviate the intestinal microflora disorder of IBD, and the effect is proportional to the dose.

Based on the above results, we expected to determine the intestinal microflora taxa and their relative abundance to determine the exact species with significant differences between the two groups. Here, draw a microbial classification hierarchy tree and show the composition of all taxa. As shown in Fig. 3A, Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the phyla with a significant difference. Prevotella was the noticeably varied bacteria at the genus level in the DSS group, and the relative abundance of bacteria increased in the DSS compared to the CON group. Next, a heatmap figured out the exact species with a significant difference in the two groups. There were 50 species with significant variations and was a significant difference between DSS and CON groups (Additional file 5: Figure S5). LEfSe (LDA Effect Size) analysis finds the marker species significantly different from each group. As illustrated in Fig. 3B, Olsenella is the landmark species of the CON group, which belongs to a group of bacteria under the subclass Red Hemiptera of actinomycetes; the iconic species in the DSS group: Peptostreptococcaceae Clostridium is a bacterium belonging to the genus Clostridium of Digestive Streptococcaceae; the signature species of BBr group: Ruminococcus and Shrektonia.

Last but not least, based on the network analysis of the relationships among microbial members, we try to explore the following questions: Is there a specific group of microorganisms to perform specific ecological functions? Whether or not there is a keystone change is enough to move the composition of the entire community. As shown in Fig. 3C, Lactobacillus was the most dominant species in the experimental group, and there was a negative correlation between BBr and DSS group.

Functional prediction of intestinal microbiota community after BBr treatment

We employed KEGG and MetaCyc to analyze the metabolic and biosynthesis pathway and figure out the possible functions of intestinal microbiota after BBr treatment. As shown in Fig. 4A, the metabolism and biosynthesis of amino acids have the highest abundance, followed by carbohydrate metabolism and nucleotide biosynthesis. Meanwhile, the abundance of species associated with fatty acid and lipid biosynthesis increased remarkably. Furthermore, we attempted to identify metabolic pathways with significant differences between groups. As illustrated in Fig. 4B, after DSS treatment, the relative abundance of species whose functions are associated with the metabolism of linoleic acid and sphingolipid were significantly down-regulated, whereas BBr treatment was upregulating. It indicates that BBr can control metabolic disorders.

BBr rebuilt the intestinal tight junction barrier

The intestinal epithelium consists of intestinal epithelial cells and tight junction proteins, maintaining mucosal homeostasis[15]. Therefore, we next investigated the therapeutic effect of BBr on DSS-induced intestinal barrier dysfunction. We assessed the mRNA expression level of colonic tight junction proteins (ZO-1, ZO-2, E-cadherin, ZEB1, and occluding) by RT-PCR. As shown in Fig. 5A, in contrast to the CON group, BBr significantly upregulated the mRNA expression level of tight junction proteins. Meanwhile, Western blot results demonstrated that BBr could repair the intestinal mucosal barrier. As illustrated in Fig. 5B and C, increased E-cadherin and slug expression in H-BBr-treated cats and down-regulated the expression level of N-cadherin.

BBr reduces inflammation by regulating the TLR4/NF-KB signaling pathway in the colon

Studies have demonstrated that TLR4/NF-KB signaling pathway is the key mechanism leading to persistent inflammation [16, 17]. TLR4/NF-KB signaling pathway was assayed in our research to understand the anti-inflammatory mechanism of BBr deeply. Compared with the CON group, the mRNA expressions of TLR4 and IKB-β significantly increased in the DSS group (Fig. 6A). However, there were no significant changes in NF-kB mRNA and protein levels in the three groups (Fig. 6A and C). More interestingly, protein levels of TLR4 were lower in the DSS group than in the other groups. It should note that TLRs have both protective and harmful effects on inflammation[18]. The host activates the defense system and controls TLR4 protein expression to avoid excessive uncontrolled inflammation. Meanwhile, BBr can inhibit the mRNA expression of IL-1β, TNFα, and IL-6 (Fig. 6B).

BBr activates both MTORC and autophagy

The cellular process of autophagy is required to maintain intestinal homeostasis [19]. BBr activated autophagy, as evidenced by the expression of autophagy indexes such as LC3, Atg5, Atg7, and P62. As shown in Fig. 7A and B, after BBr treatment, the expression of Atg5, Atg7, and LC3 proteins increased and downregulated the expression of P62. MTORC is a negative regulator of autophagy in IBD, composed of mTOR and several adaptors, including Raptor, Rictor, Raptor, and GβL [20, 21]. In contrast, our study found that MTORC and P-mTOR activated in the BBr group (Fig. 7C and D). Hence, the relationship between MTORC and autophagy is elusive and requires further study. To sum up, MTORC and autophagy are essential mechanisms of BBr in the treatment of IBD.

As the status of cats increased in the family, so did their health and welfare. Changes in the environment increase the risk of IBD in cats. Similarly, the number of people with IBD has increased over the years due to deviant lifestyles. Human IBD consists of 2 subtypes: Crohn's disease(CD) and Ulcerative colitis(UC) [5]. Despite being recognized for decades, IBD remains a clinical challenge today. Currently, IBD treating with antibiotics or immunosuppressant drugs. However, unwanted side effects and poor efficacy are inherent problems associated with these drugs [9]. As a result, many traditional plant-based therapies have to explore as alternatives. Moreover, the mechanism of Traditional Chinese Medicine on IBD has increasingly raised interest in recent years.

BBr, as one of the most representative and most studied natural alkaloids, has been shown to display numerous pharmacological activities [5]. There has been a significant and increasing interest in exploring the IBD-preventive effects BBr during the last decades. Our results show that BBr reduces the expression of IL-1β, TNF-α, and IL-6 in serum, suggesting a reduced inflammatory response [20]. What is more, we found that BBr maintains the homeostasis of the antioxidant system in the gut [22]. Studies have shown that BBr can produce oxidized berberine (OBB) under the action of intestinal flora[23], and oxidized berberine can significantly reduce colon shortening and histological damage in IBD patients [24]. Therefore, BBr may also interact with the intestinal flora of cats to generate oxidized berberine, which is anti-inflammatory, alleviates diarrhea symptoms of cats with colitis, and makes the intestinal microenvironment conducive to the colonization of good bacteria.

Abnormal homeostasis of intestinal flora is one necessary pathologic mechanism for IBD[8]. Our study based on 16S rRNA sequence analysis verified that the diversity and composition of intestinal flora in cats with IBD altered. In contrast to previous studies, the diversity of bacteria in this study increased after DSS treatment. The reason may be that the diversity of cats is lower than that of mice and dogs and disrupts the homeostasis more quickly. It should note that the DSS group had a single and small amount of bacteria. It further explains that DSS treatment can promote the propagation of harmful bacteria and temporarily cause an increase in bacterial diversity. We treated IBD cats with BBr and found that their gut microbiota was roughly the same as healthy cats. Firmicutes and Bacteroidetes are the major bacterial phyla in the gastrointestinal tract, and the Firmicutes/Bacteroidetes (F/B) ratio associating with maintaining homeostasis[25]. Our results are consistent with previous studies showing a reduced F/B ratio in IBD cats compared to the CON group. However, Firmicutes and Bacteroidetes were more abundant than in the CON group. The increase of other phyla may influence the F/B ratio.

Significant differences in microbiota composition exist among species[26, 27]. One should remember that changes in this ratio can cause ecological disorders, thereby leading to various pathologies. Here, we found that the F/B ratio increased after BBr treatment to that of healthy cats. Specific probiotics can restore the gut microbial balance by influencing the F/B ratio, as the genus Lactobacillus[26]. Our results imply that BBr treatment dramatically increased the abundance of Lactobacillus, suggesting that certain probiotic strains can manage IBD. Nevertheless, the therapeutic effect of Lactobacillus is limited, and their proliferation can cause pathological damage[28, 29].

A dysbiotic microbiome can affect the host not only directly but also indirectly by altering metabolic processes. Linoleic acid, a component of many cat diets, is degraded less in IBD cats, suggesting an abnormal digestive process that leads to weight loss in IBD cats. Notably, abnormal linoleic acid metabolism increases toxoplasma susceptibility[29]. The abundance of Clostridium spp. is significantly reduced in IBD cats, whose primary function is to deconjugate bile acids and promote fat absorption in the small intestine[27]. However, certain beneficial bacteria (e.g., Blautia spp., Faecalibacterium spp.) that produce immune metabolites such as SCFA increased in IBD cats, caused by the body's defense mechanism[27]. It also illustrates that the intestinal tracts of cats harbor a highly complex microbiota.

Enterotoxins produced by pathogenic bacteria can destroy villous effacement and dysfunction the mucosal barrier[25, 27]. The physical barrier of intestinal epithelial cells and tight junction proteins is part of the intestinal barrier[26]. This study demonstrated that DSS increases intestinal paracellular permeability, alters tight junction proteins' expressions, and destroys colon tissue morphology, suggesting that DSS induces intestinal tight junction barrier dysfunction in vivo. In line with the results obtained in patients with IBD, disrupted the intestinal barrier in cats with 5%DSS. Here, BBr treatment could improve the harsh pathological environments.

Intestinal microflora dysbiosis induces inflammatory reactions through Toll-like receptors (TLRs) [27]. When properly activated, Toll-like receptors protect the gut, but when overactivated, they can cause persistent inflammation that can lead to severe disease[18]. TLR4, the best-characterized member of the toll-like receptors, is known to activate adaptive immune responses and upregulate the expression of inflammatory genes by activating the NF-kB pathway[30]. Notably, more expression of TLR4 in the colon in healthy cats[31]. It may explain why the protein level of TLR4 in the DSS group was lower than that in the other two groups. Hence, BBr can maintain intestinal function and repair the intestinal barrier by controlling TLR4/NF-kB pathway.

Dysfunction of autophagy is associated with the pathogenesis of Crohn's disease [32]. Autophagy is an intracellular degradation pathway and an essential cell survival mechanism. The index of autophagy induction mainly includes microtubule-associated protein one light chain 3 (LC3), Atg7, P62, and Atg5[33]. We found that BBr treatment activated autophagy in IBD cats, as evidenced by increased LC3, Atg5, and Atg7 expression and inhibited P62 expression. Studies have suggested a tight, inverse coupling of autophagy induction and MTORC activation[34]. The components of MTORC include Rictor, Raptor, and GβL[35]. We found that both autophagy and MTORC were activated in IBD cats with BBr treatment, suggesting that the cross-talk between autophagy and MTORC has contradictory results. A recent study has revealed autophagy-induced MTORC activation to avoid excessive myofibroblast accumulation [36]. Long-term stimulation of intestinal mucosa with DSS can induce chronic inflammation, typically characterized by excessive myofibroblasts accumulation. They were reversing this direct interaction under BBr treatment further. Taken together, BBr activates autophagy and MTORC to work together to repair the intestinal barrier.

In conclusion, the principal finding of this study is that BBr could severely recover intestinal microbiome homeostasis, including composition and function. More importantly, BBr reduces inflammation by inhibiting the colon's TLR4/NF-KB signaling pathway. Furthermore, BBr activates MTORC and autophagy in the intestine to promote intestinal epithelial cell proliferation and maintain cell metabolism, restoring intestinal barrier function. These findings provide new insights into the mechanisms of BBr therapy for IBD, in which activated MTORC and autophagy may be involved. Indeed, understanding the composition of intestinal flora in cats is beneficial to finding targeted drugs to treat intestinal diseases in cats, which is of great significance for treating pet gastroenteropathy.

IBD: Inflammatory bowel disease

BBr: Berberine

TLR4: Toll-like receptors 4

TNF-α: tumor necrosis factor α

mTOR: mammalian target of rapamycin

MTORC: mTOR complex

DAI: activity index score

DSS: dextran sulphate sodium

Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Acknowledgments

Not applicable.

Funding

No Applicable Funding.

Author information

Affiliations

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People's Republic of China.

JingWen Cao, XueYing Li

Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.

MiaoYu Chen

Contributions

Conceptualization, Methodology, JW.C., MY.C.; Writing—Original Draft and Review and Editing, JW.C.; Data Curation, JW.C., MY.C., XY.L. The authors read and approved the final manuscript.

Ethics declarations

Ethics approval and consent to participate

This experiment conforms to the Animal Care and Use Committee of Northeast Agricultural University (SRM-11).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests

Crowley SL, Cecchetti M, McDonald RA. Our Wild Companions: Domestic cats in the Anthropocene. Trends in ecology & evolution 2020; 35:477-483.
Grzeskowiak L, Endo A, Beasley S, Salminen S. Microbiota and probiotics in canine and feline welfare. Anaerobe 2015; 34:14-23.
Makielski K, Cullen J, O'Connor A, Jergens AE. Narrative review of therapies for chronic enteropathies in dogs and cats. Journal of veterinary internal medicine 2019; 33:11-22.
Marsilio S, Chow B, Hill SL, Ackermann MR, Estep JS, Sarawichitr B, et al. Untargeted metabolomic analysis in cats with naturally occurring inflammatory bowel disease and alimentary small cell lymphoma. Scientific reports 2021; 11:9198.
Habtemariam S. Berberine and inflammatory bowel disease: A concise review. Pharmacological research 2016; 113:592-599.
de Mattos BR, Garcia MP, Nogueira JB, Paiatto LN, Albuquerque CG, Souza CL, et al. Inflammatory Bowel Disease: An Overview of Immune Mechanisms and Biological Treatments. Mediators of inflammation 2015; 2015:493012.
Levine A, Sigall Boneh R, Wine E. Evolving role of diet in the pathogenesis and treatment of inflammatory bowel diseases. Gut 2018; 67:1726-1738.
Glassner KL, Abraham BP, Quigley EMM. The microbiome and inflammatory bowel disease. The Journal of allergy and clinical immunology 2020; 145:16-27.
Ianiro G, Tilg H, Gasbarrini A. Antibiotics as deep modulators of gut microbiota: between good and evil. Gut 2016; 65:1906-1915.
Chen C, Yu Z, Li Y, Fichna J, Storr M. Effects of berberine in the gastrointestinal tract - a review of actions and therapeutic implications. The American journal of Chinese medicine 2014; 42:1053-70.
Chen DP, Xiong YJ, Lv BC, Liu FF, Wang L, Tang ZY, et al. Effects of berberine on rat jejunal motility. The Journal of pharmacy and pharmacology 2013; 65:734-44.
Jergens AE, Crandell JM, Evans R, Ackermann M, Miles KG, Wang C. A clinical index for disease activity in cats with chronic enteropathy. Journal of veterinary internal medicine 2010; 24:1027-33.
Oktar BK, Gulpinar MA, Bozkurt A, Ghandour S, Cetinel S, Moini H, et al. Endothelin receptor blockers reduce I/R-induced intestinal mucosal injury: role of blood flow. American journal of physiology Gastrointestinal and liver physiology 2002; 282:G647-55.
Li H, Fan C, Lu HM, Feng CL, He PL, Yang XQ, et al. Protective role of berberine on ulcerative colitis through modulating enteric glial cells-intestinal epithelial cells-immune cells interactions. Acta Pharmacol Sin B 2020; 10:447-461.
Parikh K, Antanaviciute A, Fawkner-Corbett D, Jagielowicz M, Aulicino A, Lagerholm C, et al. Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature 2019; 567:49-+.
Rathinam VAK, Chan FK. Inflammasome, Inflammation, and Tissue Homeostasis. Trends in molecular medicine 2018; 24:304-318.
Tang J, Xu L, Zeng Y, Gong F. Effect of gut microbiota on LPS-induced acute lung injury by regulating the TLR4/NF-kB signaling pathway. International immunopharmacology 2021; 91:107272.
Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 2004; 118:229-41.
Elshaer D, Begun J. The role of barrier function, autophagy, and cytokines in maintaining intestinal homeostasis. Seminars in cell & developmental biology 2017; 61:51-59.
Cosin-Roger J, Simmen S, Melhem H, Atrott K, Frey-Wagner I, Hausmann M, et al. Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation. Nature communications 2017; 8:98.
Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation. The Journal of clinical investigation 2015; 125:25-32.
Parikh K, Antanaviciute A, Fawkner-Corbett D, Jagielowicz M, Aulicino A, Lagerholm C, et al. Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature 2019; 567:49-55.
Zhang HY, Tian JX, Lian FM, Li M, Liu WK, Zhen Z, et al. Therapeutic mechanisms of traditional Chinese medicine to improve metabolic diseases via the gut microbiota. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2021; 133:110857.
Li C, Ai G, Wang Y, Lu Q, Luo C, Tan L, et al. Oxyberberine, a novel gut microbiota-mediated metabolite of berberine, possesses superior anti-colitis effect: Impact on intestinal epithelial barrier, gut microbiota profile and TLR4-MyD88-NF-kappaB pathway. Pharmacological research 2020; 152:104603.
Deng J, Zhao L, Yuan X, Li Y, Shi J, Zhang H, et al. Pre-Administration of Berberine Exerts Chemopreventive Effects in AOM/DSS-Induced Colitis-Associated Carcinogenesis Mice via Modulating Inflammation and Intestinal Microbiota. Nutrients 2022; 14.
Stojanov S, Berlec A, Strukelj B. The Influence of Probiotics on the Firmicutes/Bacteroidetes Ratio in the Treatment of Obesity and Inflammatory Bowel disease. Microorganisms 2020; 8.
Suchodolski JS. Diagnosis and interpretation of intestinal dysbiosis in dogs and cats. Veterinary journal 2016; 215:30-7.
Liu J, Song JH, Yang QF, Wang YM. Correlation between Lactobacillus and expression of E-cadherin, beta-catenin, N-cadherin, and Vimentin in postmenopausal cervical lesions. Ann Palliat Med 2022.
D'Alessandro M, Parolin C, Bukvicki D, Siroli L, Vitali B, De Angelis M, et al. Probiotic and Metabolic Characterization of Vaginal Lactobacilli for a Potential Use in Functional Foods. Microorganisms 2021; 9.
Ciesielska A, Matyjek M, Kwiatkowska K. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cellular and molecular life sciences : CMLS 2021; 78:1233-1261.
Asahina Y, Yoshioka N, Kano R, Moritomo T, Hasegawa A. Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats. Vet Immunol Immunop 2003; 96:159-167.
Larabi A, Barnich N, Nguyen HTT. New insights into the interplay between autophagy, gut microbiota and inflammatory responses in IBD. Autophagy 2020; 16:38-51.
Klionsky DJ, Abdel-Aziz AK, Abdelfatah S, Abdellatif M, Abdoli A, Abel S, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy 2021; 17:1-382.
Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation. Journal Of Clinical Investigation 2015; 125:25-32.
Szwed A, Kim E, Jacinto E. Regulation And Metabolic Functions Of Mtorc1 And Mtorc2. Physiol Rev 2021; 101:1371-1426.
Bernard M, Yang B, Migneault F, Turgeon J, Dieude M, Olivier MA, et al. Autophagy drives fibroblast senescence through MTORC2 regulation. Autophagy 2020; 16:2004-2016.

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Berberine maintains intestinal homeostasis through modulating TLR4/ NF-κB / MTORC pathway and autophagy in cats

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Abstract

Figures

Background

Materials And Methods

Animal

Drug administration

Fecal collection

Clinical monitoring and determination of the histological score

Oxidative stress measurement

16S rRNA sequence analysis

Enzyme-linked immunosorbent assay (ELISA)

RNA extraction and Quantitative PCR

Western blot

Statistical

Results

BBr alters the experimental features in DSS-induced IBD

BBr modulates DSS-Induced gut microbiota dysbiosis

Functional prediction of intestinal microbiota community after BBr treatment

BBr rebuilt the intestinal tight junction barrier

BBr reduces inflammation by regulating the TLR4/NF-KB signaling pathway in the colon

BBr activates both MTORC and autophagy

Discussion

Abbreviations

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Data availability

Acknowledgments

Funding

Author information

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Contributions

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Competing interests

References

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Supplementary Files

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