Total flavone of Abelmoschus Manihot improves colitis by promoting the growth of Akkermansia in mice

The total flavone of Abelmoschus manihot (TFA), a compound extracted from the flowers of Abelmoschus manihot (L.) Medic, has been widely used for the treatment of Crohn's disease, chronic glomerulonephritis and other diseases. The aim of this study was to investigate the effect of TFA on the gut microbiota and intestinal barrier in dextran sulfate sodium (DSS)-induced experimental colitis. C57BL/6J mice were treated with 2.5% DSS in drinking water to induce colitis. Mice were orally administered TFA (62.5 mg/kg, 125 mg/kg) or prednisone acetate (PAT, 2.5 mg/kg) once daily for 7 days. Biological samples were collected for analysis of inflammatory cytokines, gut microbiota and intestinal barrier integrity. TFA-H (125 mg/kg) markedly attenuated DSS-induced colon shortening and histological injury in experimental colitis. The therapeutic effect was similar to that of PAT administration. TFA-H notably modulated the dysbiosis of gut microbiota induced by DSS and greatly enriched Akkermansia muciniphila (A. muciniphila). Moreover, TFA-H remarkably ameliorated the colonic inflammatory response and intestinal epithelial barrier dysfunction. Interestingly, TFA directly promotes the growth of A. muciniphila in vitro. Taken together, the results revealed for the first time that TFA, as a prebiotic of A. muciniphila, improved DSS-induced experimental colitis, at least partly by modulating the gut microflora profile to maintain colonic integrity and inhibit the inflammatory response.


Induction of colitis and treatment. As shown in
, colitis was induced by 2.5% DSS in the drinking water ad libitum for 7 consecutive days (days 1-7). The mice were randomly allocated after modelling (n = 6-8). The mice were supplemented daily with 200 µL of phosphate buffered saline (vehicle), TFA (125 mg/kg, 62.5 mg/ Quantitative real-time polymerase chain reaction (qPCR). Total RNA was extracted from colon tissues using TRIzol reagent, and the concentration of RNA was measured and then reverse transcribed according to the manufacturer's instructions using a HiScript 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). cDNA was used for qPCR using SYBR Green Master Mix (Service, Wuhan, China) on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). Relative amounts of mRNA were calculated using the 2 −ΔΔCT method, and GAPDH served as the housekeeping gene. The primer sequences are shown in Table 2. The abundance of A. muciniphila in stool samples was quantified by quantitative PCR. All procedures were performed according to Everard et al. 8 .
16S rDNA gene high-throughput sequencing. The V3-V4 variable region of the bacterial 16S rRNA gene was amplified by F338 (5′-ACT CCT ACG GGA GGC AGC A-3′) and R806 (5′-GGA CTA CHVGGG TWT CTA AT-3′). On the Illumina MiSeq platform, the extracted PCR products were analysed by isomolecular 250bp double-terminal sequencing. The original pyrophosphate sequence was uploaded to the NCBI Data Center database SRA (Sequence Read Archive). High-quality sequence merge overlaps generated fastq files. QIIME (version 1.9.1, https:// qiime. org/) software was used to multichannel decode and quality control filter the fastq file output. All sequencing and bioinformatics analysis were performed using the Omicsmart online platform (http:// www. omics mart. com).
Bacterial strains and growth curve. A. muciniphila strain ATCC was cultured in brain heart infusion (BHI) medium in tubes at 37 °C in an anaerobic chamber. A. muciniphila were collected in log phase and diluted with sterile phosphate-buffered saline (PBS) to 3 × 10 8 colony-forming units/mouse for gavage. A. muciniphila freshly prepared every day for gavage. To acquire the growth curve of A. muciniphila, different concentrations of TFA were added to BHI medium, and then an A. muciniphila suspension was added to a final concentration of bacteria 10 6 cfu /ml. The growth profile was evaluated by intermittently measuring absorbance at 600 nm every 5 hours 9 . Mucin was added to the final concentration of 4 g/L. TFA was dissolved in PBS. Each experiment was repeated three times. www.nature.com/scientificreports/ Statistical analysis. Graphing was performed using GraphPad Prism (version 9.0, https:// www. graph pad. com). One-way ANOVA analysis of variance was applied to compare differences between multiple groups. When only two groups were compared, Student's t-test was conducted. Non-parametric, were tested by the Mann-Whitney test. A value of P < 0.05 indicated that the difference was statistically significant. All plots are shown as the mean ± standard error of the mean (S.E.M). P < 0.05 was considered statistically significant.

Statement.
We confirm that all methods in our study are reported in accordance with ARRIVE guidelines.

Result
Effects of TFA on damage in the colon of DSS-induced colitis mice. As shown in Fig. 1, the DSS group showed significant weight loss, diarrhoea, haematochezia and other colitis symptoms (Fig. 1B,C). Treatment with TFA (62.5 and 125 mg/kg) significantly improved weight loss and decreased the DAI score in a dosedependent manner. As shown in Fig. 1D,E, compared to that of the control group, the colon length was markedly shortened in the DSS group (P < 0.01). In contrast, TFA-H (P < 0.01) and PAT (P < 0.01) were used to significantly improve the colonic shortening induced by DSS. As shown in Fig. 1F, in the control group, the colon tissue structure was intact. In contrast, the DSS group was characterized by inflammatory cell infiltration, epithelial cell destruction and mucosal thickening. Consistent with the symptom observations, compared to the DSS group, the TFA-H and PAT groups had significantly restored intestinal epithelial structure and reduced severe inflammation. These results indicated that TFA-H has an obvious protective effect on DSS-induced colitis, which was similar to that of PAT and superior to that of TFA-L.
Effects of TFA on the production of inflammatory cytokines. Inflammatory molecules are involved in the processes that occur in colitis. To elucidate the inflammatory response in DSS-induced mice, various proinflammatory cytokines were measured in colon tissues at the mRNA level. As illustrated in Fig. 2A-F, the levels of proinflammatory cytokines, including TNF-α, IL-6, interferon-γ (IFN-γ), interleukin-18 (IL-18) and interleukin-17a (IL-17a), were significantly increased in DSS-induced colitis mice (P < 0.05 vs. CON). These elevated proinflammatory cytokines were all decreased by TFA in a dose-dependent manner. Chemokine ligand www.nature.com/scientificreports/ 2 (CCL2) was significantly increased by DSS treatment compared with that in the control group. Moreover, TFA and PAT significantly decreased CCL2 compared with the DSS group (Fig. 2F). Proinflammatory cytokines were also measured in serum at the protein level. Quantification of specific cytokines using ELISA showed the same patterns in the regulation of the production and secretion of proinflammatory cytokines (   www.nature.com/scientificreports/ The most abundant taxa at the phylum, family and genus levels are shown in Fig. 4D-F. Compared with the control group, the abundance of Verrucomicrobia was lower in the DSS group, while the abundances of Tenericutes and Proteobacteria were higher, which was consistent with a previous report 10,11 . At the genus level, the abundances of Clostridium, Parabacteroides, Ruminococcus and Romboutsia were remarkably increased, whereas those of A. muciniphila and Lactococcus were significantly decreased in the DSS group compared to the control group (P < 0.05). Following treatment with TFA, the abundance of Tenericutes and Proteobacteria nearly returned to normal levels. TFA treatment significantly increased the relative abundance of A. muciniphila, which belongs to Verrucomicrobia. PCR also proved the increase in absolute abundance of A. muciniphila in the TFA group (Fig. 4C). Spearman correlation analysis showed that the abundance of A. muciniphila is negatively correlated with IL-6, IFN-γ and CCL2, and positively correlated with MUC2, ZO-1 and KLF4 ( Supplementary Fig. S2A). However, the low-dose TFA (62.5 mg/kg) did not increase the abundance of A.muciniphila ( Supplementary  Fig. S2B,C). The microbial flora structure was favourably harmonized by treatment with TFA-H.
Furthermore, LefSe (LDA effect size) analysis was used to identify dominant flora in each group (Fig. 5).

TFA promoted A. muciniphila growth in vitro.
To examine whether TFA directly promoted the growth of A. muciniphila in vitro, the growth curve of A. muciniphila was monitored in BHI medium with mucin (Fig. 6). TFA (1 µg/mL and 0.5 µg/mL) directly promoted the growth of A. muciniphila in vitro. However, the much lower(0.1 μg/ml) concentrations cannot promote the growth of A. muciniphila. When TFA (1 µg/mL or 0.5 µg/mL) was added, A. muciniphila grew faster at log phase and plateaued at a much higher cell density. We added much higher concentration of TFA (10 µg/mL and 100 µg/mL) and the results showed that the promotion effect of TFA (10 µg/mL) is inferior to that of TFA(1 µg/mL). TFA (100 µg/mL) inhibited the growth of A. muciniphila (Supplementary Fig. S3). Thus, we inferred that TFA promoted the growth of A. muciniphila in a dose-dependent manner within a certain concentration range.

A. muciniphila alleviated colitis in mice.
To confirm whether A. muciniphila played an essential role in DSS-induced colitis. We treated DSS-induced mice daily with A. muciniphila (3×10 8 ) or PBS for 1 week after www.nature.com/scientificreports/ DSS modelling. Treatment with A. muciniphila relieved DSS-induced colitis, which was evidenced by reduced weight loss, colon length shortening and histological damage (Fig. 7A-E). Serum and colon tissue levels of inflammatory cytokines (TNF-α, IL1β, IL6) decreased as a result of A. muciniphila treatment (Fig. 7F-G). A. muciniphila treatment increased the expression of the tight junction proteins ZO-1, MUC2 and KLF4, supporting a potential role in the regulation of intestinal barrier integrity (Fig. 7H-K). In summary, A. muciniphila ameliorated DSS-induced colitis, improved macroscopic and histological damage, decreased inflammatory cytokines, and protected intestinal barrier integrity.

Discussion
Studies have shown that the gut microbiota of UC patients is out of balance 12 . Metagenomic research has shown that the abundance and diversity of the gut microbiota of UC patients are reduced 2 . Recent studies have shown that certain dietary agents, spices, oils, and dietary phytochemicals that are consumed regularly possess beneficial effects in regulating gut microbiota 13,14 . The aim of this study was to characterize the effects of TFA on DSS-induced colitis, mainly focusing on the composition of the gut microbiota and intestinal barrier in mice with DSS-induced colitis. In our study, TFA relieved the symptoms of weight loss and colon length shortening in DSS-induced mice. Compared with PAT, TFA-H has advantages in weight and DAI score. In terms of colonic histopathology, DSSinduced colitis in mice exhibited serious injuries, with the loss of histological structure, disruption of the epithelial barrier, a pronounced decrease in the number of crypts, and marked infiltration of granulocytes and mononuclear cells into the mucosa and submucosa. Compared to the DSS group, the TFA group was observed to effectively reduce histologic inflammation. Notably, TFA-H exhibited a similar effect to PAT and exerted a superior effect to TFA-L.
Animal experiments and clinical studies have found that an increase in proinflammatory cytokines further damages intestinal mucosal barrier function through activation of the NF-κB signalling pathway. Our previous study showed that TFA could markedly inhibit the release of intestinal inflammatory cytokines in Crohn's disease rats induced by 2,4,6-trinitrobenzene sulfonic acid, improve intestinal inflammation and ameliorate colitis symptoms 6 . In this study, we demonstrated that supplementation with TFA significantly decreased the mRNA expression of inflammatory cytokines (TNF-α, IL-6, IL-18, IL-17a, IFN-γ) and chemokines (CCL2) in colon tissue. TFA also decreased the protein expression of inflammatory factors (TNF-α, IL-6, IL-1β) in the serum. Notably, TFA-H exerted superior anti-inflammatory effects to TFA-L.
The intestinal mucosal barrier plays an important role in the pathogenesis of colitis 15 . The function of the intestinal mucosal barrier mainly refers to the isolation of the intestinal lumen from the environment to prevent the invasion of bacteria and toxic substances. The intestinal mucus layer is a protective gel-like substance covering the surface of the intestinal mucosa, which is the first barrier in the intestinal lumen. A large amount of mucin synthesized and secreted by goblet cells is the most important substance that constitutes the mucus layer of the intestine 16 . The destruction of the mucus layer and the pathological changes of goblet cells are closely related to the progression of colitis 17 . Our experiment showed that DSS-induced colitis decreased the mRNA expression of KLF4, MUC2 and ZO-1 in colon tissue. KLF4 is a zinc finger transcription factor expressed in differentiated www.nature.com/scientificreports/ epithelial cells of the intestine and is widely involved in the regulation of cell proliferation, differentiation and embryonic development, especially in the proliferation of goblet cells 18 . Studies have shown that goblet cells are not fully developed and that the expression of mucin MUC2 is abnormal in the colon tissue of KLF4 −/− mice 19 . The number of goblet cells in the colon tissue of intestine-specific KLF4 deletion mice was significantly reduced 20 .
In the mucus layer secreted by goblet cells, the highest content of intestinal epithelial cell proliferation and differentiation is mucin MUC2. MUC2 effectively blocks pathogens in the intestinal lumen from invading intestinal epithelial cells. MUC2 also provides a habitat and nutrients for symbiotic bacteria in the intestine. The mucus layer disappeared in the colon of MUC2 −/− mice, direct contact between the gut microbiota and intestinal epithelial cells triggered an inflammatory response, and spontaneous colitis eventually formed 21,22 . ZO-1 is crucial for connecting individual epithelial cells and maintaining the integrity of the epithelium. The reduction of ZO-1 interrupts the assembly of tight junctions by inhibiting the recruitment of other components 23 . Destruction of intestinal barrier integrity causes pathogens in the intestinal lumen to invade intestinal epithelial cells, increases the immune response of intestinal epithelial cells, and ultimately strengthens the intestinal inflammatory response. Treatment with TFA improved intestinal mucosal barrier integrity by promoting the production of MUC2, KLF4 and ZO-1.
A previous study showed that the composition of the intestinal flora is altered in UC patients or in DSS-treated mice 24 . In addition, the bacteria associated with the mucosa increase the mucus layer thickness and promote the repair of the intestinal barrier 25 . Several studies have focused on the clinical improvement of DSS-induced colitis by using probiotics and antibiotics to modulate the commensal microbiota 26 . Dietary flavone could contribute to the maintenance of intestinal health by preserving the gut microbial balance through the stimulation of the growth of beneficial bacteria and the inhibition of pathogenic bacteria 27 . We found that the gut microbiota of mice with DSS was substantially changed during TFA-H or PAT treatment. In our study, a lower abundance of A. muciniphila and Bifidobacterium and a higher abundance of Actinomyces, Escherichia coli and Proteobacteria were observed in the DSS group than in the control group, which was consistent with previous reports. Studies have shown that the abundance of Actinomycetes in UC patients is significantly increased, and Actinomyces is also significantly increased in intramucosal carcinomas 28 . Escherichia coli, a conditional pathogenic bacterium, causes the disease to worsen by destroying intestinal barrier integrity 29 . Treatment with TFA-H reversed the intestinal dysbacteriosis caused by DSS. In addition, the abundance of A. muciniphila in TFA-H was significantly increased.
Our previous study found that in patients with UC, the abundance of A. muciniphila was significantly reduced. This is consistent with the results reported in the literature [30][31][32] . A prospective study unequivocally showed that 3 months of administration of A. muciniphila (10 10 cfu/day) was safe and verified the feasibility and tolerance of A. muciniphila supplementation in humans. In our study, 1 week of treatment with A. muciniphila (3×10 8 cfu/ mouse/day) improved the colitis induced by DSS. Treatment with A. muciniphila significantly reduces the expression of inflammatory cytokines (TNF-α, IL-6, IL-1β) and protects the intestinal barrier by increasing the thickness of the mucus layer and the expression of tight junction proteins. Studies have shown that A. muciniphila is a protein-degrading anaerobic bacterium attached to the mucus layer of the intestine. It can promote the metabolism of the mucous layer, thereby creating a healthy environment for intestinal epithelial cells 8,33 . In vitro experimental studies have shown that A. muciniphila enhances the integrity of the intestinal epithelium and repairs the damaged intestinal mucosal barrier. This may be related to the metabolites of A. muciniphila. A. muciniphila maintains the homeostasis of the intestinal epithelium and inhibits the immune response of intestinal epithelial cells by degrading the host's intestinal mucus into short-chain fatty acids 34 . Previous study has shown that A. muciniphila, its outer membrane protein Amuc_1100 and the extracellular vesicles derived from A. muciniphila could protect the progression of DSS-induced colitis and maintain the integrity of the intestinal mucosal barrier. The mechanism by which A. muciniphila relieves colitis may be related to the interaction of Amuc_1100 with Toll-like receptor 2. Extracellular vesicles derived from A. muciniphila promote tight junction expression by activating AMPK 35,36 . A study certified that A. muciniphila promotes intestinal inflammation in a germ-free IL10 −/− mouse model of IBD 37 . However, a subsequent study showed that A. muciniphila was examined in gnotobiotic IL10 −/− mice and did not promote intestinal inflammation 38 . A. muciniphila plays a regulatory role in maintaining intestinal barrier integrity, host metabolism and other biological functions 39 .
In the in vivo experiment, PCR confirmed the increase (P < 0.01) in absolute abundance of A. muciniphila in mice treated with TFA-H (Fig. 4C). In an in vitro experiment, TFA promoted the growth of A. muciniphila in a dose-dependent manner within a certain concentration range. A. muciniphila grew faster at the logarithmic phase and at a higher cell density in the platform period. TFA may improve the intestinal barrier to relieve colitis through its prebiotic effect on A. muciniphila.

Conclusion
The study showed that TFA treatment improved the colitis caused by DSS, at least partly through modulating gut microbiota and restoring the integrity of the intestinal barrier. Our study further suggested that TFA administration promotes the growth of A. muciniphila, which may be associated with this protective effect. We reveal that TFA, as a prebiotic of A. muciniphila, effectively treats colitis.

Data availability
Illumina amplicon sequences were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession number PRJNA765530. www.nature.com/scientificreports/