Diets containing berries and pomegranates are believed to have potentially manifold benefits to human health, and scientists in India and the U.S. have now demonstrated in mice how one pomegranate-derived metabolite that is produced by microorganisms residing naturally in the gut can help to protect against and reduce the severity of inflammatory bowel disease (IBD). The studies, by researchers at the Institute of Stem Cell Biology and Regenerative Medicine (inStem) in Bangalore, and the University of Louisville, showed that the pomegranate polyphenol-derived metabolite Urolithin (UroA), and a synthetic UroA analog UAS03, both effectively reduced inflammation and restored gut barrier integrity in the mouse models.

Reporting their findings in Nature Communications, the team suggests that as well as having therapeutic uses in the control of inflammatory bowel diseases, UroA and its stable synthetic analog UAS03 may also have clinical applications in other disorders involving gut barrier dysfunction and inflammation. “Restoring the gut barrier and reducing the inflammation using a small-molecule will provide a better therapeutic output in the treatment of IBDs,” said Praveen Kumar Vemula, PhD, at inStem, who is one of the senior authors on the team’s published paper. “A synthetic analog overcomes the stability limitation that a microbial metabolite poses, thus enhancing the efficacy.” The researchers reported their findings in a paper titled, “Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway.”

Crohn’s disease and ulcerative colitis are IBDs that result in intestinal inflammation and changes to gut microbiota. The key role of gut microbiota and their metabolites in biological processes are well recognized, so any alterations to the gut microbiome could be linked with adverse outomes in diseases such as IBD, as well as in cancer, neurological disorders, diabetes, and obesity, the authors wrote.

The gut microbiome is closely associated with the gut epithelium, a single layer of cells lining the gut that acts as a barrier between the gut lumen and the environment outside the gut. Integrity of this gut barrier depends on tight junctions between the epithelial cells, which are maintained by a number of proteins including claudins (Cldn), Zona occlude-1 (ZO1), and occluding (Ocln). “Microbiota in our gut has evolved to generate beneficial microbial metabolites in the proximity of the gut barrier,” notes co-senior author Venkaakrishna Rao Jala, PhD, an assistant professor at the University of Louisville, “However, the exact role of these metabolites have not been identified and the mechanism in which they exert their function is elusive.”

Research has suggested that tight junction proteins are downregulated in IBD, which could impact on barrier integrity. “Previously, it has been reported that levels of tight junction proteins are significantly down-regulated under IBD conditions leading to increased gut permeability to microbial ligands and noxious metabolites resulting in systemic inflammatory responses,” the researchers commented. “Despite the availability of large metagenomics data, the functional dynamics of microbiota and their metabolites in IBDs are unknown.”

tightening of gut barrier cells
Illustration showing tightening of gut barrier cells and reduced inflammation due to gut metabolite. [Praveen Kumar Vemula, inStem, India.]
Pomegranate juice contains high levels of polyphenols including ellagitannins (ETs) and ellagic acid (EA), which prior studies have indicated may have health benefits including preventing hypertension. “They have been recognized as potential nontoxic chemo-preventive compounds against chronic diseases such as cancer, diabetes, and cardiovascular and neurodegenerative disorders,” the authors reported.

The gut microbiome further metabolizes EA to downstream metabolites, including urolithins, which are also thought to have health benefits. Studies indicate that the microbiome-derived EA metabolite UroA, for example, has anti-inflammatory, anti-oxidative and anti-ageing properties, the researchers continued. “The general belief thus far in the field is that urolithins have a beneficial effect only through their anti-inflammatory property,” commented co-author Rajbir Singh, PhD., a postdoctoral fellow at the University of Louisville.

The team set out to investigate the effects of UroA and a potent, stable synthetic UroA analog UAS03 both in vitro and in mouse models of IBD. Their initial results showed that both compounds enhance barrier function by inducing the expression of tight junction proteins, via a mechanism that involved activation of aryl hydrocarbon receptor (AhR)-nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways. “Overall, these results suggest that treatment with UroA/UAS03 increased the expression of tight junction proteins potentially enhancing the gut barrier integrity,” they wrote.

The researchers then tested both UroA and UAS03 in a mouse model of colitis induced by the chemical 2,4,6-trinitrobenzenesulfonic acid (TNBS). The results showed that oral administration of either compound protected the animals from chemically induced weight loss, reduced disease activity index scores and intestinal permeability, as well as reducing markers of inflammation and ulcerative colitis, and protecting against the TNBS-induced downregulation of the tight junction protein Cldn4 in the animals’ colons.

Detailed tissue analyses confirmed reduced colon tissue damage and inflammation scores in animals treated using either UroA or UAS03. In the therapeutic setting, “treatment with UroA/UAS03 also significantly reversed the colitis phenotype by reducing shortening of colons, gut permeability, and inflammation compared to vehicle treatment,” the team reported.

Encouragingly, a single pretreatment with UroA or its analog also demonstrated protective effects against TNBS-induced colitis, with evidence of enhanced gut barrier function and reduced TNBS-induced inflammation. “Despite not receiving further treatments post-TNBS administration, these mice were protected from disease development suggesting prophylactic effects of these compounds through enhanced barrier function,” they wrote. “These results suggest that UroA/UAS03 mediated enhanced gut barrier function will likely have long-term beneficial effects in preventing colitis.”

The team then investigated therapeutic use of UroA or UAS03 in a different, dextran sodium sulfate (DSS)-induced mouse model of colitis. DSS disrupts the epithelial cell barrier, the team explained, which lets bacteria penetrate and leads to inflammation and colonic tissue damage. Again, the results showed that the UroA/UAS03-treated mice demonstrated lower damage and inflammation scores, reduced colon shortening, decreased gut permeability, and reduced inflammation. There was also evidence of treatment-related increases in tight junction proteins. “These results highlight the model independent beneficial activities of UroA/UAS03 in preserving the barrier integrity and mitigating colonic inflammation,” the team stated.

In a final set of tests in NRf2- and AhR-knockout mice, the team demonstrated that protection against colitis afforded by UroA and UAS03 therapy was reliant on the integrity of Ahr-Nrf2 pathways. “Based on these results we propose that UroA/UAS03 exert protective barrier functional activities through AhR-Nrf2-dependent pathways by inducing tight junction proteins,” they wrote.

Existing treatments for IBD include the use of anti-TNF-α antibodies to reduce inflammation, the researchers pointed out. “Here we suggest that enhancing gut barrier functions in addition to inhibiting inflammation might provide better therapeutic options for control of IBDs.”

“We have for the first time discovered that their major mode of function is repairing the gut barrier dysfunction,” Singh commented. “Overall, UroA/UAS03 will not only be efficacious in IBD-related diseases but may also have significant translational implications in other disorders involving barrier dysfunction and inflammation such as alcohol liver diseases, neurological disorders, and colon cancers,” the team concluded.

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