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A protective effect of rosmarinic acid in animal model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic, relapsing-remitting systemic disease of the gastrointestinal tract, triggered by a complex interplay of genetic variability, the host immune system, and environmental factors. Previous studies have shown that altered gut flora, essential for maintaining intestinal homeostasis, is important in triggering chronic inflammation, including IBD. In this study, the Chinese authors investigated the protective effect of rosmarinic acid on the intestinal tract in an animal model of inflammatory bowel disease

The human gastrointestinal tract contains around 100 trillion microorganisms that form a complex microbial ecosystem. This symbiotic relationship between the gut microbiota and the host is mutually beneficial. 

Rosmarinic acid is a natural phenolic acid compound, an ester of caffeic acid and 3, 4-dihydroxyphenyl lactic acid, which was isolated for the first time from Rosmarinus officinalis L. leaves and later found in other species of Labiatae and Boraginaceae. Rosmarinic acid has antioxidant, anti-cancer, anti-apoptotic, antiviral, antibacterial, antimutagenic, and anti-inflammatory activities. For more details see Luo C, Zou L. Front. Pharmacol. 28 February 2020. https://doi.org/10.3389/fphar.2020.00153

About the study

A total of 40 mice of both sexes were randomly assigned to four groups. The first group was treated with rosmarinic acid every 24 hours, the second group was treated with rosmarinic acid and 5% dextran sulfate sodium salt every 24 hours, the third group was treated with 5% dextran sulfate sodium salt alone, and the fourth control group was fed with normal food and water.

The intestinal flora in all groups was determined by metagenomic technology. The changes in the intestinal tract were examined by histologic staining. To evaluate the effects of rosmarinic acid on the intestinal flora, the authors used 16S rRNA sequencing, a quantitative PCR, and a Western blot.

Results

The first group of animals treated with rosmarinic acid had the highest abundance in the bacterial genera Lactobacillus (59.4%), Dubosiella (10.2%), and Candidatus Arthromitus (6.9%). The abundance of Dubosiella and Lactobacillus was higher in this group than in other groups.

In the second group treated with rosmarinic acid and dextran sulfate sodium salt, the genera Turicibacter (21.4%), Streptococcus (16.9%), and Clostridium sensu stricto1 (9.9%) had the highest abundance. The abundance of Clostridium sensu stricto1, Sarcina, Streptococcus, and Turicibacter was higher in this group than in the other groups.

In the third group of animals that were given dextran sulfate sodium salt alone, the genera Bifidobacterium (17.6%), Faecalibaculum (17.4%), and Turicibacter (7.9%) had the highest abundance. Compared to other groups, the abundance of Bifidobacterium and Faecalibaculum was higher, whereas the abundance values of Lactobacillus were lower in this group.

The control group had the highest abundance of Lactobacillus (56.3%), Limosilactobacillus (14.7%), and Candidatus Arthromitus (10%). The abundance values in Bifidobacterium pseudolongum, Escherichia coli, and Romboutsia ilealis were higher in this group than in the other groups.

The findings demonstrated that rosmarinic acid, at the species level, could maintain the balance of intestinal flora by reducing the abundance of Bifidobacterium pseudolongum, Escherichia coli, and Romboutsia ilealis, and increasing the abundance of Lactobacillus johnsonii. Lactobacillus johnsonii was shown to reduce inflammation and endoplasmic reticulum stress in mice.

The effect of rosmarinic acid on damage to the small intestine tissue

The administration of dextran sulfate sodium salt produced obvious signs of IBD in mice. Histological examination revealed reduced height or even breakage of the small intestine villi, a severe deformation of cup cells, the absence of muscle-arranged cells, and inflammatory infiltration. Rosmarinic acid reduced damage to the small intestine tissue caused by dextran sulfate sodium salt, and alleviated the severe weight loss, diarrhea, and blood in the stools. The analysis of tight junction damage and inflammation confirmed a protective effect of rosmarinic acid.

Animals treated with dextran sulfate sodium salt had higher mRNA levels of E-cadherin, occlindin, ZEB, and ZO-1, ZO-2 genes, encoding tight junction proteins.  According to the authors, the upregulation of E-cadherin, ZEB, ZO-1, ZO-2, and occlindin at the gene level, the upregulaion of tight junction-related genes, and abnormal expression of proteins suggest a disruption of the intestinal barrier. In mice treated with rosmarinic acid and dextran sulfate sodium, mRNA levels of the genes E-cadherin, occlindin, ZO-1, ZO-2, and ZEB were lower than in mice treated with dextran sulfate sodium salt alone. 

The mice treated with rosmarinic acid and dextran sulfate sodium salt had decreased expression of (IL)-6, IL-10, IL-1β, and tumor necrosis factor (TNF)-α, and increased expression of anti-inflammatory factor IL-10 compared to mice treated with dextran sulfate sodium alone.

The increase in protein levels of caspase8, p-RIPK1, p-RIPK3, p-MLKL, Bax, Bcl-2, caspase12, caspase9, and caspase3 confirmed cell necrosis, apoptosis of the mitochondrial pathway and the endoplasmic reticulum stress pathway. The mice treated with dextran sulfate sodium salt alone had the highest of these proteins, but they were significantly lower in mice treated with rosmarinic acid and dextran sulfate sodium salt.

In addition, the mice treated with rosmarinic acid and dextran sulfate sodium salt had decreased expression of p-MLC, RhoA, and ROCK, indicating that rosmarinic acid could inhibit the abnormal smooth muscle contraction in the mouse intestine induced by dextran sulfate sodium salt.

Conclusion

This study has shown a protective effect of rosmarinic acid in inflammatory bowel disease. The rosmarinic acid effectively reduces intestinal inflammation, intestinal flora dysbiosis, endoplasmic reticulum stress, cell death, and intestinal smooth muscle contraction abnormalities through the regulation of gut microbiota.

These findings provide new insights into the potential treatment of IBD with rosmarinic acid, which acts as a natural antioxidant.

This article was published in Microbiology Spectrum.

Journal Reference

Li K et al. Rosmarinic acid alleviates intestinal inflammatory damage and inhibits endoplasmic reticulum stress and smooth muscle contraction abnormalities in intestinal tissues by regulating gut microbiota. Microbiol Spectrum 2023; September/October 2023 Volume 11 Issue 5.  (Open Access) https://doi.org/10.1128/spectrum.01914-23

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