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 (IBD). The results showed that rosmarinic acid has a protective effect in an animal model of IBD by regulating intestinal flora. 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.

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. IBD is a chronic, relapsing-remitting systemic disease of the gastrointestinal tract, that is triggered by a complex interplay of genetic variability, the host immune system, and environmental factors. It was found that altered gut flora, which is essential for maintaining intestinal homeostasis, plays an important role in triggering chronic inflammation and regulating chronic diseases, including IBD.

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. A Review of the Anti-Inflammatory Effects of Rosmarinic Acid on Inflammatory Diseases) 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 of mice was fed with normal food and water. The administration of dextran sulfate sodium salt showed 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.

Metagenomic technology was used to determine the composition of the intestinal flora in all groups. The changes in the intestinal tract were examined by histologic staining. 16S rRNA sequencing was used to evaluate the effects of rosmarinic acid on the intestinal flora in mice, and the effects of rosmarinic acid on the intestinal mechanism of action were examined through quantitative PCR and Western blot.

The results

Metagenomic technology showed that 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%). Moreover, abundance in Dubosiella and Lactobacillus was higher in this group than in other groups.

In the second group of mice 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 values. The abundance values in Bifidobacterium and Faecalibaculum were higher in this group, whereas the abundance values of Lactobacillus were lower in this group than in other groups.

The control group had the highest abundance in Lactobacillus (56.3%), Limosilactobacillus (14.7%), and Candidatus Arthromitus (10%). The abundance values in Bifidobacterium pseudolongum, Escherichia coli, and Romboutsia ilealis was 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 and Candidatus Arthromitus sp SFB-mouse-NL. In mice, Lactobacillus johnsonii can reduce inflammation and endoplasmic reticulum stress.

The results also showed that rosmarinic acid reduced a damage of the small intestine tissue caused by dextran sulfate sodium salt, and alleviated the severe weight loss, diarrhea, and blood in the stool. The protective effect of rosmarinic acid on tissue was confirmed by the analysis of tight junction damage, inflammation, and endoplasmic reticulum stress. Animals treated with dextran sulfate sodium salt had the highest mRNA levels of the genes E-cadherin, occlindin, ZEB, and ZO-1, ZO-2 (these genes encode tight junction proteins) compared to the other groups. This group also had the highest levels of mRNA expression for GRP78, IRE1, PERK, ATF6, and CHOP compared to other groups. According to the authors, the upregulation of E-cadherin, ZEB, ZO-1, ZO-2, occlindin at the gene level, the upregulation of tight junction-related genes, and abnormal expression of proteins indicate a disruption of the intestinal barrier. 

In contrast, mRNA levels of the genes E-cadherin, occlindin, ZO-1, ZO-2, and ZEB were lower in mice treated with rosmarinic acid and dextran sulfate sodium than in mice treated with dextran sulfate sodium salt alone. In addition, the treatment of mice with rosmarinic acid and dextran sulfate sodium salt decreased mRNA expression for GRP78, IRE1, PERK, ATF6, and CHOP compared to mice treated with dextran sulfate sodium salt alone.

To assess the dextran sulfate sodium salt-induced inflammation of the small intestine, the researchers used Elisa kits, and determined the expression of interleukin (IL)-6, IL-10, IL-1β, and tumor necrosis factor (TNF)-α. The mice treated with rosmarinic acid and dextran sulfate sodium salt had a decrease in the expression of IL-6, IL-1β, and TNF-α, and an increase in the expression of anti-inflammatory factor IL-10 compared to mice treated with dextran sulfate sodium alone.

The differences in the expression of inflammatory factors and related genes between the mice treated with rosmarinic acid and control group were not significant.

Evaluation of the effect of rosmarinic acid on dextran sulfate sodium salt-induced cell death in mouse intestinal tissue revealed that mice treated with dextran sulfate sodium salt alone had the highest mRNA levels for caspase8, RIPK1, RIPK3, MLKL, Bax, Cytc, caspase12, caspase9, and caspase3. In contrast, the expression levels of these genes were reduced in mice treated with rosmarinic acid and dextran sulfate sodium salt.

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

Since dextran sulfate sodium salt causes abnormal contraction of small intestinal smooth muscle, researchers analyzed the gene expression of CaM, MLC, MLCK, RhoA, and ROCK, as well as the protein expression of p-MLC, RhoA, and ROCK. The findings revealed an increase in the mRNA levels of CaM, MLC, MLCK, RhoA, and ROCK in the group treated with dextran sulfate sodium salt, compared to the other groups. In mice treated with rosmarinic acid and dextran sulfate sodium salt, the protein expressions of p-MLC, RhoA, and ROCK were significantly decreased, indicating that rosmarinic acid could inhibit the abnormal smooth muscle contraction in the mouse intestine induced by dextran sulfate sodium salt.

The above-mentioned results showed that rosmarinic acid downregulated the expression of ROCK, RhoA, CaM, MLC, MLCK, ZEB1, ZO-1, ZO-2, occludin, E-cadherin, IL-1β, IL-6, TNF-α, GRP78, PERK, IRE1, ATF6, CHOP, caspase12, caspase9, caspase3, Bax, Cytc, RIPK1, RIPK3, MLKL, and upregulated the expression of IL-10 and Bcl-2.

The results showed that rosmarinic acid has a protective effect in an animal model of IBD by regulating intestinal flora. Intestinal flora dysbiosis caused by dextran sulfate sodium salt led to inflammatory damage in the small intestine. Rosmarinic acid increased the abundance values of Lactobacillus johnsonii and Candidatus Arthromitus sp SFB-mouse-NL and decreased the abundance values of Bifidobacterium pseudolongum, Escherichia coli, and Romboutsia ilealis. The ability of rosmarinic acid to regulate the intestinal flora led to a reduction in intestinal damage and alleviated endoplasmic reticulum stress. 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.  https://doi.org/10.1128/spectrum.01914-23