Gut Microbiota and Inflammation Modulation in a Rat Model for Ulcerative Colitis after the Intraperitoneal Administration of Apigenin, Luteolin, and Xanthohumol

Abstract

Ulcerative colitis (UC) is a chronic inflammatory disorder affecting the colon, with symptomatology influenced by factors including environmental, genomic, microbial, and immunological interactions. Gut microbiota dysbiosis, characterized by bacterial population alterations, contributes to intestinal homeostasis disruption and aberrant immune system activation, thereby exacerbating the inflammatory state. This study assesses the therapeutic efficacy of intraperitoneal (IP) injected flavonoids (apigenin, luteolin, and xanthohumol) in the reduction of inflammatory parameters and the modulation of the gut microbiota in a murine model of ulcerative colitis. Flavonoids interact with gut microbiota by modulating their composition and serving as substrates for the fermentation into other anti-inflammatory bioactive compounds. Our results demonstrate the effectiveness of luteolin and xanthohumol treatment in enhancing the relative abundance of anti-inflammatory microorganisms, thereby attenuating pro-inflammatory species. Moreover, all three flavonoids exhibit efficacy in the reduction of pro-inflammatory cytokine levels, with luteolin strongly demonstrating utility in alleviating associated physical UC symptoms. This suggests that this molecule is a potential alternative or co-therapy to conventional pharmacological interventions, potentially mitigating their adverse effects. A limited impact on microbiota is observed with apigenin, and this is attributed to its solubility constraints via the chosen administration route, resulting in its accumulation in the mesentery.

Keywords: anti-inflammatory; flavonoid; gut microbiota; inflammatory bowel disease.

Figure 1

Comparison of the body weight increase in UC-induced animals from the four studied cohorts. The reductions in body weight observed around days 9 to 11 in UC-induced animals are due to the peak in UC symptoms (reduced feed ingest due to colon inflammation). Note that this reduction was minimal in luteolin and xanthohumol cohorts, thus showing a protective effect exerted by these treatments. PBS (red); apigenin (yellow); luteolin (green); xanthohumol (blue).

Figure 2

Measurements of several UC parameters in UC-induced animals from the four studied cohorts: (a) DAI index, (b) stool consistency score at day 12, (c) progression of the DAI index during the last five days of the experiment, and (d) colon ulceration at day 12. Comparisons were performed between the PBS control cohort and each flavonoid treatment cohort. In all cases, luteolin treatment showed a strong protective effect. Circle: control cohort; square: apigenin treatment cohort; upward triangle: luteolin treatment cohort; downward triangle: xanthohumol treatment cohort. Asterisks indicate statistically significant differences (** p < 0.005).

Figure 3

Measure of different pro-inflammatory biomarkers in UC-induced animals from the four studied cohorts: (a) IL-6, (b) IL-1β, and (c) MPO. Comparisons were performed between the PBS control cohort and each flavonoid treatment cohort. All three flavonoids demonstrated efficacy in reducing the levels of the pro-inflammatory cytokine IL-6, whereas only apigenin and luteolin exhibited effectiveness in modulating IL-1β. In contrast, flavonoids did not affect levels of MPO. PBS (red); apigenin (API, yellow); luteolin (LUT, green); xanthohumol (XAN, blue). Asterisks indicate statistically significant differences (** p < 0.005, *** p < 0.0005).

Figure 4

Comparisons of gut microbiota beta diversity metrics between the four studied cohorts: (a) unweighted Unifrac PCoA plot and (b) Permanova test for the unweighted Unifrac beta diversity measure. As ascertained by the unweighted Unifrac beta diversity analysis, the animals within the luteolin and xanthohumol cohorts exhibited greater similarity to each other, while they were more distinct from both the control (PBS) and apigenin cohorts. Conversely, the control and apigenin cohorts demonstrated similarity in terms of microbial community structure. In the PCoA plot, each dot represents one animal, and distances between dots represent the ecological distances between samples. PBS (red); apigenin (yellow); luteolin (green); xanthohumol (blue). Asterisks indicate statistically significant differences (* p < 0.05).

Figure 5

Barplot representation of the relative abundance (%) at the phylum level in the gut microbiota between animals induced with UC across the four experimental cohorts. Bacillota and Bacteroidota constitute 90% of the relative abundance in all cohorts. Higher differences between PBS and flavonoid treatment cohorts could be observed regarding the phyla Pseudomonadota and Verrucomicrobiota.

Figure 6

Barplot representation of the relative abundance (%) at the family level in the gut microbiota populations between the animals induced with UC across the four experimental cohorts. See Table 2 for a detailed list of statistically significant differences between the PBS control animals and each of the flavonoid treatments in the UC-induced animals.