Anti-Inflammatory Effects of Heritiera littoralis Fruits on Dextran Sulfate Sodium- (DSS-) Induced Ulcerative Colitis in Mice by Regulating Gut Microbiota and Suppressing NF- κ B Pathway

Abstract

Materials and methods: The chemical compositions of EFH were identified using LC-ESI-MS. The mice with 3% DSS-induced UC were administered EFH (200, 400, and 800 mg/kg), sulfasalazine (SASP, 200 mg/kg), and azathioprine (AZA, 13 mg/kg) for 10 days via daily gavage. The colonic inflammation was evaluated by the disease activity index (DAI), colonic length, histological scores, and levels of inflammatory mediators. The gut microbiota was characterized by 16S rRNA gene sequencing and analysis.

Results: LC-ESI-MS analysis showed that EFH was rich in alkaloids and flavones. The results indicated that EFH significantly improved the DAI score, relieved colon shortening, and repaired pathological colonic variations in colitis. In addition, proteins in the NF-κB pathway were significantly inhibited by EFH. Furthermore, EFH recovered the diversity and balance of the gut microbiota.

Conclusions: EFH has protective effects against DSS-induced colitis by keeping the balance of the gut microbiota and suppressing the NF-κB pathway.

Figure 1

The LC-ESI-MS chromatograms of EFH. (a) The total ion currents of EFH. (b) Positive mode in black and negative mode in red. Peak assignments are listed in Table 2.

Figure 2

EFH ameliorated inflammatory indices of DSS-induced colitis. (a) The daily body weight changes from 1^st day to 10^th day. (b) The disease activity index (DAI) in mice. (c) The length of colons from a macroscopic perspective. (d) Quantitative measurement of colon length. Data are presented as the means ± S.E.M. (n = 12). ^##p < 0.01 vs. the control group; ∗p < 0.05, ∗∗p < 0.01 vs. the DSS group.

Figure 3

EFH suppressed inflammatory infiltration in DSS-induced colitis. H&E staining of the formalin-fixed sections of colon (scale bars, 100 μm). (a) Magnification ×200 (the parts in red circles were enlarged), (b) magnification ×400, (c) histological scores, and (d) MPO activity of colon tissue in DSS-induced colitis mice. Data are presented as the means ± S.E.M. (n = 10). ^##p < 0.01 vs. the control group; ∗p < 0.05, ∗∗p < 0.01 vs. the DSS group.

Figure 4

Effects of EFH on colonic level of proinflammatory cytokines TNF-α (a), IFN-γ (b), IL-1β (c), and IL-6 (d) as determined by ELISA in DSS-induced colitis mice. Data are presented as the means ± S.E.M. (n = 8). ^##p < 0.01 vs. the control group; ∗p < 0.05, ∗∗p < 0.01 vs. the DSS group.

Figure 5

Effects of EFH on mRNA expression levels of COX-2 (a), iNOS (b), IL-12 (c), IL-17 (d), and IL-4 (e) in colorectums as determined by q-PCR in DSS-induced colitis mice. Data are presented as the means ± S.E.M. (n = 3). ^#p < 0.05, ^##p < 0.01 vs. the control group; ∗p < 0.05, ∗∗p < 0.01 vs. the DSS group.

Figure 6

Effects of EFH on colonic expressions of the NF-κB pathway proteins determined by western blot in DSS-induced colitis mice. Densitometry analyses of western blots were determined by quantifying the protein levels of p-IKKα/IKKα (a), p-IκBα/IκBα (b), and NF-κB p65 nuclear/cytoplasm (c). Data are presented as the means ± S.E.M. (n = 3). ^##p < 0.01 vs. the control group; ∗p < 0.05, ∗∗p < 0.01 vs. the DSS group.

Figure 7

Effects of EFH on the overall structure and microbial diversity of the gut microbiota in DSS-induced colitis mice. (a) OTU rank curve. (b) The histogram of OTU number. (c) Shannon's rarefaction curve of samples. (d) Multiple sample PCA analysis. (e) Ace and (f) Chao of alpha diversity analysis. Data are presented as the means ± S.E.M. (n = 3). ^##p < 0.01 vs. the control group; ∗p < 0.05, ∗∗p < 0.01 vs. the DSS group.

Figure 8

Effects of HQD on structural segregation of the gut microbiota in DSS-induced colitis mice. (a) Phylum, (b) class, (c) order, (d) family, and (e) genus (n = 5).