Periplaneta americana Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Rats by Keap1/Nrf-2 Activation, Intestinal Barrier Function, and Gut Microbiota Regulation

Abstract

Periplaneta americana, a magic medicinal insect being present for over 300 million years, exhibits desirable therapeutic outcome for gastrointestinal ulcer treatment. Nowadays, P. americana ethanol extract (PAE) has been shown to ameliorate ulcerative colitis (UC) by either single-use or in combination with other therapeutic agents in clinics. However, its underlying mechanisms are still seldom known. Herein, we investigated the anti-UC activity of PAE by alleviating intestinal inflammation and regulating the disturbed gut microbiota structure in dextran sulfate sodium (DSS)-induced UC rats. Based on multiple constitute analyses by HPLC for quality control, PAE was administrated to DSS-induced UC rats by oral gavage for 2 weeks. The anti-UC effect of PAE was evaluated by inflammatory cytokine production, immunohistochemical staining, and gut microbiota analysis via 16S rRNA sequencing. As a result, PAE remarkably attenuated DSS-induced UC in rats. The colonic inflammatory responses manifested as decreased colonic atrophy, intestinal histopathology scores and inflammatory cytokines. In addition, PAE improved the intestinal barrier function via activating Keap1/Nrf-2 pathway and promoting the expressions of tight junction proteins. It was observed that the UC rats showed symptoms of gut microbial disturbance, i.e., the increased Firmicutes/Bacteroidetes ratio and the significantly decreased probiotics such as Lactobacillus, Roseburia, and Pectobacterium, which were negatively correlated with these detected pro-inflammatory cytokines (secreted by immune CD⁴⁺ T cells, and including IFN-γ, TNF-α, IL-6, IL-8, IL-17, IL-1β). Besides, PAE administration regulated the abnormal intestinal microbial composition and made it similar to that in normal rats. Therefore, PAE could attenuate the DSS-induced UC in rats, by means of ameliorating intestinal inflammation, improving intestinal barrier function, and regulating the disturbed gut microbiota, especially improving beneficial intestinal flora growth, modulating the flora structure, and restoring the intestinal-immune system.

Keywords: 16S rRNA; Periplaneta americana; gut microbiota; intestinal immunity; ulcerative colitis.

FIGURE 1

HPLC chromatograms of mixed standard substances (A) and PAE samples (B). Peaks 1∼9 are derived from cytimidine, uracil, cytidine, hypoxanthine, uridine, thymine, adenine, inosine, and guanosine, respectively.

FIGURE 2

Effects of PAE on the production of NO (A), TNF-α (B), IL-Iβ (C), and PGE₂ (D) in LPS-stimulated RAW 264.7 macrophages. RAW 264.7 cells were pre-incubated in FBS-free medium containing a series of amounts of PAE for 2 h, and then co-treated with 1 μg/ml of LPS stimulation for 12 h. ^∗P < 0.05 blank control group or PAE vs. LPS-stimulated group, P < 0.05 LPS + PAE groups vs. LPS-stimulated group (n = 6 per group).

FIGURE 3

Effects of PAE treatment on the of DSS-induced UC rats after 14 days of continuous gavage. (A) The medication regimen. (B) Rats’ body weight changes from day 1 to day 21 throughout the experiment. (C) DAI scores of rats in various groups. (D) Representative photographs of rats’ colons in various groups. (E) Colon length of rats in various groups. ^∗P < 0.05 untreated control group vs. DSS-induced UC model group, P < 0.05 model group vs. PAE-H group (n = 6 per group).

FIGURE 4

Effects of PAE on DSS-induced UC rats by HE staining and alcian blue staining of colorectum sections (A and C with magnification ×100, B and D with magnification ×200).

FIGURE 5

Effects of PAE on the amounts of inflammatory cytokines, including IFN-γ, TNF-α, IL-6, IL-8, IL-17, IL-1β, in the colorectum tissues of DSS-induced UC rats by ELISA measurement. ^∗P < 0.05 untreated control group vs. DSS-induced UC model group, P < 0.05 model group vs. PAE-H group (n = 6 per group).

FIGURE 6

Effects of PAE on the intestinal mucosal barrier function. mRNA expressions of Keap1 (A) and Nrf-2 (B) determined by real-time quantitative PCR analysis. Expressions of ZO-1 (C), occludin (D), and claudin-1 (E) over GAPDH determined by Western blot analysis. ^∗P < 0.05 untreated control group vs. DSS-induced UC model group, P < 0.05 model group vs. PAE-treated group (n = 6 per group).

FIGURE 7

PAE regulation on the disturbed gut microbiota in DSS-induced UC rats. (A) Rarefaction curves determined at the 97% similarity level. (B) Alpha diversity analyzed by Shannon diversity index. (C) Venn diagram of OTUs in the three groups. (D) PCA analysis of variation. (E) PCoA analysis of variation based on the weighted UniFrac distance. (F) Cluster dendrogram of the three groups based on Jaccard distance. Significant difference among two group was set as ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 (n = 6 per group).

FIGURE 8

Gut microbial community structures of rats from various groups. Microbial community bar plot by phylum (A) and genus (B). a: normal rats serve as the control group; b: UC rats induced by DSS; c: UC rats treated with high-dose PAE (n = 6 per group).

FIGURE 9

Correlation analysis between 40 microflora genera with high abundance in all samples and environmental variables, i.e., the above-mentioned six pro-inflammatory cytokines. Red and blue blocks represent the positive and negative correlations, respectively. Gradation of color indicates the correlation degree. ^∗∗∗P < 0.001; ^∗∗P < 0.01; ^∗P < 0.05 (n = 6 per group).