The ethanol extract of Periplaneta Americana L. improves ulcerative colitis induced by a combination of chronic stress and TNBS in rats

Abstract

Purpose: To investigate the effects of Periplaneta americana L. on ulcerative colitis (UC) induced by a combination of chronic stress (CS) and 2,4,6-trinitrobenzene sulfonic acid enema (TNBS) in rats.

Methods: The experiment UC model with CS was established in rats by a combination of chronic restraint stress, excess failure, improper, and TNBS. The body weight, disease activity index (DAI), colonic mucosal injury index (CMDI), histopathological score (HS) and pro-inflammatory mediators were measured. The content of corticotropin-releasing hormone (CRH) in hypothalamus or adrenocorticotropic hormone (ACTH) and corticosteroids (CORT) in plasma were evaluated by enzyme-linked immunosorbent assay. The proportion of T lymphocyte subsets was detected by flow cytometry, and gut microbiota was detected by 16S rDNA amplicon sequencing.

Results: Weight loss, DAI, CMDI, HS and proinflammatory mediators were reversed in rats by P. americana L. treatment after UC with CS. Increased epidermal growth factor (EGF) was observed in P. americana L. groups. In addition, P. americana L. could reduce the content of CRH and ACTH and regulate the ratio of CD3+, CD3+CD8+ and CD3+CD4+CD25+/CD4+ in spleen. Comparably, P. americana L. changes composition of gut microbiota.

Conclusions: The ethanol extract of Periplaneta Americana L. improves UC induced by a combination of CS and TNBS in rats.

Figure 1. Experimental design.

Figure 2. HPLC analysis of Periplaneta americana L., xanthine, hypoxanthine, inosine, uracil, and uridine. (a) The dried insect body of P. americana L. (b) Ethanol extract of P. americana L. (c) HPLC fingerprint chromatogram of Periplaneta americana L. (d) Peaks identified.

Figure 3. Effect of Periplaneta americana L. on weight loss, colonic length, colonic mucosal injury index in ulcerative colitis model induced by a combination of chronic stress and 2,4,6-trinitrobenzenesulfonic acid (TNBS). Rats continuously received intragastric of sulfasalopyridine or P. americana L. for 14 days. At the end of the experimental period, rats were sacrificed, and colons were removed. (a) Weight loss. (b) Thymus index. (c) Spleen index. (d) Disease activity index. (e) Colonic morphology. (f) Colonic index. (g) Length of the colon. (h) Length-width ratio of the colon. (i) Colonic mucosal injury index scores of each group were determined. Data were presented as mean ± standard deviation (n = 10 per group);

Figure 4. Periplaneta americana L. reduces release of proinflammatory cytokine, inhibits infiltration of inflammatory cell and damage of epithelial cell. These cytokines, such as (a) IL-6, (b) iNOS, (c) COX-2, (d) EGF, (e) IL-17, and (f) MPO were detected by ELISA. (g) The photomicrographs of hematoxylin & eosin staining on colonic sections of rats. (h) Epithelial cell scores and inflammatory cell infiltration were determined. (i) Histopathological scores of each group were determined. Scale bars: 50 μm; magnification: 200×. Data were presented as mean ± standard deviation (n = 6 per group).

Figure 5. The effect of Periplaneta americana L. on the HPA axis in rats after chronic stress with ulcerative colitis. The level of (a) ACTH, (b) CORT, and (c) CRH was measured by ELISA.

Figure 6. Effects of Periplaneta americana L. on T cell subtypes in the spleen in rats after chronic stress with ulcerative colitis. CD3⁺CD4⁺ T lymphocytes, CD3⁺CD8⁺ T lymphocytes, CD3⁺CD4⁺CD25⁺ regulatory T lymphocytes percentage were measured by flow cytometry. (a) Histogram of flow cytometry in each group; (b) CD3⁺ T lymphocytes; (c) CD3⁺CD4⁺ T lymphocytes and CD3⁺CD8⁺ T lymphocytes in spleen; (d) the ratio of CD3⁺CD4⁺ T lymphocytes to CD3⁺CD8⁺ T lymphocytes; (e) the ratio of CD3⁺CD4⁺CD25⁺ regulatory T lymphocytes to CD3⁺CD4⁺ T lymphocytes. Data were presented as mean ± standard deviation (n = 4 per group).