Ethyl acetate extract of Terminalia chebula alleviates DSS-induced ulcerative colitis in C57BL/6 mice

Abstract

Background: The Chinese pharmacopeia records Terminalia chebula as effective in treating prolonged diarrhea and dysentery, blood in the stool, and prolapse. Modern pharmacological research proves it has multiple pharmacological benefits, including antioxidant, anti-inflammatory, analgesic, hepatoprotective, neuroprotective, and other properties. Objectives: This study aims to clarify the role of Terminalia chebula's ethyl acetate extract (TCEA) on ulcerative colitis (UC) induced by dextran sodium sulfate (DSS) in mice, as well as explore the potential mechanism of action. Materials and methods: The variation of different extracts of T. chebula was detected using the HPLC technique, and the main components in TCEA were identified. DSS was used to establish a mouse model to mimic the physiological state of UC in humans; the alleviating effect of TCEA and positive control 5-ASA on UC mice were evaluated by gavage treatment. Disease progression was assessed by monitoring the mouse's weight change and disease activity index (DAI). The changes in colon tissue were estimated by measuring colon length, HE, and AB-PAS staining and detecting oxidative stress parameters. The results draw from Western blot and real-time PCR showed the TLR4/MyD88/NF-κB pathway may involve in the anti-inflammatory activity of TCEA. Furthermore, the gut flora sequencing technique was employed to monitor the differentiation of intestinal microbiota of mice induced by DSS and TCEA treatment. Results: TCEA significantly lowered DAI scores and inhibited the weight loss and colonic shortening induced by DSS. The colon histomorphology and oxidative stress levels were enhanced after TCEA treatment compared with DSS induced UC group. TCEA attenuated the inflammatory response by regulating TLR4/MyD88/NF-κB pathway activation. Intestinal flora sequencing showed that DSS and TCEA greatly impacted mice's composition and diversity of intestinal microorganisms. But TCEA increased the abundance of Bacteroidetes and decreased the abundance of Firmicutes and Proteobacteria compared with the DSS group, which contributed a lot to returning the intestinal flora to a balanced state. Conclusion: This study confirms the alleviating effect of TCEA on UC and provides new ideas for developing TCEA into a new drug to treat UC.

Keywords: TLR4/MyD88/NF-κB; Terminalia chebula; inflammation; intestinal flora; ulcerative colitis.

FIGURE 1

The HPLC chromatogram of different extracts of Terminalia chebula. (A) 95% ethanol extract. (B) Petroleum ether extract. (C) Dichloromethane extract. (D) Ethyl acetate extract. (E) n-Butanol extract. (F) Aqueous phase extract.

FIGURE 2

The HPLC chromatogram of TCEA and its main components, including Chebulic acid, Gallic acid, Corilagin, Chebulagic acid, Ellagic acid, and Chebulinic acid. (A) Standards. (B) TCEA Sample.

FIGURE 3

Effect of TCEA on the symptoms of DSS-induced colitis in mice. (A) Schematic diagram of animal experiment design. (B) The weight change of mice in each group during the experiment. (C) DAI index of colitis mice. (D) Pictures of the colon in different groups of mice. (E) Colonic length in different groups. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, compared with the control group. *p < 0.05; **p < 0.01; ***p < 0.001, compared with the DSS group.

FIGURE 4

Effect of TCEA on histopathological damage in mice colons. (A) H&E staining of colons. (B) Histological score of colon tissues. (C) AB-PAS staining of colons. (D) Goblet cell count in colon tissue. (i, a) Control. (ii, b) DSS. (iii, c) 5-ASA. (iv, d) TCEA-L. (v, e) TCEA-H. Upper case letters represent ×40magnification, and lower case letters represent ×200magnification. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, compared with the control group. *p < 0.05; **p < 0.01; ***p < 0.001, compared with the DSS group.

FIGURE 5

Effect of TCEA on parameters related to oxidative stress in the tissues of mice with DSS-induced colitis. (A) CAT levels. (B) GSH levels. (C) MDA levels. (D) SOD levels. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, compared with the control group. *p < 0.05; **p < 0.01; ***p < 0.001, compared with the DSS group.

FIGURE 6

The protein and mRNA expression of TLR4, MyD88, and NF-κB p65 in colonic tissues of various groups of mice. (A) TLR4 mRNA expression (B) MyD88 mRNA expression. (C) NF-κB p65 mRNA expression. (D) Representative Western blotting images for TLR4/MyD88/NF-κB signal pathway. (E) TLR4 protein expression. (F) MyD88 protein expression. (G) NF-κB p65 protein expression. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, compared with the control group. *p < 0.05; **p < 0.01; ***p < 0.001, compared with the DSS group.

FIGURE 7

Effect of TCEA on the intestinal microflora of mice with DSS-induced UC. (A) Venn diagram of species in control, DSS, and TCEA-L group. (B) PCA analysis of three groups. (C) Chao index. (D) ACE index. (E) Shannon index. (F) Simpson index. (G) Relative abundance of species at the phylum level for three groups of gut flora. (H) Relative abundance of species at the genus level for three groups of gut flora. ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, compared with the control group. *p < 0.05; **p < 0.01; ***p < 0.001, compared with the DSS group.

FIGURE 8

Analysis of the variability of intestinal flora in three groups and prediction of flora function. (A) Evolutionary branching diagram based on OTU. (B) Differences in the microbial taxa shown by LEfSe analysis. (C) Heat map of PICRUSt2 predicting the function of each sample in control, DSS, and TCEA-L groups.

FIGURE 9

Heatmap of Pearson correlation analysis between the changes in the gut microbiota and UC parameters. Red and blue represent positive and negative correlations, respectively.