Korean J Physiol Pharmacol.  2021 Sep;25(5):439-448. 10.4196/kjpp.2021.25.5.439.

Anti-inflammatory effects of DA-9601, an extract of Artemisia asiatica, on aceclofenac-induced acute enteritis

• Kim JH
• Shin CY
• Jang SW
• Kim DS
• Lee W
• Kim HG
• Kim HR

• KMID: 2519406
• DOI: http://doi.org/10.4196/kjpp.2021.25.5.439

Abstract

DA-9601 is an extract obtained from Artemisia asiatica, which has been reported to have anti-inflammatory effects on gastrointestinal lesions; however, its possible anti-inflammatory effects on the small intestine have not been studied yet. Therefore, in this study, we investigated the protective effects of DA-9601 against the ACF-induced small intestinal inflammation. Inflammation of the small intestine was confirmed by histological studies and the changes in the CD4 ⁺ T cell fraction induced by the inflammation-related cytokines, and the inflammatory reactions were analyzed. Multifocal discrete small necrotic ulcers with intervening normal mucosa were frequently observed after treatment with ACF. The expression of IL-6, IL-17, and TNF-α genes was increased in the ACF group; however, it was found to have been significantly decreased in the DA-9601 treated group. In addition, DA-9601 significantly decreased the levels of proinflammatory mediators such as IL-1β, GMCSF, IFN-γ, and TNF-α; the anti-inflammatory cytokine IL-10, on the other hand, was observed to have increased. It is known that inflammatory mediators related to T cell imbalance and dysfunction continuously activate the inflammatory response, causing chronic tissue damage. The fractions of IFN-γ ⁺ Th1 cells, IL-4 ⁺ Th2 cells, IL-9 ⁺ Th9 cells, IL-17 ⁺ Th17 cells, and Foxp3 ⁺ Treg cells were significantly decreased upon DA-9601 treatment. These data suggest that the inflammatory response induced by ACF is reduced by DA-9601 via lowering of the expression of genes encoding the inflammatory cytokines and the concentration of inflammatory mediators. Furthermore, DA-9601 inhibited the acute inflammatory response mediated by T cells, resulting in an improvement in ACF-induced enteritis.

Keyword

DA-9601; Inflammation; NSAIDs; Small intestine; T cell

Figure

• 

  Fig. 1 Representative images depicting histological appearance of small intestine tissues after ACF-induced enteropathy. (A) Small intestine section from the normal control group. Small intestine section from (B) ACF treated group (C) ACF + DA-9601 treated group, (D) ACF + ESO treated group, and (E) ACF + ESO + DA-9601 treated group. Multifocal discrete small necrotic ulcers with intervening normal mucosa were observed. Frequent necrotic ulcers through small intestine were also observed, and the degrees of necrotic damage were considered very severe with accompanying intestinal micro perforations with peritonitis (H&E, magnification: ×40 and ×100). ACF, aceclofenac; ESO, esomeprazole.

• 

  Fig. 2 Expression levels of IL-6, IL-17, and TNF-α mRNA in small intestine after ACF-induced enteropathy. Expression of IL-6 (A), IL-17 (B), and TNF-α (C) was quantified using qRT-PCR. The relative mRNA levels of IL-6, IL-17, and TNF-α were calculated by normalizing them to the expression level of GAPDH by using the 2–∆∆Ct method. The expression values obtained from the drug-treated small intestine were normalized to those of the control. Data are represented as the mean ± SEM. A, aceclofenac (ACF); D, DA-9601; E esomeprazole. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

• 

  Fig. 3 Concentrations of IL-6, IL-1β, IL-10, GM-CSF, IFN-γ, and TNF-α in small intestine after ACF-induced enteropathy. Quantification of (A) IL-6, (B) IL-10, (C) IL-1β, (D) GM-CSF, (E) IFN-γ, and (F) TNF-α in the small intestine by using ELISA. The concentration of whole lysates homogenized from each sample was normalized, and the lysates were diluted within the measurable range. Statistical significance was evaluated using one-way ANOVA. Data are represented as mean ± SEM. A, aceclofenac (ACF); D, DA-9601; E, esomeprazole. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

• 

  Fig. 4 Flow cytometric characteristics of CD4+T cells in small intestine after ACF-induced enteropathy. (A) Numbers inside quadrant indicate the percentage of the cell population. Only 30% of the cell populations were presented in the flow cytometric characteristics graph. (B) The proportion of CD4+ T cells from lamina propria was determined according to intracellular levels of cytokines for (a) interferon (IFN)-γ+ Th1 cell, (b) interleukin (IL)-4+ Th2 cells, (c) IL-9+ Th9 cells, (d) IL-17+ Th17 cells, and (e) Foxp3+ Treg cells. A, aceclofenac (ACF); D, DA-9601; E, esomeprazole.

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