A potential herbal therapeutic for trichinellosis

Abstract

Background: Trichinellosis is a helminthic disease caused by Trichinella spiralis via the ingestion of raw or undercooked meat of infected animals. Current estimates indicate that 11 million humans have trichinellosis, worldwide. The effective use of anti-trichinella medications is limited by side effects and resistance which highlight the critical need for safe and effective drugs, particularly those derived from medicinal plants. Therefore, in the present study, we aimed to evaluate the efficacy of the ethanolic extract of Artemisia annua (A. annua) in treatment of experimentally induced trichinellosis.

Materials and methods: Trichinellosis was induced experimentally in male 6-8 weeks BALB/c mice. BALB/c mice were divided into four groups, 10 mice each. One group was left uninfected and untreated, whereas three groups were infected with T. spiralis. One infected group of mice was left untreated (negative control) while the remaining two infected groups received either 300 mg/kg of the ethanolic extract of A. annua or 50 mg/kg of albendazole (positive control). All treatments started from the third day post-infection (dpi) for 3 successive days. All animals were sacrificed on the 7th dpi for evaluation of treatment efficacy.

Results: Our findings showed that A. annua treatment reduced the T. spiralis adult-worm count in the intestine of infected animals. Moreover, treatment with A. annua restored the normal intestinal architecture, reduced edema, alleviated inflammation as demonstrated by reduced inflammatory infiltrate and expression of TGF-β in intestinal tissues of A. annua-treated animals compared to infected untreated animals.

Conclusions: Our findings show that A. annua extract is effective in treating experimentally induced trichinellosis which highlight the therapeutic potential of A. annua for intestinal trichinellosis.

Keywords: Artemisia annua; T. spiralis; TGF-β; goblet cells; trichinellosis.

Figure 1

Effect of A. annua treatment on the number of adult T. spiralis worm in the small intestine of infected mice. Treatment with A. annua extract significantly reduced worm count in the intestine of infected mice, an effect that was comparable to albendazole (positive control). Data are expressed as mean ± SD (N = 10) and analyzed by using ANOVA followed by LSD as a post hoc test. Asterisk (*) indicate significant difference; p < 0.05.

Figure 2

Treatment with A. annua restored villi architecture and alleviated inflammation in intestinal tissues of T. spiralis-infected mice. (A) Representative image of small intestinal tissue of uninfected mice stained with H&E showing uniform villi and goblet cells (100x). (B) Representative image of small intestinal tissue of T. spiralis-infected untreated mice showing distorted degenerated edematous villi (black arrows) with significant mucosal inflammation (red arrows) (100x). (C) Higher magnification of (B) (400x) demonstrating edema in villi and lamina propria (blue arrows) and also high goblet cell numbers (black arrows) and inflammatory infiltrate composed mainly of eosinophils (red arrows) (D) Higher magnification of (B) (400x) with parasite on surface (red arrows) of distorted villi (black arrows). (E) Representative image of small intestinal tissue of infected mice treated with A. annua extract showing restoration of villi outline and reduced edema (black arrows). (F) Higher magnification (400x) of (E) with similar number of goblet cells (black arrows) and lower inflammatory infiltrate than infected untreated mice (red arrows). (G). Representative image of small intestinal tissue of infected mice treated with albendazole showing protected villi and reduced edema (black arrows) compared to infected untreated mice. (H) Higher magnification (400x) of (G) showing reduced inflammatory infiltrate (red arrows) and similar frequency of goblet cell number (black arrows) compared to infected untreated mice.

Figure 3

Eosinophil cell numbers in intestinal tissues of different groups of mice. Infection of mice with T. spiralis significantly increased eosinophil cell number in intestinal tissue/high power field (HPF) compared to uninfected mice. Treatment with either A. annua or albendazole (positive control) did not result in significant change (ns) in eosinophil cell numbers infiltrating the mucosa and submucosa of small intestine compared to infected untreated mice. Asterisk (*) indicate significant difference between uninfected and infected untreated mice (p < 0.007), whereas ns, non-significant.

Figure 4

Representative microphotographs of intestinal tissue of different groups of mice showing goblet cells. Periodic Acid Schiff's (PAS) staining was performed to demonstrate goblet cells in intestinal tissues. (A) Uninfected mice. (B) Infected untreated mice. (C) Intestinal tissue of mice treated with A. annua ethanolic extract. (D) Intestinal tissue of mice treated with albendazole (positive control). Black arrows point to goblet cells. Magnification, 400x.

Figure 5

Goblet cell numbers in intestinal tissues of different groups of mice. High magnification images (400x) of intestinal tissues stained with PAS and hematoxylin were used to count goblet cells. All infected mice groups (treated and untreated) showed significant high numbers of goblet cells/HPF in comparison to uninfected mice group. Asterisk (*) indicate significant difference, whereas ns, non-significant.

Figure 6

A. annua treatment markedly reduced TGF-β expression in intestinal tissues of T. spiralis-infected mice. (A–D) Immunohistochemical staining of TGF-β in intestinal tissues of different mice groups A. Representative image of intestinal tissue of uninfected mice showing weak expression of TGF-β (black arrows). (B) Representative image of intestinal tissue of infected untreated animals showing high expression of TGF-β in submucosal inflammatory cells (black arrows) and mucosal stromal cells (arrow heads). (C) Representative image of intestinal tissue of albendazole-treated animals showing reduction of TGF-β-positive mucosal stromal cells (black arrows). (D)Representative image of intestinal tissue of A. annua-treated mice showing marked reduction in TGF-β -positive mucosal stromal cells (black arrows). Magnification is 200x.