A Complementary Herbal Product for Controlling Giardiasis

Abstract

Giardiasis is an intestinal protozoal disease caused by Giardia lamblia. The disease became a global health issue due to development of resistance to commonly used drugs. Since many plant-derived products have been used to treat many parasitic infestations, we aimed to assess the therapeutic utility of Artemisia annua (A. annua) for giardiasis. We showed that NO production was significantly reduced whereas serum levels of IL-6, IFN-γ, and TNF-α were elevated in infected hamsters compared to uninfected ones. Additionally, infection resulted in increased numbers of intraepithelial lymphocytes and reduced villi heights, goblet cell numbers, and muscularis externa thickness. We also showed that inducible NO synthase (iNOS) and caspase-3 were elevated in the intestine of infected animals. However, treatment with A. annua significantly reduced the intestinal trophozoite counts and IEL numbers, serum IL-6, IFN-γ, and TNF-α, while increasing NO and restoring villi heights, GC numbers, and ME thickness. Moreover, A. annua treatment resulted in lower levels of caspase-3, which indicates a protective effect from apoptotic cell death. Interestingly, A. annua therapeutic effects are comparable to metronidazole. In conclusion, our results show that A. annua extract is effective in alleviating infection-induced intestinal inflammation and pathological effects, which implies its potential therapeutic utility in controlling giardiasis.

Keywords: IFN-γ; IL-6; NO; TNF-α; giardiasis.

Figure 1

Treatment with A. annua reduced trophozoite counts in the small intestine of infected hamsters. Trophozoites were counted in infected untreated hamsters (IGL) and compared to counts in animals treated with metronidazole (Met) or A. annua (AA) extract. Data are expressed as mean ± SD (n = 8) and were analyzed using ANOVA with Bonferroni corrections for pairwise comparison. Letter “b” indicates significant reductions in trophozoite counts after treatments compared to IGL group (p < 0.001). Letter “c” indicates a significant difference in trophozoite count between Met and AA group (p < 0.001).

Figure 2

The effect of A. annua treatment on NO and proinflammatory cytokine levels in hamsters infected with G. lamblia. We measured NO end products (NOx) in serum of different groups (a) (“a” indicates a significant difference versus UI group (p < 0.001), while “b” indicates a significant difference versus IGL (p < 0.001)). Serum levels of IL-6 (b), IFN-γ (c), and TNF-α (d) were also quantified (“a” indicates a significant difference versus UI group, “b” indicates a significant difference versus IGL group, and “c” indicates a significant difference versus Met group (p < 0.05)). Values represent means ± SD (n = 8), and data were analyzed using ANOVA with Bonferroni corrections as a post hoc test. UI = uninfected animal group, while IGL = infected and untreated animals. Met = infected animals treated with metronidazole and AA = infected animals treated with A. annua extract.

Figure 3

Representative photomicrographs of small intestinal sections from different groups of hamsters stained with hematoxylin and eosin. (a) Representative tissue from uninfected animals showing intact villi (arrowheads), crypts (arrows), connective core of the villi (stars). (b) Representative tissue from infected animals showing shortening and disruption of villi (arrowheads) and retraction of the connective tissue core of the villi (stars). (c) Representative tissue from infected animals that are treated with metronidazole with well-formed mucosal epithelial lining of the villi (arrowheads) and intact villi cores (stars). (d) Representative tissue from infected animals that are treated with A. annua extract showing well-formed villi (arrowheads) with regular epithelial lining and intact connective tissue core (stars). Scale bars = 100 µm.

Figure 4

Treatment withA. annua preserved villi length in infected hamsters.A. annua extract treatment protected villi in infected hamsters. This effect was comparable to metronidazole treatment. “a” indicates significant difference versus UI group and “b” indicates significant difference versus IGL group (p < 0.001). Data are presented as means ± SD (n = 3) and were analyzed using ANOVA test to compare the mean differences between groups with Bonferroni corrections as a post hoc test.

Figure 5

Representative photomicrographs of small intestinal tissues of different groups of hamsters stained with hematoxylin and eosin. (a) Representative tissue from uninfected animals showing intact intestinal villi that are covered with simple columnar epithelium (arrowheads) with goblet cells (asterisks), villi core (stars), and a few IELs (arrows). (b) Representative tissue from infected untreated animals showing disruption of the mucosal epithelium (double-headed arrows), marked increase in IELs (arrows), retracted villi core with a few scattered connective tissue cells (stars), and presence of giardia trophozoites between villi (arrowheads). (c) Representative tissue from infected animals treated with metronidazole showing mucosal epithelium with a few desquamated cells (double-headed arrows), regeneration of the villi core (stars), a few IELs (arrows), and giardia trophozoites in the intervillous spaces (arrowheads). (d) Representative tissue from infected animals treated with A. annua showing preserved intestinal villi with a few desquamated cells (double-headed arrows) and intraepithelial lymphocytes (arrows). Stars point to intact villi core, whereas arrowheads point to a few giardia trophozoites found between the villi. Scale bar = 30 µm.

Figure 6

Treatment withA. annua reduced the number of intestinal IELs in infected hamsters. IELs were counted in images of intestinal tissues stained with hematoxylin and eosin. Infection of hamsters with G. lamblia significantly increased IEL count compared to uninfected animals (IGL versus UI). Treatment with A. annua significantly decreased IEL count compared to infected untreated animals (AA versus IGL). No significant difference was found between metronidazole or A. annua treatment (Met versus AA). “a” indicates significant difference versus UI group and “b” indicates significant difference versus IGL group (p < 0.001). Data are expressed as means ± SD (n = 3) and were analyzed using ANOVA test with Bonferroni corrections as a post hoc test.

Figure 7

Representative photomicrographs of small intestinal tissues of different groups of hamsters stained with Periodic Acid Schiff (PAS) and hematoxylin to demonstrate goblet cells. (a) Representative tissue from uninfected animals showing intestinal villi with goblet cells scattered between epithelia cells. (b) Representative tissue from infected untreated animals showing depletion of goblet cells. (c) Representative tissue from infected animals treated with metronidazole showing restored goblet cells. (d) Representative tissue from infected animals treated with A. annua with restored goblet cells. Scale bar = 50 µm.

Figure 8

Treatment withA. annua restored goblet cell numbers in small intestine of infected hamsters. Images of intestinal tissues stained with PAS and hematoxylin were used to count goblet cells. A. annua extract significantly increased the goblet cell number compared to infected untreated animals (AA versus IGL). A. annua effect was comparable to that of metronidazole. “a” indicates significant difference versus UI group and “b” indicates significant difference versus IGL group (p < 0.001). Data are means ± SD (n = 3) and were analyzed using ANOVA test with Bonferroni corrections as a post hoc test.

Figure 9

Treatment withA. annua restored pathological changes in muscularis externa due to G. lamblia infection. (a) Representative intestinal tissue from uninfected animals showing well-formed muscularis externa composed of smooth muscle fibers arranged in two layers; inner circular (CM) and outer longitudinal (LM) layers. (b) Representative intestinal tissue from infected untreated animals with extensively vacuolated muscle fibers (arrows) and decreased thickness of muscularis externa. (c) Representative intestinal tissue from infected animals treated with metronidazole with a few muscle fibers with vacuolation (arrows). (d) A representative tissue from small intestine of animals infected and treated with A. annua extract showing well-formed muscularis externa layers and a few fibers with vacuolation (arrows). In all panels, the vertical lines with brackets indicate the muscularis externa thickness. Scale bar = 30 µm.

Figure 10

Treatment withA. annua reversed the pathological effect of G. lamblia infection on muscularis externa thickness. Infection of hamsters with G. lamblia (IGL) significantly decreased the muscularis externa thickness compared to uninfected animals (UI). Treatment of infected hamsters with A. annua extract (AA) significantly increased the muscularis externa thickness compared to infected untreated animals, an effect which was comparable to that after metronidazole treatment (Met). (a) indicates significant difference versus UI group. (b) indicates significant difference versus IGL group (p < 0.001). Data are expressed as means ± SD (n = 3) and were analyzed using ANOVA test with Bonferroni corrections as a post hoc test.

Figure 11

A. annua treatment of infected hamsters modulatediNOS expression in small intestine. (a) iNOS expression was localized in core intestinal cells of infected untreated animals (arrows) with no iNOS immunoreactivity in enterocytes. (b) Enterocytes showed high expression of iNOS in infected untreated animals (arrowheads). (c) Moderate iNOS expression was detected in core cells (arrows) as well as enterocytes (arrowheads) of intestines taken from infected animals that were treated with metronidazole. (d) Low iNOS expression was detected in villi core mononuclear cells of infected animals that were treated with A. annua extract (arrows) and was barely detected in enterocytes (arrowheads). Scale bar = 30 µm.

Figure 12

Treatment with A. annua inhibited apoptosis of intestinal cells that is induced by G. lamblia infection. (a,b) Representative intestinal tissue sections of small intestine, from uninfected hamsters, showing weak caspase-3 signals (arrows). (c) Representative intestinal tissue, from infected animals, showing strong caspase-3 signals in enterocytes and lamina propria cells (arrows) and in (d) crypts and muscularis externa (arrows). Representative intestinal tissue, from infected animals treated with metronidazole, showing lower levels of caspase-3 in the enterocytes and villi core cells (e) (arrows) and in crypt cells and muscularis externa (f). (g,h) showing a very low level of caspase-3 in intestinal tissue of infected animals that are treated with A. annua. Scale bar = 30 µm.