Pumpkin seeds (Cucurbita pepo subsp. ovifera) decoction promotes Trichinella spiralis expulsion during intestinal phase via "Weep and Sweep" mechanism

Abstract

Trichinosis is a zoonotic disease of communal health concern as it instigated human outbreaks in several countries. Besides, the development of resistance, traditional therapy has numerous antagonistic effects. Thereby, finding efficient natural alternatives is required. In comparison to albendazole, this study evaluated the impact of pumpkin decoction on Trichinella spiralis in experimentally infected mice. The anthelmintic action of pumpkin decoction (500 mg/kg) was determined using T. spiralis infected mice in enteric phase for 5 days. Pumpkin decoction anthelmintic activity fortified by mixing with honey (1:1). Pumpkin decoction and Pumpkin decoction-honey mixture were evaluated by comprising with reference drug, albendazole (50 mg/kg). The T. spiralis adult count was significantly lower in all treated groups, with the pumpkin decoction-honey mixture showing the largest reduction (83.2%) when compared to the infected group (P ≤ 0.001). The intestinal histological changes and the level of COX-2 expression in the intestinal tissue were both significantly reduced in the same group. The pumpkin decoction improved the immune response, as evidenced by a significant decrease in nitric oxide (NO) and tumor necrosis factor (TNF-α) and a significant increase in the expression of the transforming growth factor (TGF-1β) and interleukin-17 (IL-17). The pumpkin decoction's anthelmintic action was facilitated by the TGF-1β and IL-17-driven Weep and Sweep mechanism. Both administration of pumpkin decoction beside honey showed the best treatment group that resulted in high infection reduction besides amelioration of biochemical markers and restoration of histological to normal state. In conclusion, pumpkin decoction is highly effective against T. spiralis which could be a promising alternative herbal drug and the pumpkin decoction effect was higher in the case of combination with honey.

Figure 1

TNF-α of trichinosis mice treated with pumpkin decoction and its mixture with honey. Results are shown as the means ± SE of six duplicates. The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Figure 2

TGF-β1 of trichinosis mice treated with pumpkin decoction and its mixture with honey. Results are shown as the means ± SE of six duplicates. The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Figure 3

IL-17 of trichinosis mice treated with pumpkin decoction and its mixture with honey. Results are shown as the means ± SE of six duplicates. The results were construed using one-way ANOVA using BIM-SPSS version 25, P ≤ 0.001, Value with the same letter has no significance but the value with different letter has significant at 0.001.

Figure 4

Sections of ileum of studied groups; (a,b): uninfected control group showing normal appearance of intestinal layers mucosa comprised in columnar cells (red arrowhead) with goblet cells (wavy arrows), lamina propria (curved arrows); submucosa (green arrow), musculosa (red arrow) and serosa covered with mesothelium (arrowhead). Also, intestinal villi (arrows) with normal architecture and length, shallow intestinal crypts are visible (blue arrow). (c,d): Infected, non- treated group showing malformation of the intestinal villi (arrowhead) and marked inflammation (I) and mature T. spiralis lodged in the intramucosal and in the lumen (arrows). (e,f): Pumpkin decoction treated mice with moderate inflammatory cell infiltrates and slight necrosis could be noticed in villi (curved arrows). (g,h): Honey treated mice showed moderate inflammatory infiltration (I) and necrotic and damaged villi (curved arrows). (i,j): Pumpkin decoction + honey mixture administrated group displayed an improved appearance, with the majority of the villi returning to their typical design with hyperplasia of goblet cells and an apparent decrease in the inflammatory infiltration. (k,l): Albendazole manipulated group exhibited T. spiralis adults in the core of villi (black arrows) with obvious inflammatory infiltration (red arrow), also villi appeared detached and atrophied (arrowheads). Original magnifications were 200 × (Scale-bar: 100 µm), 400× (Scale-bar: 50 µm). Quantitative estimation of mean number of goblet cells from 4 images/sample/group using Image-Pro Plus software is charted down the images. ∗∗ ∗p < 0.05 compared to uninfected. ∗∗ p < 0.05 compared to infected. ∗p < 0.05 compared to pumpkin. #p < 0.05 compared to honey. $p < 0.05 compared to standard.

Figure 5

(a) Ileum tissue of negative control group (GI) showing no immunostaining, (b): infected control group (GII), COX-2 is expressed moderately in the mucosal epithelium and diffusely in inflammatory cells at the center of intestinal villi with moderate expression cells. (c) Subgroup (I) that received pumpkin decoction showing focused expression of COX-2 in inflammatory cells and no expression in epithelial cells. (d): COX-2 expression in the epithelial cells of the subgroup (II) that received honey was moderate, and it was widely expressed in inflammatory cells. (e): COX-2 expression was null in the epithelium and inflammatory cells in subgroup (III) that had combination treatment (f) The subgroup (IV) that only received albendazole revealed that COX-2 was moderately expressed in the cytoplasm of inflammatory cells and the inner core of intestinal villi. Quantitative estimation of immunohistochemical staining based on the determination of the % positive-stained area from 4 images/sample/group using Image-Pro Plus software is charted down the images. ∗∗∗p < 0.05 compared to uninfected. ∗∗p < 0.05 compared to infected. ∗p < 0.05 compared to pumpkin. #p < 0.05 compared to honey. $p < 0.05 compared to standard. Original magnification was 400× (Scale-bar: 50 µm).