Targeting the nervous system of the parasitic worm, Haemonchus contortus with quercetin

Abstract

Prevalence of infection, limited choice of drugs, and emerging resistance against contemporary medications lead to a pressing need to develop new anthelmintic drugs and drug targets. However, little understanding of worms' physiology has substantially delayed the process. Here, we are reporting the tissue morphology of Haemonchus contortus, intestinal parasitic helminths found in small ruminants, and targeting its nervous system with quercetin, a naturally occurring flavonoid. Quercetin showed anthelmintic activity against all of the developmental stages of H. contortus. Further, histological analysis demonstrated damage to various body parts, including isthmus, brut, pseudocoele, and other organs. Mechanistic studies revealed the generation of oxidative stress and alterations in the activities of the stress response enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase. Moreover, the time-dependent imaging of reactive oxygen species (ROS) generated due to quercetin treatment disclosed neuropils as the primary targets of quercetin in adult worms, which eventually lead to the paralysis and death of the worms. Thus, this work demonstrates that the nervous system of the parasitic helminth, H. contortus, is a novel target of the drug quercetin.

Keywords: Catalase; Glutathione peroxidase; Haemonchus contortus; Nervous system; Oxidative stress; Parasitic nematodes; Quercetin; ROS; Superoxide dismutase.

Graphical abstract

Fig. 1

Paralysis and death time analysis in the adult H. contortus. Adult H. contortus worms were exposed to different doses of quercetin (0.125, 0.25, 0.5, 1, and 2 mM), 0.2 mM albendazole, and RPMI media (control condition) for 24 h. In male worms, quercetin was most active in 1 mM, where approx 80% of the worms were paralyzed in 12 h, and nearly 100% of the worms died within 24 h of Quercetin treatment. Whereas with quercetin in 1 mM till 12 h of treatment, only 60% of the female worms got paralyzed, and 100% died in 24 h of quercetin treatment.

Fig. 2

Percent viability of L3 larval stage and percentage of eggs hatched of H. contortus after treatments with different concentrations of quercetin and Alb. The blue bars are showing the effect after 24 h of treatment at different concentrations of quercetin (0.125, 0.25, 0.50, 1, and 2 mM). The green and red-colored bars are representing the treatment of the control set (RPMI media) and albendazole (0.2 mM). Quercetin treatment showed concentration-dependent effects on (A) the survival of the L3 larvae and higher mortality than Alb and (B) the reduced hatching of eggs. The results of the three experiments are plotted as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Study of morphological damage in the adult H. contortus due to the treatment with quercetin. Scanning electron microscopic images are showing the morphology of adult H. contortus A, B, and C are showing the whole body, anterior and posterior ends in the control group, respectively. D, E, and F are showing partial disruptions of the body ends and cuticle due to the treatment with 0.2 mM Alb of the same body parts. G, H, and I are showing the same for 1 mM quercetin-treated worms.

Fig. 4

Histopathological changes in the adult H. contortus after the treatment with quercetin. A shows the tissue morphology (H&E) of the control adult female H. contortus at 20×. Higher magnified (100×) images are showing detailed structures of the isthmus, brut, pseudocoele, globular leukocytes, muscle cells, intestinal epithelial region, ovary, and intact skin tissue in ai–aviii, respectively. Due to the treatment with Alb, partial disorganizations in some of these body parts were observed (B). Treatment with 1 mM of quercetin resulted in extensive damage to the body part, such as loss of egg count, wrecked globular leukocytes, etc. . (C) bi–bviii and ci–cviii are showing magnified images of the body parts as before; the same for Alb (0.2 mM) and Quercetin (0.1 mM) treated worms, respectively.

Fig. 5

Generation of the reactive oxygen species (ROS) in the adult worms' nervous system due to quercetin exposure. Adult worms were treated for 3 h separately with Alb and quercetin and processed with DCFDA (100 nM) to detect the ROS. The images from A-C are showing the differences in staining (10× magnification) in the anterior end, after 7 min of DCFDA treatment (due to differences in ROS generation) respectively in control (treated with RPMI media), Alb (0.2 mM) and quercetin (1 mM)-treated worms. The following three images (D–F) are showing the differences in staining (10× magnification), at the posterior end, in the same three experimental groups. In subfigure F, the blue arrowhead indicates the eggs of the quercetin-treated worm. Generations of ROS respectively in the nerve ring (I), marked with white arrowhead; (40×), commissural connections (L), marked with red arrowheads; (40×) and ventral cord, marked with yellow arrowhead; (40×) were detected in the adult worms treated with quercetin. No such structural discrimination and elevation of ROS were observed in the control (G, H) and Alb-treated (J, K) worms. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)