Linearolactone (1) and kaempferol (2) have amebicidal activity in in vitro studies. The type of cell death induced by 1 and 2 and their effects on the virulence of E. histolytica were analyzed by transmission and confocal electron microscopy, reactive oxygen species (ROS) production, and apoptosis, detected by flow cytometry with dichlorofluorescein 2′,7′-diacetate and annexin-V binding, respectively, and confirmed by TUNEL. The interaction of 1 and 2 with actin was analyzed by docking, and the in vivo amoebicidal activity was established with the Mesocricetus auratus model; amebic liver abscess (ALA) development was evaluated by magnetic resonance (MR) and validated post mortem. In vitro, compounds 1 and 2 caused chromatin condensation, intracellular ROS, and loss of actin structures. Coupling analysis showed that they bind to the allosteric and catalytic sites of actin with binding energies of −11.30 and −8.45 kcal/mol, respectively. Treatments with 1 and 2 induced a decrease in ALA formation without toxic effects on the liver and kidney. Thus, compound 1, but not 2, was able to induce apoptosis-like effects in E. histolytica trophozoites by intracellular production of ROS that affected the actin cytoskeleton structuration. In vivo, compound 1 was more active than compound 2 to reduce the development of ALA.
BACKGROUND
Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown.
OBJECTIVE
To analyse the cytotoxic effects of KPF on
Giardia duodenalis
trophozoites and to identify a likely parasite target of this compound.
METHODS
We used inhibitory concentrations of KPF (IC
25
, IC
50
and IC
100
) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a
G. duodenalis
aldose reductase (
GdAldRed
) homolog.
RESULTS
KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that
GdAldRed
is a viable KPF target (ΔG = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog.
CONCLUSIONS
KPF exerted a proapoptotic effect on
G. duodenalis
trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures.
GdAldRed
is a likely target underlying its antigiardial activity.
Linearolactone (LL) is a neo-clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on Giardia intestinalis trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand–protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from G. intestinalis. LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.
This work study the expression of leptin ObRb receptor and the functional changes in the vascular reactivity in thoracic and abdominal aorta and isolated heart, obese and control Sprague Dawley rats were study, in one group was induced intrauterine malnutrition (E) due to the 50% food restriction in the pregnant rats and was compare with another group with any food restriction, control (C). The newborns with low weight at birth (BPN) group E had been feed with hypercaloric diet (60% lipids) until 6 months older. Recognize the vascular effect of angiotensin II and phenylephrine in thoracic and abdominal rings and isolated heart rat, and in the other hand we analyze the RNA total expression and RT‐PCR for ObRb receptor.ResultsIntrauterine malnutrition showed BPN in the group E (p<0.01) compare with group C, and at 6 months older the difference between 2 groups was ±100g for group E with the hypercaloric diet. Group E showed a hyperglycemic (p<0.01), like an increment in the leptin receptor ObRb expression (p<0.01) compared with the group C. On the other hand both groups showed differences in the vascular reactivity and heart responses for angiotnesin II, it's possible that those could be by the changes in the leptin receptor expression in the obese rat.
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