Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action.

BACKGROUND: Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE: The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS: Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS: The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS: Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

Effects of Disodium Cromoglycate Treatment in the Early Stage of Diabetic Nephropathy: Focus on Collagen Deposition.

Inflammation is part of the pathophysiology of diabetic nephropathy (DN), and mast cells (MCs) appear to increase in number within the kidney of humans and animals with diabetes. Disodium cromoglycate (CG) not only inhibits the degranulation of MCs but also has several secondary effects that may improve inflammation. However, little is known about the effects of CG treatment on kidney collagen deposition and myofibroblast population in animals with type I diabetes (DM1). Data presented here suggest that the increases in the density and activity of MCs within the kidney in the early stages of DN contribute to tubulointerstitial collagen deposition, even in the absence of alterations in the renal myofibroblast population. Moreover, CG treatment showed renoprotective effects in rats with DM1, which appear to be linked to its mast cell stabilizing property and its ability to avoid some detrimental morphofunctional alterations.

Mitochondrial dysfunction on Leishmania (Leishmania) amazonensis induced by ketoconazole: insights into drug mode of action

BACKGROUND Leishmania parasites cause leishmaniasis that range from self-limiting cutaneous lesions to more serious forms of the disease. The search for potential drug targets focusing on biochemical and metabolic pathways revealed the sterol biosynthesis inhibitors (SBIs) as a promising approach. In this class of inhibitors is found ketoconazole, a classical inhibitor of 14α-methysterol 14-demethylase. OBJECTIVE The present study aimed to better understand the biological response of Leishmania (Leishmania) amazonensis promastigotes at the cellular level after ketoconazole treatment. METHODS Herein, techniques, such as fluorimetry, flow cytometry, fluorescence microscopy, electron and scanning microscopy were used to investigate the cellular structures and to identify organelles affected by ketoconazole treatment. FINDINGS The study demonstrated, for the first time, the effect of ketoconazole on mitochondrion functioning and its probable relationship to cell cycle and death on L. (L.) amazonensis promastigotes (IFLA/BR/67/PH8 strain). MAIN CONCLUSIONS Ketoconazole-induced mitochondrial damages led to hyperpolarisation of this single organelle and autophagic vacuoles formation, as a parasite survival strategy. These damages did not reflect directly on the parasite cell cycle, but drove the parasites to death, making them susceptible to ketoconazole treatment in in vitro models.

Effects of maternal hypothyroidism in the gastrointestinal system of male young offspring from Wistar rats.

Alterations in the maternal environment may impact on the fetal development. The objective of this study was to investigate the gastrointestinal consequences of maternal hypothyroidism for the male offspring from Wistar rats. The pregnant rats were divided into three groups: control (C - received water), experimental 1 [E1 - received methimazole (MMI) solution] during gestation and lactation, and experimental 2 (E2 - received MMI solution) during gestation. Maternal parameters evaluated: free T3 and T4, bodyweight variation, and water/food intake. Offspring parameters evaluated: litter size, number of male/female, free T3 and T4, stomach area, gastric ulcer susceptibility, small intestine length and weight, small intestine and distal colon motility, the stomach and intestinal weight-body weight ratio (SW/BW-IW/BW), and the accumulation of intestinal fluid. Maternal T3 and T4 from E1 were decreased when compared to the other groups. There were no differences for maternal water/food intake and weight gain, litter size, and number of males and females. Regarding to offspring, free T3, SW/BW, IW/BW, and intestinal fluid accumulation were not different between the groups, but T4 was decreased in E1. However, 30-day-old pups from E1 and E2 were smaller with lower stomach and small intestine. Even more, E1 presented a lower ulcer index when compared to the C, while E2 had a higher distal colon transit. It can be concluded that maternal hypothyroidism impaired the total body development, as well as gastric and intestinal development, besides interfering with the susceptibility to the ulcer and intestinal transit of male offspring from Wistar rats.

Hypotensive activity of Campomanesia xanthocarpa leaf extract: beyond angiotensin II type 1 receptor blockage.

The ability of Campomanesia xanthocarpa leaf extract (CXLE) to alter blood pressure and heart rate was evaluated in anesthetized rats. The CXLE-induced hypotension was evaluated before and after losartan, methylatropine, L-N(ω)-nitro-L-arginine methyl ester (L-NAME), hexamethonium, indomethacin, glibenclamide, or nifedipine administration. The constituents of CXLE were identified by LC-DAD-MS. CXLE decreased blood pressure in a dose-dependent manner; only the highest dose decreased heart rate. The hypotension induced by CXLE was sensitive only to losartan, nifedipine, and glibenclamide. L-NAME decreased the time to recover 50% of the hypotensive effect of CXLE without altering its magnitude. Flavan-3-ols, proanthocyanidins (dimers and trimers), and glycosylated flavonols were identified from CXLE. The chemical constituents of CXLE seem to induce not only angiotensin II type 1 receptor blockage, but also ATP-sensitive potassium channels activation and L-type voltage-dependent Ca2+ channels inactivation. Nitric oxide is involved in the maintenance of the hypotensive effect of CXLE.

Hypotensive effect of Eugenia dysenterica leaf extract is primarily related to its vascular action: The possible underlying mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia dysenterica (ED) leaves are used in Brazil to treat cardiac diseases; however, there are no scientific data describing the effects of this species on cardiac activity. AIM OF THE STUDY: To investigate the effect of ED aqueous leaf extract (EDLE) on hear rate (HR) and mean arterial pressure (MAP) of anaesthetised rats and its underlying mechanism of action. MATERIAL AND METHODS: EDLE was analysed, and its proanthocyanidin composition was determined. After performing dose-effect curves for EDLE on HR and MAP, EDLE-induced hypotension was evaluated before and after atropine (AT), L-N(ω)-nitro-L-arginine methyl ester (L-NAME), hexamethonium (HXT), indomethacin (IND), carbenoxolone (CBX), or nifedipine (NFD) administration. The effect of proanthocyanidin-depleted extract (EDLE/P-) was also determined and compared to that of the EDLE with proanthocyanidins. RESULTS: EDLE decreased the MAP in a dose-dependent manner; HR was decreased only with the highest and most toxic dose. Only CBX and NFD decreased EDLE-induced hypotension. Five polymeric series of proanthocyanidins were identified, which were mainly constituted by procyanidin and prodelphinidin units with B-type linkage and up to 12 flavan-3-ol units. EDLE/P- induced hypotension did not differ from that induced by EDLE. CONCLUSIONS: The cardiovascular effects of EDLE were primarily related to its vascular action. EDLE-induced hypotensive effect appeared to involve L-type calcium channel blockage as well as myoendothelial gap junction signalling. The higher molecular weight proanthocyanidins from EDLE are unlikely to contribute to its cardiovascular effect.

Antidiarrhoeic effect and dereplication of the aqueous extract of Annona crassiflora (Annonaceae).

We investigated the antidiarrhoeic effect of the aqueous extract of Annona crassiflora leaves (AEAC). The AEAC decreased the diarrhoeic stools and enteropooling induced by castor oil, without altering total faecal output; moreover, the distance travelled by charcoal meal in the intestine was increased. Twenty-eight compounds were identified by LC-DAD-MS in the AEAC, including flavonoids, alkaloids and proanthocyanidins. In addition, two oligomeric series of condensed tannins of up to nine flavan-3-ol units were characterised by MALDI-MS. These data suggest that the antidiarrhoeic effect of the AEAC is related to its ability to inhibit intestinal secretion and/or to increase intestinal absorption. Moreover, the prokinetic effect of AEAC, together with its inhibitory effect on enteropooling induced by castor oil, explains why this extract decreased diarrhoeic faeces without altering the total faecal output. All these effects are in agreement with the pharmacological activity reported in the literature for many of the secondary metabolites identified.

Mast cell population in the development of diabetic nephropathy: Effects of renin angiotensin system inhibition.

Considering the importance of the renin-angiotensin system (RAS) in diabetic nephropathy (DN) and the link between mast cells (MC) and the RAS, this study evaluated the effects of RAS blockade on the MC cell population in the kidneys from rats with experimental diabetes. Wistar rats were divided into six groups: control non-diabetic (C); sham (S); diabetic (D); and D treated with enalapril (EN), losartan (LO), or aliskiren (AL). Ninety days after diabetes induction, glomerular filtration rate (GFR) and urinary albumin excretion (UAE) were determined. Kidneys were collected for MC counting. RAS blockers minimized changes in morphometrical parameters (EN), cortical collagen (LO, AL), GFR (AL) and UAE (EN, LO). An increased number of MC was observed in the kidneys from D animals. Only AL treatment prevented this increase. MC may be involved in some aspects of DN pathogenesis and the possible protective effects of AL on the kidneys might involve MC modulation.

Testosterone and Mast Cell Interactions in the Development of Kidney Fibrosis after Unilateral Ureteral Obstruction in Rats.

Mast cell and testosterone interactions involved in renal fibrosis in rats subjected to unilateral ureteral obstruction (UUO) were investigated. Orchiectomized (ORX) and nonorchiectomized Wistar rats were subjected to UUO, and a nonorchiectomized group was sham-operated (control: SO). Animals from the UUO group were treated with saline or sodium cromoglycate (CG). Some ORX rats from the saline or CG groups also received testosterone propionate replacement (TR). Kidneys and blood were collected 14 d after UUO or SO. Kidney sections were stained with toluidine blue to quantify mast cells, and picrosirius red was used for collagen analysis. Immunohistochemistry for α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) expression was also performed. Plasma testosterone levels (PTLs) were measured. ORX decreased and TR normalized PTLs. UUO increased mast cell density in the kidney pelvis, but not in the kidney parenchyma. UUO increased mast cell degranulation, and CG or ORX inhibited this effect. TR partially reversed the effect of ORX on mast cell degranulation, and CG partially inhibited that effect of TR. UUO increased the collagen areas of the renal parenchyma, whereas CG or ORX abolished that alteration; TR reversed the effects of ORX, and CG partially inhibited that effect of TR. UUO increased tubulointerstitial α-SMA expression and PCNA-positive cells, and these changes were sensitive to ORX or CG to the same degree, while TR again reversed the effect of ORX. Renal fibrosis after UUO appears to be determined by interactions between testosterone and mast cells.

In vitro additive interaction between ketoconazole and antimony against intramacrophage Leishmania (Leishmania) amazonensis amastigotes.

Leishmaniasis is a group of diseases caused by protozoa of Leishmania genus. The currently available treatments for this disease are expensive, present high toxicity and are associated to difficulties of healing and parasite resistance. Therefore, the development of strategies for leishmaniasis treatment is indispensable and includes reposition of existing drugs, as well as drug combination therapy. The aim of this study was to assess the nature of ketoconazole and antimony association on the cytotoxic effect against Leishmania (Leishmania) amazonensis amastigotes. The calculated mean sum of fractional 50% inhibitory concentration ([Formula: see text]ΣFIC50) was 2.54 and 1.43 for free and intracellular amastigotes, respectively, values that suggest an additive interaction between ketoconazole and antimony concerning to Leishmania toxicity only in the intramacrophage parasite form. Despite the clinical efficacy of ketoconazole-antimony combination has been shown in the literature, our study is the first to describe the nature of ketoconazole-antimony interaction against L. (L.) amazonensis amastigotes. Moreover, our results point out the need for future in vivo studies to confirm the nature of ketoconazole-antimony interaction and also to determine possible effective dosage regimens related to ketoconazole administration in association with the optimal lower dose of antimony.

Antidiarrhoeic effect of Eugenia dysenterica DC (Myrtaceae) leaf essential oil.

Essential oil from Eugenia dysenterica leaves was able to inhibit both the diarrhoea and enteropooling induced by castor oil; however, the distance travelled by charcoal meal in the intestine was not change. These data suggest that the antidiarrhoeic effect of the essential oil from E. dysenterica leaves is related to its ability to inhibit intestinal secretion and/or to increase intestinal absorption.

The gastroprotective effects of Eugenia dysenterica (Myrtaceae) leaf extract: the possible role of condensed tannins.

We applied a taxonomic approach to select the Eugenia dysenterica (Myrtaceae) leaf extract, known in Brazil as "cagaita," and evaluated its gastroprotective effect. The ability of the extract or carbenoxolone to protect the gastric mucosa from ethanol/HCl-induced lesions was evaluated in mice. The contributions of nitric oxide (NO), endogenous sulfhydryl (SH) groups and alterations in HCl production to the extract's gastroprotective effect were investigated. We also determined the antioxidant activity of the extract and the possible contribution of tannins to the cytoprotective effect. The extract and carbenoxolone protected the gastric mucosa from ethanol/HCl-induced ulcers, and the former also decreased HCl production. The blockage of SH groups but not the inhibition of NO synthesis abolished the gastroprotective action of the extract. Tannins are present in the extract, which was analyzed by matrix assisted laser desorption/ionization (MALDI); the tannins identified by fragmentation pattern (MS/MS) were condensed type-B, coupled up to eleven flavan-3-ol units and were predominantly procyanidin and prodelphinidin units. Partial removal of tannins from the extract abolished the cytoprotective actions of the extract. The extract exhibits free-radical-scavenging activity in vitro, and the extract/FeCl3 sequence stained gastric surface epithelial cells dark-gray. Therefore, E. dysenterica leaf extract has gastroprotective effects that appear to be linked to the inhibition of HCl production, the antioxidant activity and the endogenous SH-containing compounds. These pleiotropic actions appear to be dependent on the condensed tannins contained in the extract, which bind to mucins in the gastric mucosa forming a protective coating against damaging agents. Our study highlights the biopharmaceutical potential of E. dysenterica.

The aversive, anxiolytic-like, and verapamil-sensitive psychostimulant effects of pulegone.

We investigated the psychostimulant, rewarding, and anxiolytic-like effects of pulegone. Possible interactions between pulegone and menthol concerning their psychostimulant effect were also analyzed. General mouse activity after pulegone treatment, and the interacitons between pulegone and menthol, were determined in the open field. The anxiolytic-like activity, motor coordination and strength force were evaluated using the elevated plus maze (EPM), rotarod test and grasping test, respectively. The motivational properties of pulegone were evaluated by pairing the drug effects on the mice with the least preferred compartment (previously determined) of a conditioned place preference (CPP) apparatus. Pulegone increased mouse locomotor activity and immobilization time. Verapamil, but not diltiazem, haloperidol or picrotoxin, decreased the psychostimulation induced by pulegone. Pulegone also decreased grooming and rearing behaviors and caused motor incoordination and weakness at high doses. Pulegone increased the time spent by mice in the open arms of the EPM, and flumazenil pre-treatment did not alter this effect. Pulegone either produced no CPP or induced conditioned place aversion. The changes in mouse ambulatory activity caused by the association of pulegone with menthol were either lower than those predicted by the theoretical curve or not different from the predicted values. Therefore, pulegone induces a verapamil-sensitive psychostimulant effect that appears to independ on the opening of L-type calcium channels. Pulegone has negative reinforcing properties and seems to possess anxiolytic-like actions unrelated to the benzodiazepine site of the γ-aminobutyric acid type A (GABAA) receptor. Finally, pulegone might act in an addictive or synergic way with menthol.

Effects of diethylpropion treatment and withdrawal on aorta reactivity, endothelial factors and rat behavior.

Diethylpropion (DEP) is an amphetamine-like compound used as a coadjutant in the treatment of obesity and which presents toxicological importance as a drug of abuse. This drug causes important behavioral and cardiovascular complications; however, the vascular and behavioral alterations during DEP treatment and withdrawal, have not been determined. We evaluated the effects of DEP treatment and withdrawal on the rat aorta reactivity to noradrenaline, focusing on the endothelium, and the rat behavior during DEP treatment and withdrawal. DEP treatment caused a hyporreactivity to noradrenaline in aorta, reversible after 2 days of withdrawal and abolished by both the endothelium removal and the presence of L-NAME, but not by the presence of indomethacin. Furthermore, DEP treatment increased the general activity of rats. Contrarily, DEP withdrawal caused a decrease in the locomotor activity and an increase in grooming behavior, on the 2nd and 7th days after the interruption of the treatment, respectively. DEP treatment also caused an adaptive vascular response to noradrenaline that seems to be dependent on the increase in the endothelial nitric oxide system activity, but independent of prostaglandins synthesis. The data evidenced chronological differences in the adaptive responses of the vascular and central nervous systems induced by DEP treatment. Finally, a reversion of the adaptive response to DEP was observed in the vascular system during withdrawal, whereas a neuroadaptive process was still present in the central nervous system post-DEP. These findings advance on the understanding of the vascular and behavioral pathophysiological processes involved in the therapeutic and abusive uses of DEP.