Alicia B. Penissi

Effect of dehydroleucodine on mucus production : A quantitative study

We have demonstrated that dehydroleucodine(DhL), a lactone isolated from Artemisia douglasianaBesser, prevents gastroduodenal damage induced bynecrosis-inducing agents such as absolute ethanol. Wehave also reported, in a qualitative study, thatthis effect is related to the ability of the drug tostimulate mucus production. The present study wasdesigned to quantitatively evaluate the effect of DhL on adherent mucus layer thickness, to obtain amore objective approach to the mechanism of action ofthe drug. Mice were divided into two groups: (I)controls were treated with orally administeredcarboxymethylcellulose (CMC) and (II) experimental animals receivedDhL in CMC. The thickness of the mucus gel layer wasmeasured in unfixed stomachs and duodena, using an imageanalysis system. We observed an increase in the adherent mucus layer thickness in theexperimental samples. This confirms that one of the mainmechanisms involved in the cytoprotective action of thedrug is mucus secretion.

Novel anti-ulcer α,β-unsaturated lactones inhibit compound 48/80-induced mast cell degranulation

The present study was designed to examine the effects of a sesquiterpene lactone isolated from Artemisia douglasiana Besser (dehydroleucodine), a xanthanolide sesquiterpene isolated from Xanthium cavanillesii Schouw (xanthatin) and a semisynthetic butenolide (3-benzyloxymethyl-5H-furan-2-one) on mast cell degranulation induced by compound 48/80. Peritoneal mast cells from male adult Sprague-Dawley rats were purified in Percoll, preincubated in the presence of test lactones (dehydroleucodine, xanthatin or 3-benzyloxymethyl-5H-furan-2-one) and then challenged with the mast cell activator compound 48/80 (10 microg/ml). Concentration-response and kinetic studies of mast cell serotonin release evoked by compound 48/80, evaluation of mast cell viability and morphology by light and electron microscopy, and comparative studies using ketotifen and sodium chromoglycate were carried out. Serotonin release studies, carried out together with morphological studies, showed the effectiveness of the above lactones to stabilize mast cells. The comparative study with ketotifen and sodium chromoglycate, well known mast cell stabilizers, showed the following order of potency dehydroleucodine=xanthatin>3-benzyloxymethyl-5H-furan-2-one> or =ketotifen/sodium chromoglycate to inhibit mast cell serotonin release induced by compound 48/80. The present study provides the first strong evidence in favour of the hypothesis that dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one inhibit compound 48/80-induced serotonin release from peritoneal mast cells, acting thus as mast cell stabilizers. Our findings may provide an insight into the design of novel pharmacological agents which may be used to regulate the mast cell response.

Hormonal profile and reproductive performance in lactation deficient (OFA hr/hr) and normal (Sprague-Dawley) female rats.

Lactation deficiency may have important consequences on infant health, particularly in populations of low socioeconomic status. The OFA hr/hr (OFA) strain of rats, derived from Sprague-Dawley (SD) rats, has deficient lactation and is a good model of lactation failure. We examined the reproductive performance and hormonal profiles in OFA and SD strains to determine the cause(s) of the lactation failure of the OFA strain. We measured hormonal (PRL, GH, gonadotropins, oxytocin, and progesterone) levels by RIA in cycling, pregnant, and lactating rats and in response to suckling. Dopaminergic metabolism was assessed by determination of mediobasal hypothalamic dopamine and dihydroxyphenylacetic acid (DOPAC) concentrations by HPLC and tyrosine hydroxylase expression by immunocytochemistry and western blot. OFA rats have normal fertility but 50% of the litters die of malnutrition on early lactation; only 6% of the mothers show normal lactation. The OFA rats showed lower circulating PRL during lactation, increased hypothalamic dopamine and DOPAC, and impaired milk ejection with decreased PRL and oxytocin response to suckling. Before parturition, PRL release and lactogenesis were normal, but dopaminergic metabolism was altered, suggesting activation of the dopaminergic system in OFA but not in SD rats. The number of arcuate and periventricular neurons expressing tyrosine hydroxylase was higher in SD rats, but hypothalamic expression of TH was higher in OFA rats at the end of pregnancy and early lactation. These results suggest that the OFA rats have impaired PRL release linked with an augmented dopaminergic tone which could be partially responsible for the lactational failure.

Gastroduodenal Mucosal Protection Induced by Dehydroleucodine: Mucus Secretion and Role of Monoamines

In previous work we have demonstrated thatdehydroleucodine (DhL) prevents gastric damage inducedby necrosis-inducing agents such as absolute ethanol(EtOH). In this study we examine the effects of DhL on gastroduodenal morphology and monoaminelevels by histological and biochemical methods,respectively, as an approach to elucidating thecytoprotective mechanism of the drug. Histologicalevidence shows that DhL prevents formation of gastroduodenalmucosal lesions induced by EtOH and that this protectiveeffect is related to the ability of the drug tostimulate mucus production. DhL itself does not affect the tissue concentration of NE, DA and 5-HT.However, it prevents the depletion of DA and 5-HTprovoked by EtOH. We propose that the abundant mucoidblanket secreted after treatment with DhL acts as adiffusion barrier against EtOH. It is also possible thatDhL could act as a “cell stabilizer,” byinhibiting the degranulation of cells containingmonoamines.

Dopaminergic Mechanisms Involved in Prolactin Release after Mifepristone and Naloxone Treatment during Late Pregnancy in the Rat

Background/Aims: During late pregnancy, the antiprogesterone mifepristone facilitates prolactin release. This effect is enhanced by administration of the opioid antagonist naloxone, suggesting an inhibitory-neuromodulatory role of the opioid system. Since hypothalamic dopamine (DA) is the main regulator of prolactin release, in this study we explored the role of DA on prolactin release induced by mifepristone and naloxone treatment. Methods/Results: Rats on day 19 of pregnancy were used. Naloxone treatment did not modify the 3,4-dihydroxyphenylacetic acid/DA (DOPAC/DA) ratio or serum prolactin concentration in control rats. After mifepristone treatment, DA activity diminished significantly without modifying serum prolactin levels. Naloxone administration to antiprogesterone-treated rats did not change the DOPAC/DA ratio but increased serum prolactin. Tyrosine hydroxylase (TH) expression in medial basal hypothalamus (MBH) protein extracts was lowered by pretreatment with mifepristone, with no additional effect of naloxone. While mifepristone decreased the intensity of TH immunoreactivity in the arcuate and periventricular nuclei and in fibers of the median eminence, naloxone treatment had no further effect. Conclusions: (1) A reduction of tuberoinfundibular dopaminergic (TIDA) neuron activity is suggested by the fall of the DOPAC/DA ratio and the low expression of MBH TH; (2) this reduction facilitates prolactin secretion by naloxone, indicating that progesterone stimulates DA neurons to maintain low serum prolactin; (3) naloxone action seems to depend on a previous decrease of DA tone induced by mifepristone, without involve a direct effect on neuronal DA activity, and (4) endogenous opioids may inhibit prolactin secretion through a non-dopaminergic neuronal system that regulates prolactin secretion in which as yet undetermined prolactin-releasing factors may participate.

Hydroxytyrosol and oleuropein of olive oil inhibit mast cell degranulation induced by immune and non-immune pathways

The aim of this study was to determine whether hydroxytyrosol and oleuropein, the major phenols found in olives and olive oil, inhibit mast cell activation induced by immune and non-immune pathways. Purified peritoneal mast cells were preincubated in the presence of test compounds (hydroxytyrosol or oleuropein), before incubation with concanavalin A, compound 48/80 or calcium ionophore A23187. Dose-response and time-dependence studies were carried out. Comparative studies with sodium cromoglycate, a classical mast cell stabilizer, were also made. After incubation the supernatants and pellets were used to determine the β-hexosaminidase content by colorimetric reaction. The percentage of β-hexosaminidase release in each tube was calculated and taken as a measure of mast cell activation. Other samples of cell pellets were used for cell viability studies by the trypan blue dye exclusion test, or fixed for light and electron microscopy. Biochemical and morphological findings of the present study showed for the first time that hydroxytyrosol and oleuropein inhibit mast cell degranulation induced by both immune and non-immune pathways. These results suggest that olive phenols, particularly hydroxytyrosol and oleuropein, may provide insights into the development of useful tools for the prevention and treatment of mast cell-mediated disorders.

Activation of human leukemic mast cell line LAD2 is modulated by dehydroleucodine and xanthatin

Abstract The aim of the present study was to determine whether dehydroleucodine, xanthatin and 3-benzyloxymethyl-5H-furan-2-one inhibit the activation of human leukemic LAD2 mast cells induced by compound 48/80 or the calcium ionophore A23187. LAD2 cells were preincubated in the presence of test drugs and then challenged with the secretagogues. This study provides the first evidence in favor of the view that dehydroleucodine and xanthatin inhibit the degranulation of LAD2 cells, thus acting as human mast cell stabilizers. These molecules could be effective in the treatment of human diseases associated with inappropriate mast cell activation.

Effect of dehydroleucodine on histamine and serotonin release from mast cells in the isolated mouse jejunum.

Abstract:Objective and design: DhL, a lactone isolated from Artemisia douglasiana, prevents gastrointestinal damage elicited by necrosis-inducing agents and exhibits antiinflammatory action. This work examines the effect of DhL on compound 48/80-induced histamine and serotonin release in the isolated mouse jejunum, to determine whether DhL inhibits mediator release from mast cells at the enteric level. Material: Thirty jejuna from male Balb-c mice were used for the studies. Treatment: Samples were incubated sequentially in 9 test tubes containing RBS or 10 μg/ml compound 48/80 or 1.6 mmol/l + 10 μg/ml compound 48/80 at 37°C for 90 minutes (10 min per tube). Methods: Histamine and serotonin release studies, quantification of granulated mast cells, and evaluation of mast cell ultrastructure were carried out. Differences between groups were determined using analysis of variance followed by Tukey-Kramer multiple comparisons test. Results: Compound 48/80 increased histamine and serotonin release by the tissue (141.95 ± 62.58 pg/mg tissue vs basal 5.45 ± 1.04, P<0.01 and 20.04 ± 2.81 vs basal 9.24 ± 1.56 ng/ mg tissue, P<0.01, respectively), decreased the number of granulated submucosal mast cells (0.077 ± 0.0035 vs basal 0.14 ± 0.015, P<0.05), and elicited evident granule ultraestructural changes. These effects were reduced by dehydroleucodine (19.51 ± 7.88, P<0.01; 12.69 ± 1, P<0.05 and 0.143 ± 0.014, P<0.05, respectively). Conclusion: The lactone inhibits compound 48/80-induced histamine and serotonin release from mast cells in the isolated mouse jejunum.

Changes in Duodenal Mast Cells in Response to Dehydroleucodine

Introduction: Dehydroleucodine (DhL), a sesquiterpene lactone isolated from Artemisia douglasiana Besser, prevents gastroduodenal damage elicited by necrosis-inducing agents such as absolute ethanol. Changes in the number of mast cells or evidence of activation of the cells for mediator release have been observed in a wide spectrum of disease processes involving the gastrointestinal tract. In the present study we examined the effects of DhL on duodenal mast cell population and their histamine content, with the goal of throwing more light on the mechanism of action of the drug. Materials and Methods: Male Rockland mice (n = 30) were divided into two groups and administered orally with 0.4% carboxymethylcellulose (CMC; control group) or DhL in 0.4% CMC, 40 mg/kg body weight (DhL group). The animals were killed 60 min after dosing and their duodena were removed. Mucosal and submucosal mast cells were studied by light and electron microscopy, and the duodenal histamine content was measured by high-performance liquid chromatography. Results: DhL increased the number of mast cells in the submucosal layer. This was related to an increase in the tissue histamine levels (from 324 ± 19.14 to 1,284 ± 20 pg/mg tissue, in controls and DhL-treated, respectively). The mast cells in the submucosa from the control group showed a cytoplasm containing a predominant population of homogenously dense granules, and the DhL-treated group exhibited swollen granules showing different degrees of particulation. The mucosal mast cell population showed no modifications in response to the cytoprotective agent. Conclusions: DhL induces (1) a selective increase in the number of mast cells in the submucosal layer and (2) changes in the distribution and appearance of their secretory granules. These findings, probably associated with the higher histamine levels after DhL treatment, could be involved in the cytoprotective action of the lactone, previously reported by us.

Daily morphological variations in the viscacha (Lagostomus maximus maximus) retina. Probable local modulatory action of melatonin

Given that the local melatonin levels exhibit rhythmic daily changes in the retina of the viscacha, we considered it important to study the likely daily variations in morphology and specific 2‐[125I]‐iodomelatonin binding in retinas from this rodent and to correlate these putative changes with local indole levels. Adult animals of both sexes were captured in their habitat and were kept under a natural photoperiod. For light and electron microscopic studies the viscachas were sacrificed by decapitation at 08:00, 16:00, and 24:00 hr. A computer‐assisted image analysis system was used to measure the thickness of the complete retina, the photoreceptor layer, the rod outer and inner segments, and the outer nuclear layer. The daily variation in 2‐[125I]‐iodomelatonin binding sites was followed during a 24‐hr light‐dark cycle, the animals being sacrificed at six time points. The parameters studied showed significant variations throughout the 24‐hr period. Maximal specific binding, lysosomal content in the pigment epithelium, and photoreceptor layer outer segment thicknesses were observed at 24:00 hr. Close contact between photoreceptor membranes and microvilli of the pigment epithelium was observed at 08:00 and 16:00 hr. Moreover, the minimal outer segment thickness at 16:00 hr was accompanied by a scarcity of dense bodies, such as lysosomes, a maximum dispersion of melanin pigment granules, and a minimum density of radioligand binding sites. Therefore, in the retina of the viscacha, we suggest that the interaction between melatonin and specific sites could be one of the factors or causes that participate in the regulation of the daily morphological changes observed in viscacha. Anat Rec 266:198–206, 2002.