Ferulic Acid Improves Cognitive Skills Through the Activation of the Heme Oxygenase System in the Rat.

Over the last years, many studies reported on the antioxidant effects of ferulic acid (FA) in preclinical models of dementia through the activation of the heme oxygenase/biliverdin reductase (HO/BVR) system. However, only a few studies evaluated whether FA could improve neurological function under milder conditions, such as psychological stress. The aim of this study was to investigate the effects of FA (150 mg/kg intraperitoneal route) on cognitive function in male Wistar rats exposed to emotional arousal. Animals were randomly assigned to two experimental groups, namely not habituated or habituated to the experimental context, and the novel object recognition test was used to evaluate their cognitive performance. The administration of FA significantly increased long-term retention memory in not habituated rats. Ferulic acid increased the expression of HO-1 in the hippocampus and frontal cortex of not habituated rats only, whereas HO-2 resulted differently modulated in these cognitive brain areas. No significant effects on either HO-1 or HO-2 or BVR were observed in the cerebellum of both habituated and not habituated rats. Ferulic acid activated the stress axis in not habituated rats, as shown by the increase in hypothalamic corticotrophin-releasing hormone levels. Pre-treatment with Sn-protoporphyrin-IX [0.25 µmol/kg, intracerebroventricular route (i.c.v.)], a well-known inhibitor of HO activity through which carbon monoxide (CO) and biliverdin (BV) are generated, abolished the FA-induced improvement of cognitive performance only in not habituated rats, suggesting a role for HO-derived by-products. The CO-donor tricarbonyldichlororuthenium (II) (30 nmol/kg i.c.v.) mimicked the FA-related improvement of cognitive skills only in not habituated rats, whereas BV did not have any effect in any group. In conclusion, these results set the stage for subsequent studies on the neuropharmacological action of FA under conditions of psychological stress.

Ferulic Acid Regulates the Nrf2/Heme Oxygenase-1 System and Counteracts Trimethyltin-Induced Neuronal Damage in the Human Neuroblastoma Cell Line SH-SY5Y.

Over the past years, several lines of evidence have pointed out the efficacy of ferulic acid (FA) in counteracting oxidative stress elicited by ß-amyloid or free radical initiators, based on the ability of this natural antioxidant to up-regulate the heme oxygenase-1 (HO-1) and biliverdin reductase (BVR) system. However, scarce results can be found in literature regarding the cytoprotective effects of FA in case of damage caused by neurotoxicants. The aim of this work is to investigate the mechanisms through which FA exerts neuroprotection in SH-SY5Y neuroblastoma cells exposed to the neurotoxin trimethyltin (TMT). FA (1-10 µM for 6 h) dose-dependently increased both basal and TMT (10 µM for 24 h)-induced HO-1 expression in SH-SY5Y cells by fostering the nuclear translocation of the transcriptional activator Nrf2. In particular, the co-treatment of FA (10 µM) with TMT was also responsible for the nuclear translocation of HO-1 in an attempt to further increase cell stress response in SH-SY5Y cells. In addition to HO-1, FA (1-10 µM for 6 h) dose-dependently increased the basal expression of BVR. The antioxidant and neuroprotective features of FA, through the increase of HO activity, were supported by the evidence that FA inhibited TMT (10 µM)-induced lipid peroxidation (evaluated by detecting 4-hydroxy-nonenal) and DNA fragmentation in SH-SY5Y cells and that this antioxidant effect was reversed by the HO inhibitor Zinc-protoporphyrin-IX (5 µM). Among the by-products of the HO/BVR system, carbon monoxide (CORM-2, 50 nM) and bilirubin (BR, 50 nM) significantly inhibited TMT-induced superoxide anion formation in SH-SY5Y cells. All together, these results corroborate the neuroprotective effect of FA through the up-regulation of the HO-1/BVR system, via carbon monoxide and BR formation, and provide the first evidence on the role of HO-1/Nrf2 axis in FA-related enhancement of cell stress response in human neurons.

Endocrine disruptors and human reproductive failure: the in vitro effect of phthalates on human luteal cells.

OBJECTIVE: To evaluate the influence of phthalates on human luteal cell function. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S): Twenty-three normally menstruating patients in the midluteal phase. INTERVENTION(S): Human luteal cells isolated from corpora lutea for primary cultures. MAIN OUTCOME MEASURE(S): Progesterone (P4) and prostaglandin release assayed by enzyme immunoassay, vascular endothelial growth factor (VEGF) secretion by enzyme-linked immunosorbent assay (ELISA), and VEGF mRNA expression by real-time polymerase chain reaction. RESULT(S): We investigated the effect of di(2-ethylhexyl)phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) on basal and hCG-induced progesterone (P4) release, as well as DEHP effect on the balance between prostaglandin (PG) E2, vascular endothelial growth factor (VEGF)-luteotrophic factors, and the luteolitic PGF2α in isolated human steroidogenc cells. Phthalates influence on VEGF expression has been also evaluated. DEHP, DBP, and BBP were able to reduce both basal and hCG-stimulated P4 as well as PGE2 release. PGF2α release was reduced after DEHP incubation. VEGF protein release was decreased by the incubation with the tested phthalates. VEGF mRNA expression was not affected by DEHP, DBP, and BBP. As expected, both hCG and cobalt chloride were able to induce P4 release and VEGF release and mRNA expression in human luteal cells respectively. CONCLUSION(S): The results show the ability of phthalates to affect luteal steroidogenesis as well as the balance between luteotrophic and luteolytic factors suggesting an interference of phthalates in human luteal function. These data may contribute to clarify the classically known impaired reproductive health observed after phthalates exposure.

Endocrine disruptors and human corpus luteum: in vitro effects of phenols on luteal cells function.

Endocrine disruptors are well known to impair fertility. The aim of the present study was to investigate the effects of bisphenol A (BPA) and nonylphenol (p-NP) on human luteal function in vitro. In particular, in luteal cells isolated from 21 human corpora lutea progesterone, prostaglandin (PG) F2α, PGE2 and vascular endothelial growth factor (VEGF) release, as well as VEGF expression were evaluated. BPA and p-NP negatively affected both luteal steroidogenesis and luteotrophic/ luteolytic factors balance, without influencing VEGF mRNA expression. Actually, BPA and p-NP impaired human luteal cells function in vitro, underlining the already suggested correlation between phenols and reproductive failure.

In vitro effect of unacylated ghrelin and obestatin on human luteal cell function.

OBJECTIVE: To evaluate whether unacylated ghrelin and obestatin were able to influence human luteal cell function. The effect of these two ghrelin-related peptides on progesterone (P4), prostaglandin (PG) F(2α), PGE(2), and vascular endothelial growth factor (VEGF) release and on VEGF expression in isolated human steroidogenic cells has been investigated. DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENT(S): Corpora lutea were obtained from 23 normally menstruating patients in the midluteal phase of the menstrual cycle. INTERVENTION(S): Human luteal cells were isolated from corpora lutea, and primary cultures were established. MAIN OUTCOME MEASURE(S): P4 and PGs release was assayed by enzyme immunoassay, VEGF secretion by ELISA, and VEGF mRNA expression by real-time polymerase chain reaction. RESULT(S): P4 and VEGF release were significantly reduced by both unacylated ghrelin and obestatin. Moreover, the highest concentration of obestatin was able to reduce the release of PGE(2) and PGF(2α). VEGF mRNA expression was not affected by the incubation with any of these ghrelin-related peptides. As expected, CoCl(2) was able to induce VEGF release and mRNA expression in luteal cells. CONCLUSION(S): Our results suggest that, similar to ghrelin, both unacylated ghrelin and obestatin might play a role in regulating the luteal cell function that affects both luteal steroidogenesis and luteotrophic/luteolytic imbalance. These results further underline the pivotal correlation between the ghrelin system and reproduction.

Estrogens and androgens affect human luteal cell function.

OBJECTIVE: To evaluate estrogens (Es)--E2, estrone (E1), and estriol--and androgens--T and androstendione (A)-effect on P, prostaglandin (PG) F2α, PGE2, and vascular endothelial growth factor (VEGF) release and on VEGF expression in human luteal cells. To elucidate whether androgens effects were direct or mediated by their conversion in Es, an aromatase inhibitor was used. Finally, the luteal effect of the non-aromatizable dihydrotestosterone was evaluated. DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENT(S): Corpora lutea (CLs) were obtained from 36 normally menstruating patients in the midluteal phase of the menstrual cycle. INTERVENTION(S): The human luteal cells were isolated from CLs and primary cultures were established. MAIN OUTCOME MEASURE(S): P and PG release were assayed by enzyme immunoassay; VEGF secretion by ELISA; VEGF messenger RNA (mRNA) expression by real-time polymerase chain reaction (PCR). RESULT(S): P and PGF2α secretion were decreased by Es and androgens. The VEGF release was increased by Es and androgens, whereas VEGF mRNA expression was not. The aromatase inhibitor counteracted T and A luteal effects. CONCLUSION(S): Both Es and androgens could participate in the regulation of human luteal function. The effect of T and A seems to be mediated by their conversion to Es, whereas for dihydrotestosterone, both direct androgenic and indirect estrogenic luteal effects could coexist.

Paracrine regulation of endometriotic tissue.

Endometriosis is a chronic estrogen-dependent gynecological disease, characterized by pelvic pain and infertility, defined as the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites. In the peritoneal cavity endometrial cells adhere, proliferate and induce an inflammatory response. Despite a long history of clinical and experimental research, the pathogenesis of endometriosis is still controversial. Abnormal immunological activation, the endocrine milieu and the peritoneal environment all dramatically affect endometriotic tissue function. Recent studies suggest that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages and other immune cells that secrete various local products, such as growth factors and cytokines, which exert a paracrine action on endometriotic cells. Since the peculiar biological characteristics of eutopic endometrium from women with endometriosis differ from endometrium of normal subjects, an important role in the pathogenesis of this complex disease has been suggested. All of these factors contribute to enhanced proliferative and angiogenic activity and a number of functional and structural changes, resulting in the particular behavior of this tissue.

Ghrelin affects the release of luteolytic and luteotropic factors in human luteal cells.

CONTEXT: Ghrelin, well-known modulator of food intake and energy balance, is a rather ubiquitous peptide involved in several endocrine and nonendocrine actions. A possible as-yet-unknown role for ghrelin in modulating luteal function has been suggested because both ghrelin and its receptor (GRLN-R) have been immunohistochemically detected in human corpus luteum. OBJECTIVE: We first investigated GRLN-R mRNA expression in midluteal phase human luteal cells. Ghrelin effect on basal and human chorionic gonadotropin (hCG)-stimulated progesterone (P) release was then analyzed. Finally, we investigated whether ghrelin could affect luteal release of vascular endothelial growth factor (VEGF), prostaglandin (PG) E(2), both luteotropic factors, and PGF(2alpha), luteolytic modulator. Ghrelin effect on both basal and hypoxia-stimulated VEGF luteal expression was analyzed. METHODS: Human luteal cells were incubated for 24 h with ghrelin (10(-13) to 10(-7) m) or hCG (100 ng/ml) or CoCl(2) (10 microm), chemical hypoxia, or with hCG or CoCl(2) in combination with ghrelin. Both GRLN-R mRNA and VEGF mRNA were evaluated by real-time RT-PCR. PGs and P release was assayed by RIA, whereas VEGF release by ELISA. RESULTS: GRLN-R mRNA expression was demonstrated in human luteal cells. Both basal and hCG-stimulated P release was significantly decreased by ghrelin, which was able to reduce PGE(2) and increase PGF(2alpha) luteal release. Both basal and hypoxia-stimulated VEGF release was significantly decreased by ghrelin, which did not affect VEGF mRNA luteal expression. CONCLUSIONS: The present in vitro study provides the first evidence of a direct inhibitory influence of ghrelin on human luteal function.

Ghrelin in vitro modulates vasoactive factors in human umbilical vein endothelial cells.

OBJECTIVE: To determine whether Ghrelin could affect prostaglandins (PGs) and nitric oxide synthesis in human umbilical vein endothelial cells (HUVEC). The effect of Ghrelin on endothelial cell proliferation was also evaluated. DESIGN: In vitro research report. SETTING: Third-level referral academic centers, including molecular and cellular biology laboratories. PATIENT(S): Human umbilical cords were obtained from healthy female volunteers at term of uncomplicated pregnancies. INTERVENTION(S): HUVEC were cultured with Ghrelin (from 10(-11) to 10(-7) M). After 24 hours supernatants were collected and HUVEC were treated for total RNA extraction. MAIN OUTCOME MEASURE(S): In the culture medium PGs release was evaluated by RIA. Prostaglandin-endoperoxide synthase 2 (COX2) and both the constitutive and the inducible isoforms of nitric oxide synthases (ECNOS and INOS) mRNA expressions were evaluated by retrotranscriptase polymerase chain reaction. Endothelial cell proliferation was evaluated by bromo-deoxy-uridine incorporation and by cell counting. RESULT(S): Ghrelin negatively affected PGs release as well as COX2, ECNOS, and INOS mRNA expressions in HUVEC. Furthermore, Ghrelin increased bromo-deoxy-uridine incorporation in HUVEC without affecting cell counting. CONCLUSION(S): Our in vitro results allowed to hypothesize that Ghrelin could be involved in the modulation of vascular tone by affecting nitric oxide-related protein synthesis and PGs production in endothelial cells.

Regulation of vascular endothelial growth factor synthesis and release by human luteal cells in vitro.

CONTEXT: Vascular endothelial growth factor (VEGF) is essential for normal luteal development and function, but little is still known about the regulation of its production by human midluteal phase luteal cells. OBJECTIVE: We investigated whether human chorionic gonadotropin (hCG) or local factors, including chemical hypoxia, IGF-I and IGF-II, prostaglandin (PG)E(2), and PGF(2alpha) prevail in modulating VEGF mRNA and protein production in human midluteal phase luteal cells. The effect of progesterone (P) on luteal VEGF mRNA expression and protein secretion was also evaluated. Finally, we investigated whether VEGF could directly affect luteal P secretion. INTERVENTIONS: In human midluteal phase luteal cells, VEGF mRNA expression was evaluated by semiquantitative RT-PCR, whereas VEGF and P release was evaluated by ELISA and RIA, respectively. RESULTS: hCG was unable to significantly affect luteal VEGF mRNA and protein synthesis, which in turn was significantly increased by both chemical hypoxia and IGFs. Conversely, VEGF mRNA and protein production was reduced by PGs and P. Finally, VEGF did not affect P luteal secretion. CONCLUSIONS: Our results suggest that local ovarian factors, rather than hCG, predominate in regulating VEGF mRNA and protein production by human midluteal phase luteal cells. For VEGF, a lack of a direct luteal steroidogenic effect was also demonstrated.

Effects of nicotine on human luteal cells in vitro: a possible role on reproductive outcome for smoking women.

We investigated the effect of nicotine and its methylated metabolite, N-methyl-nicotine (M-nicotine), on human luteal cells by measuring release of progesterone and prostaglandins (PGs) from cultured cells and by testing gene expression of vascular endothelial growth factor (VEGF), an angiogenic factor strictly involved in luteal pathophysiology. Primary cultures of human luteal cells were treated for 24 h with nicotine and M-nicotine (from 10(-6) to 10(-11) M) either alone or combined with hCG (25 ng/ml); progesterone and PGs were assayed in the culture medium. In another group of experiments, luteal cells were treated for 24 h with nicotine and M-nicotine (10(-7) M) to perform reverse transcriptase-polymerase chain reaction on VEGF mRNA. Nicotine and M-nicotine negatively affected basal luteal steroidogenesis at all tested concentrations, but neither was able to affect hCG-induced progesterone release. Both substances were able to significantly increase PGF2alpha release from luteal cells, with a dose-related efficacy for M-nicotine. On the contrary, PGE2 release was significantly inhibited by both nicotine and its metabolite. Finally, nicotine was able to increase VEGF mRNA expression significantly, whereas M-nicotine was not. In conclusion, nicotine and M-nicotine can induce a sort of luteal insufficiency by inhibiting progesterone release, probably through modulation of the PG system.

Endometrial evaluation in superovulation programs: relationship with successful outcome.

It is well known that an adequate endometrial receptivity is required for successful implantation in both natural and assisted reproductive cycles. In particular, a brief "implantation window", during which endometrium undergoes anatomical and molecular changes necessary for embryo implantation, has been observed. The hormonal treatment applied to induce ovulation seems to be able to modify the normal development of the prenidatory endometrium, with possible negative effect on the implantation rate. For this reason, several attempts have been made to identify specific markers of endometrial receptivity, useful for predicting implantation outcome in clinical practice. Even if different histological, immunohistochemical, and ultrasonographic parameters are studied, none unfortunately has been univocally shown to be predictive of pregnancy outcome. Therefore, the evaluation of endometrial receptivity remains a challenge in clinical practice.