PROLACTIN REDUCES HIPPOCAMPAL PARVALBUMIN AND GABAA RECEPTOR EXPRESSION IN FEMALE MICE

Introduction: Parvalbumin (PV)-positive cells are strategic elements of neuronal networks capable of influencing memory and learning processes. However, it is not known whether pituitary hormones may be related to parvalbumin expression in the hippocampus — a part of the limbic system with important functions in learning and memory. Objective: Since previous studies indicate that prolactin (PRL) plays a significant role in hippocampal-dependent learning and synaptic plasticity, we hypothesized that a rise in PRL levels can modify PV expression in the hippocampus. Methods: We employed biochemical, immunohistochemistry and densitometry techniques —as well as a behavioural assay— in a hyperprolactinemia model using subcutaneous osmotic pumps in female mice. Results: PRL treatment via osmotic pump induced an increase in PRL receptor (PRLR) expression in most regions of hippocampus analysed by Western blotting and immunohistochemistry methods. Fluorescent densitometry analysis revealed that PV expression decreases in the same layers in the hippocampus following PRL treatment, while double labelling immunostaining indicated close localization of PV and PRLR in PV-positive interneurons. In addition, we found that PRL induced a reduction in β2/3 subunit of GABAA receptor (GABAAR) expression that was linearly correlated with the reduction in PV expression. This reduction in β2/3 subunit of GABAAR expression was maintained in trained animals in which PRL treatment improved the learning of a spatial memory task. Conclusions: These data show, for the first time, that an increase in PRL level is associated with changes in key constituent elements of inhibitory circuits in the hippocampus and may be of relevance for the alterations in cognitive function reported in hyperprolactinemia.

Corneal densitometry: a potential indicator for early diagnosis of Fabry disease

Purpose To assess corneal densitometry in patients with Fabry disease (FD) and to compare corneal densitometry differences in FD patients to different corneal manifestations. Methods Ten participants (20 eyes) with FD and 10 age-matched healthy volunteers (20 eyes) were recruited. All participants were assessed by standardized ophthalmic examinations and the corneal densitometry analysis by Pentacam HR. Densitometry measurements were analyzed in standardized grayscale units. Results Seven patients developed conjunctival vessel tortuosity, cornea verticillata appeared in 6 patients, and two patients had Fabry cataract. Retinal vessel tortuosity occurred in 4 patients, and dilation of retinal vessels appeared in 3 patients, all symptoms occurred in both eyes. The first diagnosis of FD up to examination was 4.7 ± 3.23 years, and first ERT up to examination was 2.6 ± 2.27 years. The initial time to diagnosis was negatively related to the corneal densitometry value of the 0–2-mm (r = − 0.556, p = 0.011) and 2–6-mm (r = − 0.482, p = 0.032) zones in the posterior layer. FD group have significantly higher corneal densitometry in anterior 0–2-mm zone and 2–10-mm zone anterior and posterior layer than the control group (p ≤ 0.035, respectively). When divided into two groups by the existence of cornea verticillata, there was a statistically significant difference in the anterior layer, 6–10-mm zone (p = 0.031); in the central layer, 0–2 mm (p = 0.012), 2–6 mm (p = 0.001), 6–10 mm (p = 0.002), and total (p = 0.002); and in the posterior layer, 6–10 mm (p = 0.004) and total (p = 0.002). Conclusions FD patients show higher corneal densitometry, and corneal densitometry may have potential for early diagnosis and reminding progress of FD.

173 The role of an intronic single nucleotide polymorphism within the dopamine receptor type-2 gene on pituitary prolactin protein expression in bulls

Detrimental effects to male reproductive physiology have been observed due to changes in prolactin (PRL) serum concentration. Regulation of PRL by dopamine binding to the dopamine type-2 receptor (DRD2) is well defined and associations between male physiology and single nucleotide polymorphisms (SNPs) within the DRD2 gene have been observed. The objective of the study was to evaluate association of a DRD2 SNP to PRL protein expression in bulls. Testis and epididymis were collected from bulls grazing a forage containing or lacking a dopamine agonist at the end of a 126 d study (n = 14). Bovine pituitaries (n = 587) were collected randomly over 3 mo from a local abattoir which processes cull cows and bulls. Sex of pituitaries was verified (n = 259 males) by duplex PCR for amplification of SRY and b-actin followed by Southern blotting of PCR products for selection of male. Prolactin protein expression was assessed in testis, epididymis, and pituitary by western blotting. Expression of PRL protein was below detection range in reproductive tissues but was present in pituitary, therefore experiments continued in pituitary. Restriction fragment length polymorphism genotyping was performed on pituitaries by amplification of the DRD2 SNP region followed by digestion with a Tfil enzyme. Digested of products produced 3,2, or 1 band (AG, AA, GG, respectively). A subset of male pituitaries was blotted by slot blot manifold and PRL protein expression assessed by immunodetection and densitometry analysis normalized to GAPDH expression. Pituitary genotype distribution was 17.4% AA (n = 16), 63% AG (n = 58), and 19.6% GG (n = 18). Prolactin protein expression in the pituitary was similar across genotype (P = 0.23). These findings indicate that the DRD2 SNP has no genotypic effect on PRL protein expression in bovine pituitary.

Development and validation of stability indicating HPTLC method for estimation of pirfenidone and characterization of degradation product by using mass spectroscopy

Pirfenidone is used as a novel antifibrotic agent approved for mild-to-moderate idiopathic pulmonary fibrosis. An extensive literature search revealed that, method validation by high-performance thin-layer chromatography (HPTLC) and structural determination by tandem mass spectrometry (MS/MS) method was not reported till date. Precoated silica gels plates were used as a stationary phase. Methanol: ethyl acetate: toluene (1:2:7 v/v) was delivered best separation at 315 nm (Rf 0.49±0.03) by densitometry analysis. Degradation analysis was performed as per ICH guidelines Q2 (R1). Isolation of degradation product was done by the HPTLC method and characterized by using MS/MS method. All the validation parameters were found within the range. Moreover, its possible degradation pathway was also proposed. The Proposed developed and validated HPTLC method was found to be more sensitive, simple, precise, accurate, cost-effective and robust. This method could be applied for the analysis of bulk drug and tablet formulation, degradation study. This degradation pathway of the drug will further help to identify the degradation products of Pirfenidone which may be used for the impurity profiling of the drug.

Oxyresveratrol Inhibits IL-1β-Induced Inflammation via Suppressing AKT and ERK1/2 Activation in Human Microglia, HMC3

Oxyresveratrol (OXY), a major phytochemical component derived from several plants, has been proved to have several pharmacological properties. However, the role of OXY in regulating neuroinflammation is still unclear. Here, we focused mainly on the anti-neuroinflammatory effects at the cellular level of OXY in the interleukin-1 beta (IL-1β)-stimulated HMC3 human microglial cell line. We demonstrated that OXY strongly decreased the release of IL-6 and MCP-1 from HMC3 cells stimulated with IL-1β. Nevertheless, IL-1β could not induce the secretion of TNF-α and CXCL10 in this specific cell line, and that OXY did not have any effects on reducing the basal level of these cytokines in the sample culture supernatants. The densitometry analysis of immunoreactive bands from Western blot clearly indicated that IL-1β does not trigger the nuclear factor-kappa B (NF-κB) signaling. We discovered that OXY exerted its anti-inflammatory role in IL-1β-induced HMC3 cells by suppressing IL-1β-induced activation of the PI3K/AKT/p70S6K pathway. Explicitly, the presence of OXY for only 4 h could strongly inhibit AKT phosphorylation. In addition, OXY had moderate effects on inhibiting the activation of ERK1/2. Results from immunofluorescence study further confirmed that OXY inhibited the phosphorylation of AKT and ERK1/2 MAPK upon IL-1β stimulation in individual cells. These findings suggest that the possible anti-inflammatory mechanisms of OXY in IL-1β-induced HMC3 cells are mainly through its ability to suppress the PI3K/AKT/p70S6K and ERK1/2 MAPK signal transduction cascades. In conclusion, our study provided accumulated data that OXY is able to suppress IL-1β stimulation signaling in human microglial cells, and we believe that OXY could be a probable pharmacologic agent for altering microglial function in the treatment of neuroinflammation.

EFFECT OF DIFFERENT METHODS OF TRAINING IN BODY COMPOSITION AND LIPID PROFILE IN OCCUPATIONAL MEN

Obesity is increasingly present in the population and raises the risk of death from cardiovascular diseases. The objective of this study was to investigate changes in body composition, total cholesterol, LDL, HDL and triglycerides of obese adults. The sample was costituida twelve male volunteers subjected to hold a bone densitometry analysis with body composition (DEXA) and examinations that incaram plasma concentrations of total cholesterol, LDL, HDL and triglycerides in the blood. The volunteers were inserted into three training models: aerobic training (AeT), anaerobic training (AnT) and concurrent training (CT). The training period was twelve weeks, with three sessions per week lasting 60 minutes. The AeT group took part in running sessions with cycling sessions. The AnT group performed bodybuilding exercises. The CT group combined the two previous models, with no gap between them. All variables were tested for normality of distribution by the Shapiro-Wilk test. For those who presented normality, the paired teste test was used for comparison before and after training (mean ± standard error). All analyzes were performed by the Statistical Package for the Social Sciences (SPSS) version 21.0. A significance level of α = 0.05 was used. When comparing the initial and final periods of intervention was observed reductions in variables: total weight, body fat weight and visceral fat weight in all groups. Total cholesterol decreased only in the CT group. Already, the LDL cholesterol decreased in the AnT and CT groups, being that in the CT with greater notoriety. HDL cholesterol increased in all groups, and in the AnT with greater notoriety. The plasma triglyceride concentration decreased only in the AeT and CT groups. The data suggest that the concurrent training is effective for the reduction of obesity when compared to the aerobic and anaerobic methods.