ACTA ENDOCRINOLOGICA (BUC)

Context. Endometriosis is a common gynecological disease, characterized by ectopic deposits of endometrial tissue outside of the uterine cavity, and it is associated with pelvic pain and infertility, with an important impact on the quality of life. At this point there is a controversy regarding the etiology and pathophysiology of endometriosis and it seems that pro-angiogenic growth factors might be involved, but their role is not completely understood. Objective. To evaluate the serum concentration of the main growth factors in patients with diagnosed endometriosis compared to healthy controls. S ubjects and Methods. A total of 157 women were divided into two study groups (Group I – endometriosis; Group 2 – healthy women). Serum levels of VEGF, G-CSF, GM-CSF, b-FGF, EGF, and HGF were measured with Human Multiplex Cytokine Panels. Results. VEGF serum levels were significantly lower in women with endometriosis compared to controls (1.924±0.145 compared to 1.806±0.078 pg/mL, p<0.001). Serum levels of GM-CSF, b-FGF, EGF, and HGF respectively did not differ significantly between patients with endometriosis and healthy controls. G-CSF had a very low detection rate. Conclusions. The present study showed that VEGF serum levels are significantly lower in endometriosis patients compared to healthy controls, indicating a possible role in endometriosis pathogenesis.

Introduction. Even if the last decade brings new information about the existence of the local pulmonary renin-angiotensin system (RAS), published results about its involvement in the progression of various pulmonary diseases remain contradictory. Methods. Using an experimental model of pulmonary allergic disease induced by ovalbumin (OVA) sensitization we studied effects of intratracheal (i.t.) administrated AT1 receptor blocker losartan (LOS) on inflammatory processes supposed to be mediated by pulmonary RAS. Alterations in bronchoalveolar lavage fluid (BALF) cellularity and variations of airway reactivity from OVA sensitized and challenged rats after LOS or vehicle i.t. instillation were comparatively assessed. Results. Blocking of RAS decreased the total BALF cellularity and polymorphonuclear cell count. LOS instillation also reduced the increases in specific airway resistance (sRaw) induced by inhalation of allergen (with 20%) or acetylcholine (with at least 15%). Conclusions. Our data suggest that expressly blocking of local RAS by i.t. administration of LOS have inhibitory effects on allergen - induced lung inflammation and sustain the participation of pulmonary RAS, activated by local or systemic factors, in lung diseases.

Background. Trigonella foenum graecum L. (Fenugreek, FG) is used in many countries as a medicinal plant. Evidence has suggested the hypolipidemic effect of Fenugreek; however, its actual mechanism has not been determined yet. Objectives. The purpose of our research was to investigate the effect of Fenugreek on lipid profile, liver histology and LDL receptor gene expression in male hamsters fed with high cholesterol diet. Methods. These animals were given normal diet (ND), high cholesterol diet (HCD: 2% cholesterol and 0.5% cholic acid added to ND), HCD supplemented with 2g and 8g fenugreek per 100g ND (HCD+FG2 and HCD+FG8) respectively for four weeks. At the end of treatment, serum lipids, lipoproteins and liver enzymes were measured. Finally, LDL receptor (LDLR) gene expression was determined in the liver of the studied animals using Real Time-PCR method and liver histological changes were evaluated by H&E staining method. Results. A significant reduction was observed in serum triglyceride (p<0.01), total cholesterol, low and very low density cholesterol, aspartate and alanine transaminases in HCD+FG8 group (p<0.05) compared with HCD group, while serum level of HDL-c (p<0.01) increased. In addition, LDLR gene expression in HCD+FG8 group increased 7.8 folds. The results confirm the protection effect of liver tissue in HCD+FG8 group against pathological changes. There was no significant change in LDLR gene expression in HCD+FG2 group. In conclusion, fenugreek ameliorated dyslipidemia by up-regulation of LDLR gene expression and can be used as a protective agent against hypercholesterolemia.

Introduction. Abnormal thyroid function affect spermato-genesis and male infertility. For men, the aromatase deficiency can cause infertility. In this study, the aim is to investigate the effect of maternal hypothyroidism on offspring testicular morphology and cytochrome-P450- aromatase (P450arom) immunoreactivity. Materials and Methods. Eighteen Wistar albino pregnant rats were divided into three groups, namely A, B and K groups. Hypothyroidism was induced by adding 0.01% of propyl thiouracil (PTU) in drinking water. Hypothyroid mothers, group A: given PTU for 21 days during pregnancy, group B: given PTU for 21 days prior to pregnancy; control mothers, group K, given only water. Hypothyroid and control group mothers’ pups at postnatal day (PND) 15 and 60 were sacrificed. We determined immunoreactivity intensity of P450arom and mRNA levels by RT-PCR performed in the testis tissues. ELISA method was used for thyroid function tests for T3, T4 and TSH. Structure of seminiferous tubule was evaluated by hematoxylin-eosin staining. Results. It was seen that the aromatase expression in 15-day-old maternal hypothyroid groups was similar to the one in the control group while there was a decline in the aromatase expression of 60-day-old groups. As for mRNA, it was determined that it had a tendency to increase over time in all groups but this increase was not significant. The tubule diameter and Johnsen’s Testicular Biopsy Score diminished in all hypothyroid groups in comparison to the control group. Conclusion. The changes that occur in the early period of testis development due to maternal hypothyroidism negatively affect testis development in the next stages of life. This situation leads to a decline in aromatase expression in the following years.

Background. Linagliptin (LNG) is a selective dipeptidyl peptidase-4 (DPP-4) inhibitor that ameliorates blood glucose control of patients with type 2 diabetes, without developing hypoglycemic risk and weight gain with a good clinical and biological tolerance profile. To the best of our knowledge, its cytotoxic, genotoxic and oxidative effects have never been studied on any cell line. Aim. To evaluate the in vitro cytotoxic, genotoxic damage potential and antioxidant/oxidant activity of LNG in cultured peripheral blood mononuclear cells (PBMC). Material and methods. After exposure to different doses (from 0.5 to 500 mg/L) of LNG, cell viability was measured by the MTT (3,(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) and lactate dehydrogenase (LDH) leakage tests. The antioxidant activity was assessed by the total antioxidant capacity (TAC) and total oxidative stress (TOS) assays. To evaluate the genotoxic damage potential, chromosomal aberration (CA) frequencies and 8-oxo-2’- deoxyguanosine (8-oxo-dG) levels were determined. Results. Treatment with LNG did not cause statistically significant decreases of cell viability at lower concentrations than 100 mg/L as compared to untreated cultures. However, LNG exhibited cytotoxic action at 250 and 500 mg/L. Also, IC20 and IC50 values of LNG were determined as 8.827 and 70.307 mg/L, respectively. In addition, the oxidative analysis revealed that LNG supported antioxidant capacity at concentrations of 2.5, 5, 10, 25, 50 and 100 mg/L without generating oxidative stress. Besides, the results of CA and 8-oxo-dG assays showed in vitro nongenotoxic feature of LNG. As a conclusion, our findings clearly revealed that LNG had no cytotoxic and genotoxic actions, but exhibited antioxidative activity. In conclusion, therefore it is suggested that LNG use in diabetic patients is safe and provides protection against diabetic vascular and oxidative complications.

Objective. To observe the impact of quercetin and isoquercitrin on gluconeogenesis in hepatocytes. Methods. Mouse primary hepatocytes were cultured with lactic acid and pyruvic acid. After treatment with quercetin and isoquercitrin for 24 hours, the glucose concentration in the culture supernatant was determined. RT-PCR was used to detect the mRNAs of PEPCK, G6Pase, LKB1, and AMPKα. Protein levels of LKB1, AMPKα, and Thr172 phosphorylation were evaluated by Western blot. Results. The glucose concentration in the gluconeogenesis group (GN) was significantly higher than in the control group (C), but the glucose concentrations in the high level quercetin(group 80Q) and high level isoquercitrin (group 80I) were significantly lower than in the group GN, P<0.01. In the group 80Q, and group 80I, the mRNA levels of PEPCK and LKB1were significantly lower than in the group GN (P<0.01), and the G6Pase mRNA were significantly lower than in the group GN (P<0.05). The protein levels of LKB1 and the phosphorylation of AMPKα Thr172 in the group 80Q, group 40I, and group 80I were higher than in the group GN. The effects of quercetin and isoquercitrin on LKB1 and AMPKα were similar to those of metformin. Conclusions. Quercetin and isoquercitrin inhibit gluconeogenesis in hepatocytes, which may be related to the LKB1 upregulation and phosphorylation of AMPKα.

Objective. To assess in a bicentric study the current iodine status of schoolchildren, ten years after implementation of the universal salt iodization (USI) in Romania. Subjects and methods. 102 children from 2 towns, aged between 6 and 11 years, were included in the study group: 66 children from Sibiu, a previously endemic area for iodine deficiency disorders and 36 children from Bucharest, a previously borderline iodine intake area. Body mass index (BMI), total body surface area (BSA), median urinary iodine concentration (UIC) and prevalence of goiter were evaluated. Thyroid volume was measured by ultrasonography. The study was approved by the Local Ethics Committee. An informed consent from the parents was obtained. Results. From the 102 schoolchildren in the study group, 59 were girls and 43 were boys. Median UIC in the total number of samples was 175.2 mcg/L, reflecting a sufficient iodine intake, with statistically significant differences between the two urban regions. The median UIC was 187.35 mcg/L in the Sibiu subgroup and 160.2 mcg/L in the Bucharest subgroup (p < 0.001). The total percentage of goiter in the studied subjects was 5.88%. Percentage of goiter, determined by adjusting ultrasound thyroid volume to sex and BSA, was 1.51% in Sibiu and 13.88% in Bucharest, a statistically significant difference (p = 0.011). None of the subjects showed ultrasonographic pattern suggestive of Hashimoto thyroiditis or macronodules. There was no statistically significant difference between the percentages of overweight or obesity in the two subgroups. Conclusions. Ten years after implementation of USI in Romania, a bicentric study suggests that our country is iodine sufficient in urban areas. In order to prevent recurrence of mild iodine deficiency in schoolchildren, a persistent surveillance, use of sustainable measures and public awareness are required. Recurrence of mild iodine deficiency should be avoided, because even mild iodine deficiency impairs cognition in children.

Aims. To test the effect of Hsa-miR-183-3p on cell aging and disc degeneration in lumbar intervertebral disc. Methods. This study combined clinical research with basic cell experiment, analyzing clinical data from patients with lumbar disc degeneration and traumatic lumbar spine fracture, as well as the differences in baseline data. The degree of lumbar disc injury in patients of different ages was also compared. Differentially expressed miRNAs were predicted via GEO database, and qPCR confirmation was determined by collecting cartilage endplates from two groups. ACAN, Col2A1, p16, p21, and p53 were detected by immunofluorescence, Western blot and qPCR in human nucleus pulposus cells. Changes of cell senescence were detected. The binding of Hsa-miR-183-3p to ataxiatelangiectasia mutated protein was confirmed by dual luciferase reporter assay. Results. Degenerative discs showed elevated expression of hsa-miR-183-3p, which may be upregulated by TNF-α via NF-κB signaling pathway and target ataxiatelangiectasia mutated protein regulation. Conclusion. Degeneration of the intervertebral disc can be accelerated by TNF-α. Additionally, Hsa-miR- 183-3p passed NF-κB signaling pathway is blocked via upregulation of TNF-α to reduce inflammation via targeting ataxia-telangiectasia mutated protein. As a result, this negative feedback mechanism may assist in maintaining a low degenerative load and preserving chronic disc degeneration.

Context. The inflammatory disorders of the urinary bladder represent one of the most frequent disorders associated with hormonal unbalances caused by menopause. The involvement of estrogens and mast cells in this complex mechanism mediated by neuro-hormonal pathway is well known, but the pathogenesis through which the hormonal deprivation is affecting the Estrogen Receptor expression and is predisposing to urinary bladder inflammatory changes is still argued. Objective. To determine the structural changes associated with surgically induced menopause, and the effect of estrogen replacement therapy (ERT) in the urinary bladder morphology, mast cell population and Estrogen Receptor (ERα) expression. Subjects and methods. The effect of ovariectomy and ERT was monitored by quantifying the number of mast cells and the structural changes that the urinary bladder suffers. By immunohistochemistry we assessed the changes of the Estrogen Receptor Alpha (ERα) expression in the urothelium and detrusor muscle. The study was carried out on ovariectomised female rats over a period of 42 days. Results. The main alterations associated with the hormonal deprivation were represented by the growth in number of the bladder mast cells, atrophy of the urothelium and amplification of the expression of ERα from the urothelium, but not from the detrusor muscle. ERT significantly decreased the tissue expression for ERα, reduced the severity of bladder atrophy and the number of mast cells. Conclusions. The estrogenic hormonal substitution can diminish the severity of the atrophic, inflammatory and ERα changes in bladder disorders associated with ovarectomy in rat.

Context. The undercarboxylated form of osteocalcin (ucOC) and osteoprotegerin (OPG) are bonederived molecules involved in the endocrine crosstalk governing the bone, the adipose tissue and the pancreas. In addition, glucocorticoids are major determinants of both insulin resistance and osteoporosis. Objective. We aimed to investigate the response of ucOC and OPG to dysglycemia and/or dexamethasone (DXM) in primary human osteoblastic cell (HOC) cultures. Design and methods. Third-passage sub-confluent primary HOC cultures were treated with glucose: 2.8 mmol/L, 5.6 mmol/L, 11.1 mmol/L and 28 mmol/L, respectively. Alternatively, HOC cultures were subjected to DXM 1 μmol/L. In more complex experiments, HOC cultures were pre-treated with glucose (5.6 mmol/L) with/without insulin (1 pmol/L) followed by DXM (1 μmol/L). 24-hours posttreatment, culture medium ucOC and OPG were measured by ELISA. Results. ucOC production differed significantly (p<0.05) between cell groups, decreasing in a dosedependent manner as glucose concentration in the medium increased. Insulin prevented this effect. OPG levels appeared not to be significantly influenced by the hyperglycemic culture medium and were not related to ucOC concentration (p>0.05). Addition of DXM resulted in significantly lower ucOC concentrations compared to vehicle-treated cells (p<0.05). However, the effect of insulin co-treatment on ucOC was not counteracted by DXM (p<0.05). Conclusions. An obvious alteration of OC production/metabolism was observed as glucose levels changed in the bone microenvironment, to potentially be involved in diabetes-related osteopenia. DXM suppressed ucOC levels however not in insulin-rich environment.