Protective effect of melatonin on blood-brain barrier damage caused by Endotoxemia.

OBJECTIVE: Endotoxins, products of Gram-negative bacteria, are the primary cause of blood-brain barrier (BBB) damage. In the present study, we aimed to investigate the possible neuroprotection mechanisms of melatonin on BBB damage induced by endotoxemia. METHODS: Adult, female Sprague-Dawley rats (n = 42) were separated into four random groups as a control group and three treatment groups. Lipopolysaccharide (7,5 mg/kg/day) was administrated for a single dose to generate a 24-hour sepsis model on rats. Melatonin (10 mg/kg/day) was treated a week before sepsis. Afterward, the dissected brain tissues were examined by histopathological, biochemical, and molecular analyses. RESULTS: LPS caused weight loss in the groups. As a result, degenerated neurons with cytoplasmic vacuoles and irregular pyknotic nuclei, pale stained necrotic neurons, and vascular congestion were observed in LPS-exposed rats. However, MEL decreased the number of degenerated neurons in treated groups. MEL treatment increased ZO1 and Occludin immunoreactivity while decreasing TLR4 in brain tissues. MEL effect on protein expression was recorded for ZO1 increase and TLR4 decrease in brain tissue compared to LPS groups. MEL also decreased MDA levels in brain tissue. CONCLUSIONS: MEL recovered the degenerative damage of sepsis by contributing to blood-brain barrier integrity, and by decreasing inflammation, thus the neuroprotective effects of MEL might provide an experimental basis for clinical applications.

Kaempferol treatment ameliorates memory impairments in STZ­induced neurodegeneration by acting on reelin signaling.

Many treatment initiatives, like herbal products and their active ingredients, aim to alleviate neurodegeneration to increase cognitive functions. Kaempferol may be a candidate molecule for treating neurodegeneration because of its antioxidant effects. In the present study, we examined the molecular changes associated with kaempferol's memory­enhancing effects on streptozotocin (STZ)­induced neurodegeneration. After intracerebroventricular STZ injection in Long­Evans male rats, intraperitoneal kaempferol was administered for 12 days. The Morris water maze (MWM) was used to measure learning and memory performance in the rats, and proteins related to memory formation were investigated in the hippocampi with western blotting. Kaempferol improved learning performance and memory decline in STZ­treated rats. At the molecular level, STZ­induced neurodegeneration resulted in a decrease in the expression of GAD67, reelin, and phosphorylated­NMDAR. However, kaempferol treatment ameliorated these changes by enhancing their levels similar to the controls. While neither STZ injection nor kaempferol treatment produced any significant change in phosphorylated­CAMKII levels, they increased the expression of klotho and prealbumin. These results show that kaempferol has positive effects on memory loss, affecting synaptic plasticity by ameliorating both the levels and activity of memory­relevant molecules through reelin signaling. In summary, this study provides a guide to future studies by examining in detail the healing effect of kaempferol as a candidate molecule in the treatment of neurodegeneration, such as that observed in Alzheimer's disease.

Astragalus membranaceus treatment combined with caloric restriction may enhance genesis factors and decrease apoptosis in the hippocampus of rats.

Humans have been searching for ways of extending life span, and possible underlying molecular mechanisms behind it for many years. Traditional plants and their extracts are good candidates for finding anti-aging strategies. In addition to its usage in a variety of medical treatments such as inflammation, neural diseases and cancer, Astragalus membranaceus was used to extend lifespan of C. elegans. Therefore, we aimed to show the molecular mechanisms of the possible anti-aging effects of combination of A. membranaceus and caloric restriction. Herein, Wistar rats (n = 24) were divided into Control, A. membranaceus (A) (25 mg/kg A), Caloric restriction (CR) (20% restricted-diet), and CR+A (25 mg/kg A + 20% CR diet) groups. After 18 weeks, behavioral tests were applied to observe alterations on cognitive functions. After animals were decapitated, their hippocampi and livers were dissected for molecular analysis and telomerase activity. Eventually, CR increased learning performances of rats with an increase in the telomerase activity when combined with astragalus. There was a negative correlation between learning and apoptosis parameters. In the CR group, the apoptosis rate increased, and the pyramidal neuron numbers decreased which were reached to control levels with A treatment. The CR+A treatment significantly increased the BDNF level. The A also significantly increased GDNF level independent from CR. In the combination group, the neurogenesis and angiogenesis markers increased with an increase in the anti-senescence protein klotho land a decrease in the apoptosis. In conclusion, combination of caloric restriction with A. membranaceus would become a promising strategy for healthy cognitive aging.

The role of galectin-3 and its genetic variants in tumor risk and survival of patients with surgically resected early-stage non-small cell lung cancer.

BACKGROUND: The aim of this study was to investigate the possible relationship between galectin-3 gene variants, serum level, gene expression level, and the risks and survivals of resectable non-small cell lung cancer patients. METHODS: The rs4644 and rs4652 variants of galectin-3 were genotyped by TaqMan single nucleotide polymorphism assay using genomic deoxyribonucleic acid isolated from the peripheral blood of 65 (54 males, 11 females; mean age: 60.1±11.9 years; range, 34 to 83 years) with Stage IA-IIIA non-small cell lung cancer who underwent primary surgical treatment and 95 healthy individuals (48 males, 47 females; mean age: 53.9±13.5 years; range, 32 to 87 years) between March 2017 and September 2018. Circulating galectin-3 levels in serum samples of the patient and control groups were assessed by enzyme-linked immunosorbent assay. Messenger ribonucleic acid expression of galectin-3 in tumor and surrounding tissues of the patient group was examined by real-time quantitative polymerase chain reaction. Both predictive and prognostic significance of the results were analyzed. RESULTS: The presence of angiolymphatic invasion was significant in the patients with rs4652 AA genotype (p=0.04). Serum galectin-3 levels were significantly higher in the patients than the controls (p<0.0001). The patients with rs4644 CA/CC (p<0.0001 and p<0.0001) and rs4652 AA/AC (p=0.001 and p<0.0001) genotypes had higher serum galectin-3 levels than their corresponding controls. Serum galectin-3 levels increased in the presence of vascular invasion in patients with both rs4644 AC (p=0.03) and rs4652 AC (p=0.019) genotypes. The receiver operating characteristic curve suggested serum galectin-3 level as a strong predictive marker for the patient group with a cut-off value of 17.089 ng/mL (area under the curve: 0.910±0.04; 95% confidence interval: 0.832-0.988; p<0.001). Univariate analysis revealed the association of lower serum galectin-3 levels with better survival (p=0.048). Multivariate survival analysis showed that only high serum galectin-3 levels tended to be related to survival of the patients (hazard ratio: 5.106; 95% confidence interval: 0.956-27.267; p=0.056). CONCLUSION: The presence of galectin-3 gene variants may lead to histopathological differences among patients with non-small cell lung cancer. Serum galectin-3 level may be a valuable diagnostic biomarker and be associated with survival of these patients.

Thymoquinone administration ameliorates Alzheimer's disease-like phenotype by promoting cell survival in the hippocampus of amyloid beta1-42 infused rat model.

BACKGROUND: Thymoquinone (TQ), a biologically active ingredient of Nigella sativa, has anti-inflammatory, anti-oxidative and neuroprotective properties. Therefore, it could be a good candidate in the recovery of Alzheimer`s disease (AD) pathology rather than current symptomatic reliefs. PURPOSE: In the present study, we examined the molecular healing effects of TQ in amyloid beta 1-42 (Aß1-42) peptide-infused AD rat hippocampus. STUDY DESIGN: A micro-osmotic pump containing aggregated Aß1-42 was cannulated into the hippocampus of adult female rats. After two weeks infusion, the dose of TQ (10 mg/kg or 20 mg/kg) was determined according to the HPLC results of cerebrospinal fluid and TQ was given to rats intragastrically for 15 days. METHODS: The memory performance of rats was determined by Morris water maze test. Afterwards, the acetylcholinesterase (AChE) level were measured by ELISA. Histopathological examinations of hippocampal tissue were performed for cell survival by Nissl staining, for detection of amyloid plaque deposits by Congo red staining and for determination of degenerating neurons by Fluoro Jade C staining. MicroRNA/mRNA levels and protein expressions of AD-related genes and proteins were analyzed by Real-Time Polymerase Chain Reaction and Western Blotting, respectively. RESULTS: Administration of TQ enhanced the memory performance of Aß1-42 infused rats and it also ameliorated the neuronal loss in the cornu ammonis (CA1), but not in the dentate gyrus (DG). In addition, TQ treatment decreased the fibril deposition whose accumulation was significantly higher in the Aß1-42-infused animals compared to that of the control group. The expression profiles of mir29c and Bax which significantly upregulated in the Aß1-42-infused animals were attenuated by TQ. Furthermore, administration of TQ decreased the expressions of Aß, phosphorylated-tau, and BACE-1 proteins. There was no significant therapeutic effect of TQ on the AKT/GSK3ß or MAPK signaling pathways which were affected due to Aß1-42 infusion. CONCLUSION: TQ has the capacity to recover the neuropathology by removing Aß plaques and by restoring neuron viability. All might have established the molecular basement of the consolidation in the memory observed by means of TQ treatment.

AnvirzelTMregulates cell death through inhibiting GSK-3 activity in human U87 glioma cells.

Objectives: Cardiac glycosides are used as potential anti-cancer agents due to their effects on the inhibition of proliferation and induction of apoptosis and/or autophagy in cancer cells. Herein, we aimed to study the potential signaling pathways taken role in differential cell-death properties of AnvirzelTM which is consisted of two toxic cardiac glycosides (oleandrin and oleandrigenin), in U87 human glioblastoma cells.Methods: The anti-proliferative and anti-migratory effects of AnvirzelTM were assessed in U87 cells by WST-1 assay and wound healing assay, respectively. After treatment of AnvirzelTMwith doses of 10, 25, 50, 100 and 250 µg/ml, expression levels of proteins related to cell death were investigated by Western blot.Results: Anvirzel™ markedly inhibited the growth of U87 cells in a time- and dose-dependent manner following 24 h and 48 h treatments (p < 0.05). In addition, it was found that Anvirzel™ inhibited GSK-3, NOS and HIF1-α expressions whereas activated ERK in U87 cells compared to vehicle (p < 0.05).Discussion: The results suggested that AnvirzelTM regulated cell death distinctly from apoptosis in human glioblastoma cells. Further studies are required for validation of mechanistic insights about the potential signaling pathways taken role in differential cell death properties of AnvirzelTM.

Achieving the balance: Biphasic effects of genistein on PC-3 cells.

This study examined the response of PC-3 cells to physiological (0.5, 2.5, 5, 10 µM) and pharmacological (50 µM) concentrations of genistein which is a main bioactive compound in soy. Following 48 hr genistein treatment, cell-based assays and genome-wide microarray were performed. It was evidenced that maximal physiologically achievable concentrations of genistein (0.5-10 µM) lead to significant increase in cell viability (p < 0.05) and decrease in migration at 0.5 µM (p = 0.000) and 10 µM (p = 0.001). The highest percentage of apoptotic cells was obtained at 50 µM. Microarray analysis gave the most critical pathways such as cell cycle regulation and proliferation, tumorigenesis, DNA damage and repair, stress response, and apoptosis. Physiological concentrations (≤10 µM) induced activation of CDKs, MAPKs, and RPSKs, while high concentrations of genistein (>10 µM) appeared to have a novel mechanism of action, specifically down-regulating TGF-ß by decreasing specifically SMAD 2/3,4 which are in the downstream TGF-ß signaling cascade. PRACTICAL APPLICATIONS: This study highlights for the first time that maximal physiologically achievable concentrations of genistein (0.5-10 µM) have proliferative effects evidenced by alterations in global gene expression patterns of PC-3 cells. Our results particularly represent a closer examination of dietary genistein consumption for the prevention and/or treatment of cancer that maximal physiologically achievable concentrations of genistein could have detrimental effects on individuals with prostate cancer. Further studies as in vivo would be necessary to remove shadows on the effect of genistein on prostate cancer progression.

Effect of Restraint Stress on Plasma PTH Concentration and Its Molecular Targets Expressions in Wistar Rats.

BACKGROUND: There are limited numbers of experimental studies related to the potential role of parathormone/parathyroid hormone (PTH) in response to psychological stress. In the current study, we aimed to cross-examine, for the first time, changes in PTH plasma concentration and the expression of its molecular targets mediated by restraint stress in rats. METHODS: Male Wistar rats (n = 42) were separated into control and stressed groups. They were further divided into two groups that received chronic restraint stress (CRS) for 7 and 28 consecutive days (n = 7 for each group). Elevated plus maze and tail suspension test were used to determine the anxiety- and depressive-like behaviors of a different set of rats including stress and control groups (n = 7 for each group). The plasma levels of adrenocorticotropic hormone (ACTH), corticosterone, and intact parathormone (iPTH) were measured by enzyme-linked immunosorbent assay (ELISA). In addition, alterations in the expressions of glucocorticoid receptor (GR), calcium sensing receptor (CaSR), and parathormone receptor (PTHR1) of kidney and total thyroid gland tissues were estimated by Western Blotting. RESULTS: There was no significant difference in the plasma level of iPTH while significant increases in the levels of ACTH and corticosterone were noted in the stressed-animals at day 7 and 21 (P = 0.010 and P = 0.016, respectively) of restraint stress. However, we found a negative correlation between iPTH and corticosterone levels in acute restraint stress (r = 0.771, P = 0.002). In addition, the expression of PTHR1 significantly decreased in the kidney at day 7 (P = 0.001) and in the thyroid gland at day 28 (P = 0.05) in response to CRS. CONCLUSIONS: To sum up, CRS has a significant effect on the expression of parathormone receptor rather than the iPTH concentration. The present results add a new dimension to stress research through the negative effect of chronic stress on the PTH signaling pathway.

Thymoquinone activates MAPK pathway in hippocampus of streptozotocin-treated rat model.

Streptozotocin (STZ), a glucosamine-nitrosourea compound, produces deficiencies in learning, memory, and cognitive functions when it was administered intracerebroventricularly (i.c.v). In molecular level, increase in neuroinflammation and oxidative stress in brain, and decrease in the number of surviving neurons are the outcomes of STZ administration. Herein, we aimed to investigate the effect of thymoquinone (TQ), an anti-inflammatory, immunomodulatory and neuroprotective agent, on STZ-induced neurodegeneration in rats. For this purpose, bilateral i.c.v. injection of STZ (3 mg/kg) was given to adult female rats on days 1 and 3. TQ (20 mg/kg/day in cornoil) was administered intragastrically to rats for 15 days starting from the 15th day of STZ injection. The Morris water maze test and passive avoidance test were applied to measure the learning and memory performance of animals. Following the behavioral tests, all of the rats were sacrificed for evaluation of molecular alterations. Rats in the STZ-TQ group showed higher performance in passive avoidance test than rats in the STZ group whose memory performance declined compared to control group. The worse memory performance in STZ group was correlated with low number of surviving neurons and high number of degenerating neurons. In addition, an increase in APOE expression and a decrease in NGF expression were observed with STZ injection. Administration of TQ reversed these STZ-triggered cognitive and molecular alterations. In the present study, we observed the neuroregenerative effects of TQ by activation of JNK protein, upregulation of mir-124, and downregulation of ERK1/2 and NOS enzymes. The same ameliorative effect of TQ was also observed in the pTau protein expression. To sum up, we can say that the healing effect of TQ on STZ induced neurodegeneration opens a new door for the development of Alzheimer's disease treatment using natural products as an adjuvant when their action mechanism was explained in detail.

Cellular Model of Alzheimer's Disease: Aß1-42 Peptide Induces Amyloid Deposition and a Decrease in Topo Isomerase IIß and Nurr1 Expression.

BACKGROUND: DNA topoisomerase IIß (topo IIß) plays a crucial role in neural differentiation and axonogenesis. Inhibition of topo IIß activity in vitro and in vivo results in shorter axons and increased DNA damage. These molecular events also involve in Alzheimer's disease (AD); however, the role of topo IIß in the pathogenesis of AD remains to be elucidated. OBJECTIVES: We aimed to investigate the role of topo IIß association with Nuclear receptor related 1 protein (Nurr1) in the onset of AD. METHODS: In vitro AD model was established by the incubation of fibrillar amyloid-ß 1-42 (Aß1-42) for 48 hours with cultured cerebellar granule neurons (CGNs) isolated from post-natal eight-day rats. The regulatory role of topo IIß on the transcription of Nurr1 was analyzed in topo IIß silenced CGNs, and also topo IIß silenced and overexpressed in a neurally-differentiated human mesenchymal (hMSC) cell line. RESULTS: Aß1-42 fibrils led to the upregulation of Presenilin1 and Cofilin1 genes as measured at mRNA levels and hyperphosphorylation of tau protein, all are distinctive characteristics of AD pathology. A significant decrease in topo IIß expression at mRNA and protein levels and Nurr1 at mRNA level was also observed. In both cell types, Nurr1 expression was dramatically down-regulated due to topo IIß deficiency, and was increased in topo IIß overexpressing hMSCs. CONCLUSION: Our findings suggest that topo IIß could be a down-stream target of signaling pathways contributing to AD-like pathology. However, further studies must be carried out in vivo to elucidate the precise association topo IIß with AD.