Role of hippocampal and prefrontal cortical cholinergic transmission in combination therapy valproate and cannabidiol in memory consolidation in rats: involvement of CREB- BDNF signaling pathways.

PURPOSE: Cognitive disorders are associated with valproate and drugs used to treat neuropsychological diseases. Cannabidiol (CBD) has beneficial effects on cognitive function. This study examined the effects of co-administration of CBD and valproate on memory consolidation, cholinergic transmission, and cyclic AMP response element-binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling pathway in the prefrontal cortex (PFC) and hippocampus (HPC). METHODS: One-trial, step-through inhibitory test was used to evaluate memory consolidation in rats. The intra-CA1 injection of physostigmine and atropine was performed to assess the role of cholinergic transmission in this co-administration. Phosphorylated CREB (p-CREB)/CREB ratio and BDNF levels in the PFC and HPC were evaluated. RESULTS: Post-training intraperitoneal (i.p.) valproate injection reduced memory consolidation; however, post-training co-administration of CBD with valproate ameliorated memory impairment induced by valproate. Post-training intra-CA1 injection of physostigmine at the ineffective doses in memory consolidation (0.5 and 1 µg/rat), plus injection of 10 mg/kg of CBD as an ineffective dose, improved memory loss induced by valproate, which was associated with BDNF and p-CREB level enhancement in the PFC and HPC. Conversely, post-training intra-CA1 injection of ineffective doses of atropine (1 and 2 µg/rat) reduced the positive effects of injection of CBD at a dose of 20 mg/kg on valproate-induced memory loss associated with BDNF and p-CREB level reduction in the PFC and HPC. CONCLUSION: The results indicated a beneficial interplay between valproate and CBD in the process of memory consolidation, which probably creates this interaction through the BDNF-CREB signaling pathways in the cholinergic transmission of the PFC and HPC regions.

The Protective Potential Role of ACE2 against COVID-19.

Due to the coronavirus disease 2019 (COVID-19), researchers all over the world have tried to find an appropriate therapeutic approach for the disease. The angiotensin-converting enzyme 2 (ACE2) has been shown as a necessary receptor to cell fusion, which is involved in infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is commonly crucial for all organs and systems. When ACE2 is downregulated via the SARS-CoV-2 spike protein, it results in the angiotensin II (Ang II)/angiotensin type 1 receptor axis overactivation. Ang II has harmful effects, which can be evidenced by dysfunctions in many organs experienced by COVID-19 patients. ACE2 is the SARS-CoV-2 receptor and has an extensive distribution; thus, some COVID-19 cases experience several symptoms and complications. We suggest strategy for the potential protective effect of ACE2 to the viral infection. The current review will provide data to develop new approaches for preventing and controlling the COVID-19 outbreak.

A systematic method introduced a common lncRNA-miRNA-mRNA network in the different stages of prostate cancer.

Introduction: The present study aimed to investigate the interaction of the common lncRNA-miRNA-mRNA network involved in signaling pathways in different stages of prostate cancer (PCa) by using bioinformatics and experimental methods. Methods: Seventy subjects included sixty PCa patients in Local, Locally Advanced, Biochemical Relapse, Metastatic, and Benign stages, and ten healthy subjects were entered into the current study. The mRNAs with significant expression differences were first found using the GEO database. The candidate hub genes were then identified by analyzing Cytohubba and MCODE software. Cytoscape, GO Term, and KEGG software determined hub genes and critical pathways. The expression of candidate lncRNAs, miRNAs, and mRNAs was then assessed using Real-Time PCR and ELISA techniques. Results: 4 lncRNAs, 5 miRNAs, and 15 common target genes were detected in PCa patients compared with the healthy group. Unlike the tumor suppressors, the expression levels of common onco-lncRNAs, oncomiRNAs, and oncogenes showed a considerable increase in patients with advanced stages; Biochemical Relapse and Metastatic, in comparison to the primary stages; Local and Locally Advanced. Additionally, their expression levels significantly increased with a higher Gleason score than a lower one. Conclusion: Identifying a common lncRNA-miRNA-mRNA network associated with prostate cancer may be clinically valuable as potential predictive biomarkers. They can also serve as novel therapeutic targets for PCa patients.

Innovative targets of the lncRNA-miR-mRNA network in response to low-dose aspirin in breast cancer patients.

This study aimed to investigate innovative targets in breast cancer patients by considering the interaction of the lncRNA-miR-mRNA network in response to low-dose aspirin. The candidate miRs were first taken from the GEO and TCGA databases. Then, the candidate network was constructed using the high-throughput sequencing data. The expression levels of candidate targets were finally measured using Real-Time PCR in luminal A breast cancer patients undergoing aspirin (80 mg daily for three months) and non-aspirin groups during chemotherapy after surgery. The expression levels of TGFß, IL-17, IFNγ, and IL-ß proteins were measured using the ELISA technique. 5 lncRNAs, 12 miRs, and 10 genes were obtained in the bioinformatic phase. A significant expression increase of the candidate tumor suppressor lncRNAs, miRs, and genes and a substantial expression decrease of the candidate onco-lncRNAs, oncomiRs, and oncogenes were achieved after the aspirin consumption. Unlike the non-aspirin group, the expression levels of TGFß, IL-17, IFNγ, and IL-ß proteins were significantly decreased following aspirin consumption. The Kaplan-Meier analysis indicated a longer overall survival rate in the patients after aspirin consumption. Our results showed that the lncRNA-miR-mRNA network might be a significant target for aspirin; their expression changes may be a new strategy with potential efficacy for cancer therapy or prevention.

Dual effects of atorvastatin on angiogenesis pathways in the differentiation of mesenchymal stem cells.

Atorvastatin (ATO) can improve the transplantation efficacy of mesenchymal stem cells (MSCs) after acute myocardial infarction. The present study aimed at ATO effects on the angiogenesis-signaling pathways from MSCs' differentiation to tissue angiogenesis. MSCs were first prepared from BALB/c mouse bone marrow. MTT assay was then done for the biodegradability of MSCs with the extracellular matrix. After that, the differentiation of cells into the bone and fat tissues was confirmed by Alizarin and Oil Red O staining. The extracellular matrix was then combined with the cells to the implant. Animals were intraperitoneally treated with ATO (2 and 40 mg/kg, daily) three days before cell transplantation to one week after. Finally, the assays were carried out by electron microscopy, immunocytochemistry, ELISA, Western blot, and RT-qPCR techniques. A phase-contrast microscope confirmed the morphology of cells. The cell differentiation into bone and fat tissues was confirmed by Alizarin red staining and flow cytometry, and the cell proliferation was confirmed by MTT assay. Unlike ATO 40 mg/kg group, ATO 2 mg/kg was significantly increased the CD31, eNOS, podocalyxin, von Willibrand factor, and alpha-smooth muscle actin proteins levels compared to the control group in vitro experiment. The expression of CD31 and VEGF proteins, as angiogenesis markers, and Ki-67 protein, as a proliferation marker, was significantly higher in a low dose of ATO (2 mg/kg) than that of the control group in vivo experiment. Unlike ATO 40 mg/kg, the expression levels of ERK, AKT, NF-ҝB, Rho, STAT3, Ets-1, HIF-1α, and VEGF proteins and genes were significantly increased in ATO 2 mg/kg compared to the control. A low dose of ATO can be a beneficial tool in the function of MSCs and their differentiation to tissue angiogenesis.

Ovarian Stem Cells Differentiation into Primary Oocytes Using Follicle Stimulating Hormone, Basic Fibroblast Growth Factor, and Neurotrophin 3.

BACKGROUND: In vitro obtaining oocytes can be an appropriate alternative for patients with gonadal insufficiency or cancer survivors. The purpose of the current research was isolating stem cells from ovarian cortical tissue as well as evaluating the effectiveness of follicle stimulating hormone (FSH), basic fibroblast growth factor (bFGF), and neurotrophin 3 (NT3) in differentiating to oocyte-like cells. METHODS: A human ovary was dissected and cortical tissue pieces were cultured for cell isolation. Isolated cells were divided into 8 groups (3 cases in each group) of control, FSH, NT3, bFGF, FSH+NT3, FSH+bFGF, NT3+bFGF, and FSH+NT3+ bFGF. Pluripotency specific gene (OCT4-A and Nanog), initial germ cells (c-KIT and VASA) and PF growth initiators (GDF-9 and Lhx-8) were evaluated by qRTPCR. Experiments were performed in triplicate and there were 3 samples in each group. The results were analyzed using one-way ANOVA and p-value less than 0.05 was considered statistically significant. RESULTS: Flow cytometry results showed that cells isolated from the ovarian cortex expressed markers of pluripotency. The results showed that the expression of Nanog, OCT4, GDF-9 and VASA was significantly increased in FSH+NT3 group, while treatment with bFGF caused significant expression of c-KIT and Lhx-8 (p<0.05). Also, according to the results, isolated cells treated with NT3 significantly increased c-KIT expression. CONCLUSION: According to our results, the ovarian cortex cells could be differentiated into primordial follicles if treated with the proper combination of FSH, bFGF, and NT3. These findings provided a new perspective for the future of in vitro gamete proudest.

Noscapine Modulates Neuronal Response to Oxygen-glucose Deprivation/Reperfusion Injury Via Activation of Sigma-1 Receptor in Primary Cortical Cultures.

In the present study, we investigated the effects of noscapine (0.5-2 µM), an alkaloid from the opium poppy (Papaver somniferum), on primary murine cortical neurons exposed to 60 min oxygen-glucose deprivation (OGD) in the presence of 5 µM BD-1047, a selective sigma-1 receptor antagonist. The experiments were performed on cortical neurons after 11-16 days of culture. To initiate oxygen-glucose deprivation, the culture medium was transferred to glucose-free DMEM, and placed in a humidified incubation chamber containing a mixture of 95% N2 and 5% CO2 at 37 °C for 60 min. In order to explore the effect on neurons under oxygen-glucose deprivation in this condition, some cultures were pretreated with noscapine and BD1047 together, 24 h prior to OGD followed by 24 h recovery. Cell viability, nitric oxide (NO) production and intracellular calcium concentration ([Ca2+]i) levels were evaluated by MTT assay, the modified Griess method, and Fura-2, respectively. Pretreatment of the cultures with noscapine in the presence of BD1047 significantly increased cell viability and decreased NO generation in a dose-dependent manner compared to BD1047 alone. Pretreatment with 2 µM noscapine and BD-1047 was shown to decrease the rise in [Ca2+]i induced by sodium azide (NaN3) and glucose deprivation. We concluded that noscapine in the presence of BD1047 could protect primary cortical neurons after oxygen-glucose deprivation-induced cell injury but this effect was not complete. Our results indicate that neuroprotective effects of noscapine could be mediated partially through activation of sigma-1 receptor and by decreasing NO production and [Ca2+]i levels.

Noscapine protects the H9c2 cardiomyocytes of rats against oxygen-glucose deprivation/reperfusion injury.

Noscapine is an antitumor alkaloid derived from Papaver somniferum plants. Our previous study has demonstrated that exposure of noscapine on primary murine fetal cortical neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R) has neuroprotective effects. In current study, the effects of noscapine on cardiomyocytes (H9c2 cells) damage caused by 120 minutes (min) of OGD/R were evaluated and we determined whether the addition of BD1047, sigma-one receptor antagonist, prevents the protective effects of noscapine in H9c2 cells through the production of nitric oxide (NO) and apoptosis. To initiate OGD, H9c2 cells was transferred to glucose-free DMEM, and placed in a humidified incubation chamber. Cell viability was assessed with noscapine (1-5 µM) in the presence or absence of BD1047, 24 hours (h) after OGD/R. Cell viability, NO production and apoptosis ratio were evaluated by the MTT assay, the Griess method and the quantitative real-time PCR. Noscapine considerably improved the survival of H9c2 cells compared to OGD/R. Also, noscapine was extremely capable of reducing the concentrations of NO and Bax/Bcl-2 ratio expression. While the BD1047 administration alone diminished cell viability and increased the Bax/Bcl-2 ratio and NO levels. The addition of noscapine in the presence of BD1047 did not increase the cell viability relative to noscapine alone. Noscapine exerted cardioprotective effects exposed to OGD/R-induced injury in H9c2 cells, at least partly via attenuation of NO production and Bax/Bcl-2 ratio, which indicates that the sigma-one receptor activation is involved in the protection by noscapine of H9c2 cells injured by OGD/R.

Comparative phenotypic characterization of human colostrum and breast milk-derived stem cells.

There is a diverse population of stem cells in human breast milk that can be employed for therapeutic purposes as a reservoir of cells. The current study mainly aimed to determine the nature markers expressing on stem cells. For this aim, the expression of embryonic stem cell markers, as well as the expression of endothelial, mesenchymal, neural, and hematopoietic markers were evaluated by the flow cytometry analysis in fresh colostrum, breast milk, and cultured colostrum samples. The results showed that the embryonic (OCT4, SOX2, HLA-DR), hematopoietic (CD33, CD45, CD117), neural (CD133, Nestin), and mesenchymal (CD44, SCA1) stem cell markers present in colostrum had higher expression in comparison with their counterpart markers in fresh breast milk. The expression markers of stem cells in colostrum following a 2-week culture period were significantly increased compared with their counterpart markers in colostrum before the culture process. In the culture of breastmilk, cells were not observed adherent cells and colonies. Our findings form flow cytometry and cell culture suggest that the lactation stage could be one of the factors influencing the stem cell population and, consequently, the cultivation of breastmilk cells. The present study indicates that colostrum is a tremendous source of stem cells that could be applied in cell-based research.

Evaluation of the neuroprotective effects of electromagnetic fields and coenzyme Q10 on hippocampal injury in mouse.

Electromagnetic fields (EMFs) are reported to interfere with chemical reactions involving free radical production. Coenzyme Q10 (CoQ10) is a strong antioxidant with some neuroprotective activities. The purpose of this study was to examine and compare the neuroprotective effects of EMF and CoQ10 in a mouse model of hippocampal injury. Hippocampal injury was induced in mature female mice (25-30 g), using an intraperitoneal injection of trimethyltin hydroxide (TMT; 2.5 mg/kg). The experimental groups were exposed to EMF at a frequency of 50 Hz and intensity of 5.9 mT for 7 hr daily over 1 week or treated with CoQ10 (10 mg/kg) for 2 weeks following TMT injection. A Morris water maze apparatus was used to assess learning and spatial memory. Nissl staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests were also performed for the histopathological analysis of the hippocampus. Antiapoptotic genes were studied, using the Western blot technique. The water maze test showed memory improvement following treatment with CoQ10 and coadministration of CoQ10 + EMF. The Nissl staining and TUNEL tests indicated a decline in necrotic and apoptotic cell count following treatment with CoQ10 and coadministration of CoQ10 + EMF. The Western blot study indicated the upregulation of antiapoptotic genes in treatment with CoQ10, as well as coadministration. Also, treatment with EMF had no significant effects on reducing damage induced by TMT in the hippocampus. According to the results, EMF had no significant neuroprotective effects in comparison with CoQ10 on hippocampal injury in mice. Nevertheless, coadministration of EMF and CoQ10 could improve the neuroprotective effects of CoQ10.

Evaluation of effects of morphine and ionizing radiation in cancer cell lines.

PURPOSE: Breast and cervical cancers are the two most common cancers among women worldwide. Morphine is a potent analgesic for cancer pain, and radiation therapy is a conventional treatment for cancer. Unfortunately, the combined adjuvant cellular effects of morphine and ionizing radiation in cancer cells are largely unknown. MATERIALS AND METHODS: In this study, we examined the effects of morphine and single radiation dose of 2 Gy on viability and survival fraction of human breast cancer cell line MDA-MB 231 and human cervical cancer cell line HeLa, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. We were also interested in evaluating these effects in human umbilical vein endothelial cells as well. RESULTS: We found that morphine did not have a dose- and time-dependent manner in endothelial, breast, and cervical cancer cells in vitro. It seems that pretreatment of breast and cervical cancer cells with morphine at some doses before irradiation reduces the cytotoxic effect of radiation. We also observed that endothelial cells were less sensitive than breast and cervical cancer cells to radiation or morphine + radiation. Based on the results of endothelial cells, morphine or radiation might not have a selective effect on the viability and clonogenic survival of different cell lines. CONCLUSIONS: Our data may suggest that morphine and radiotherapy could not be administered together to breast and cervical cancer patients if additional and in vivo studies confirm our results.

Neuroprotective Effects of Trolox, Human Chorionic Gonadotropin, and Carnosic Acid on Hippocampal Neurodegeneration After Ischemiareperfusion Injury.

INTRODUCTION: One of the serious complications of stroke is memory impairment, which is considered as one of the complications of reperfusion of tissue. The present study was designed to compare the effect of administration of Trolox, carnosic acid and Human Chorionic Gonadotropin (HCG) immediately after reperfusion of the stroke tissue on the memory and hippocampal histology. METHOD: Ischemia-Reperfusion Model (IRI) was created by bilateral occlusion of the common carotid artery for 15 minutes and the first dose was administered immediately after reperfusion. 10 days after ischemia, passive avoidance memory test and apoptotic protein levels were evaluated. RESULTS: Cerebral Ischemia perfusion reduced the time of latency in entering the dark box in the ischemic group. Administration of Trolox and HCG increased this latency time, while treatment with carnosic acid had no effect. Also, IRI significantly reduced the number of healthy cells in the hippocampus. Administration of Trolox, carnosic acid and HCG increased the number of healthy cells and decreased the expression of Caspase-3 and Bax, but significantly increased the expression of Bcl-2 compared to the ischemic group. CONCLUSION: Findings indicate the beneficial effects of HCG and Trolox on the improvement of memory and the number of healthy cells in the hippocampal region. It is worth noting that the amount of apoptosis in the hippocampus was significantly reduced by Trolox, HCG and Carnosic acid.

Evaluation of metformin effects in the chronic phase of spontaneous seizures in pilocarpine model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a common form of drug-resistant epilepsy that sometimes responds to dietary manipulation such as the 'ketogenic diet'. Here we have investigated the effects of metformin in the rat pilocaroin model of TLE. Male rats were treated with intra peritoneal injection of pilocarpine hydrochloride, in dose of 360 mg/kg to induce status epilepticus (SE). At 45 day after induction of SE, metformin was injected intraperitoneally in dose of 250 mg/kg/day for 5 days. We show that metformin potently reduces the progression of seizures and blocks seizure-induced over-expression of brain-derived neurotropic factor (BDNF) and its receptor, Tropomyosin receptor kinase B (TrkB). We have shown that this reduced expression pattern is mediated by the transcriptional co-repressor CtBP (C-terminal binding protein). Moreover, metformin decreased mechanistic target of rapamycin (mTOR) activation through activation of AMP-activated protein kinase (AMPK) signaling pathway. Our findings have been shown that metformin has anticonvulsant and antiepileptic properties, and suggesting that antiglycolytic compounds such as metformin may represent a new class of drugs for treating epilepsy.

The Effect of Noscapine on Oxygen-Glucose Deprivation on Primary Murine Cortical Neurons in High Glucose Condition.

In the present work we set out to investigate the neuroprotective effects of noscapine (0.5-2 µM) in presence of D-glucose on primary murine foetal cortical neurons after oxygen-glucose deprivation/24 h. recovery. Cell viability, nitric oxide production and intracellular calcium ((ca(2+))i) levels were evaluated by MTT assay, the modified Griess method and Fura-2 respectively. 25 and 100 mM D-glucose could, in a concentration dependent manner, improve cell viability and decrease NO production and (ca(2+))i level in neuronal cells after ischemic insult. Moreover, pre-incubation of cells with noscapine, noticeably enhanced protective effects of 25 and 100 mM D-glucose compared to similar conditions without noscapine pre-treatment. In fact, noscapine attenuated NO production in a dose-dependent fashion, after 30 minutes (min) OGD, during high-glucose (HG) condition in cortical neurons. Pretreatment with 2 µM noscapine and 25 or 100 mM D-glucose, was shown to decrease the rise in (ca(2+))i induced by Sodium azide/glucose deprivation (chemical OGD) model. These effects were more pronounced than that of 25 or 100 mM D-glucose alone. The present study demonstrated that the neuroprotective effects of HG before an ischemic insult were augmented by pre-treatment with noscapine. Our results also suggested that the neuroprotection offered by both HG and noscapine involve attenuation of NO production and (ca(2+))i levels stimulated by the experimental ischemia in cortical neurons.

Neuroprotective effect of noscapine on cerebral oxygen-glucose deprivation injury.

BACKGROUND: The present study aims to investigate the effect of noscapine (0.5-2.5 µM), an alkaloid from the opium poppy, on primary murine fetal cortical neurons exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. METHODS: Cells were transferred to glucose-free DMEM and were exposed to hypoxia in a small anaerobic chamber. Cell viability and nitric oxide production were evaluated by MTT assay and the Griess method, respectively. RESULTS: The neurotoxicities produced by all three hypoxia durations tested were significantly inhibited by 0.5 µM noscapine. Increasing noscapine concentration up to 2.5 µM produced a concentration-dependent inhibition of neurotoxicity. Pretreatment of cells with MK-801 (10 µM), a non-competitive NMDA antagonist, and nimodipine (10nM), an L-type Ca(2+) channel blockers, increased cell viability after 30 min OGD, while the application of NBQX (30 µM), a selective AMPA-kainate receptor antagonist partially attenuated cell injury. Subsequently, cells treated with noscapine in the presence of thapsigargin (1 µM), an inhibitor of endoplasmic reticulum Ca(2+) ATPases. After 60 min OGD, noscapine could inhibit the cell damage induced by thapsigargin. However, noscapine could not reduce cell damage induced by 240 min OGD in the presence of thapsigargin. Noscapine attenuated nitric oxide (NO) production in cortical neurons after 30 min OGD. CONCLUSIONS: We concluded that noscapine had a neuroprotective effect, which could be due to its interference with multiple targets in the excitotoxicity process. These effects could be mediated partially by a decrease in NO production and the modulation of intracellular calcium levels.