Impact of morphine on the expression of insulin receptor and protein levels of insulin/IGFs in rat neural stem cells.

Alzheimer's disease is correlated with neuronal degeneration and loss of neuronal precursors in different parts of the brain. It has been found disturbance in the homeostasis neural stem cells (NSCs) can cause neurodegeneration. Morphine, an analgesic agent, can disrupt the dynamic and normal state of NSCs. However, more investigations are required to clearly address underlying mechanisms. The current experiment aimed to investigate the effects of morphine on the cell distribution of insulin factor and receptor and insulin-like growth factors (IGF1, IGF2) in NSCs. NSCs were isolated from rats and stemness feature confirmed by antibodies against nestin and Sox2. The cells were exposed to 100µM morphine, 50µM naloxone and combination of these two drugs for 72h. The neural cell growth, changes in levels of insulin and insulin-like growth factors secreted by NSCs as well as the insulin-receptor-gene expression were assessed by flow cytometry, ELlSA, and real-time PCR, respectively. Cell cycle assay revealed the exposure of cells to morphine for 72h increased cell apoptosis and decreased neural stem cell growth. The biosynthesis of insulin, insulin-like growth factors, and insulin receptor were reduced (p<0.05) after NSCs exposure to morphine at the concentration of 100µM for 24, 48 and 72h. Naloxone is a competitive antagonist which binds MOR where morphine (and endogenous opioids) bind, and reversed the detrimental effects of morphine. It can be concluded that morphine initiated irregularity in NSCs kinetics and activity by reducing the secretion of insulin and insulin-like growth factors and down-regulation of insulin receptor.

The role of morphine on rat neural stem cells viability, neuro-angiogenesis and neuro-steroidgenesis properties.

A lack of comprehensive data exists on the effect of morphine on neural stem cell neuro-steroidogenesis and neuro-angiogenesis properties. We, herein, investigated the effects of morphine (100µM), naloxone (100µM) and their combination on rat neural stem cells viability, clonogenicity and Ki-67 expression over a period of 72h. Any alterations in the total fatty acids profile under treatment protocols were elucidated by direct transesterification method. We also monitored the expression of p53, aromatase and 5-alpha reductase by real-time PCR assay. To examine angiogenic capacity, in vitro tubulogenesis and the level of VE-cadherin transcript were investigated during neural to endothelial differentiation under the experimental procedure. Cells supplemented with morphine displayed reduced survival (p<0.01) and clonogenicity (p<0.001). Flow cytometric analysis showed a decrease in Ki-67 during 72h. Naloxone potentially blunted morphine-induced all effects. The normal levels of fatty acids, including saturated and unsaturated were altered by naloxone and morphine supplements. Following 48h, the up-regulation of p53, aromatase and 5-alpha reductase genes occurred in morphine-primed cells. Using three-dimensional culture models of angiogenesis and real time PCR assay, we showed morphine impaired the tubulogenesis properties of neural stem cells (p<0.001) by the inhibition of trans-differentiation into vascular cells and led to decrease of in VE-cadherin expression. Collectively, morphine strongly impaired the healthy status of neural stem cells by inducing p53 and concurrent elevation of aromatase and 5-alpha reductase activities especially during early 48h. Also, neural stem cells-being exposed to morphine lost their potency to elicit angiogenesis.

Effects of combination of melatonin and laser irradiation on ovarian cancer cells and endothelial lineage viability.

The main goal of anti-cancer therapeutic approaches is to induce apoptosis in tumor masses but not in the normal tissues. Nevertheless, the combination of photodynamic irradiation with complementary oncostatic agents contributes to better therapeutic performance. Here, we applied two different cell lines; SKOV3 ovarian carcinoma cells and HUVECs umbilical cord cells as in vitro models to pinpoint whether pharmacological concentration of melatonin in combination with photodynamic therapy induces cell cytotoxicity. The cells were separately treated with various concentrations of melatonin (0 to 10 mM) and photodynamic irradiation alone or in combination. Cells were preliminary exposed to increasing concentrations of melatonin for 24 h and subsequently underwent laser irradiation for 60 s with an output power of 80 mW in continuous mode at 675 nm wavelength and a total light dose of 13.22 J/cm2. Cell viability, apoptosis/necrosis rates, and reactive oxygen species levels as well as heat shock protein 70 expression were monitored after single and combined treatments. A statistical analysis was performed by applying one-way analysis of variance (ANOVA) and post hoc Tukey's test. Combination treatment of both cell lines caused a marked increase in apoptosis/necrosis rate, reactive oxygen species generation, and heat shock protein 70 expression compared to incubation of the cells with each agent alone (p < 0.05). SKOV3 cancer cells expressed higher level of heat shock protein 70 under experimental procedure as compared to HUVECs (p < 0.05). Our results introduce melatonin as a potent stimulus for enhancing the efficacy of laser on induction of apoptosis in tumor cells.

Morphine Inhibited the Rat Neural Stem Cell Proliferation Rate by Increasing Neuro Steroid Genesis.

Up to present, a large number of reports unveiled exacerbating effects of both long- and short-term administration of morphine, as a potent analgesic agent, on opium-addicted individuals and a plethora of cell kinetics, although contradictory effect of morphine on different cells have been introduced until yet. To address the potent modulatory effect of morphine on neural multipotent precursors with emphasis on endogenous sex-related neurosteroids biosynthesis, we primed the rat neural stem cells isolated from embryonic rat telencephalon to various concentrations of morphine including 10, 20, 50 and 100 µM alone or in combination with naloxone (100 µM) over period of 72 h. Flow cytometric Ki-67 expression and Annexin-V/PI based necrosis and apoptosis of exposed cells were evaluated. The total content of dihydrotestosterone and estradiol in cell supernatant was measured by ELISA. According on obtained data, both concentration- and time-dependent decrement of cell viability were orchestrated thorough down-regulation of ki-67 and simultaneous up-regulation of Annexin-V. On the other hand, the addition of naloxone (100 µM), as Mu opiate receptor antagonist, could blunt the morphine-induced adverse effects. It also well established that time-course exposure of rat neural stem cells with morphine potently could accelerate the endogenous dihydrotestosterone and estradiol biosynthesis. Interestingly, naloxone could consequently attenuate the enhanced neurosteroidogenesis time-dependently. It seems that our results discover a biochemical linkage between an accelerated synthesis of sex-related steroids and rat neural stem cells viability.