Effect of Carvacrol on histological analysis and expression of genes involved in an animal model of multiple sclerosis.

BACKGROUND: The most common non-traumatic neurological disease in young- and middle-aged adults is multiple sclerosis (MS), leading to central nervous system (CNS) atrophy and neurological disorders with loss of myelin and axonal degeneration. Due to the inadequate efficiency of common treatments, some natural products with antioxidant properties such as Carvacrol have been considered. OBJECTIVE: the present study aimed to investigate carvacrol's anti-inflammatory and therapeutic effects on MS symptoms in healthy and experimental autoimmune encephalomyelitis (EAE) induced female Lewis rats. METHODS: The study was performed in three groups of Lewis rats: control group, EAE model, and EAE treated with carvacrol (carvacrol-treated group). The treatment group received 25 mg/kg of carvacrol intraperitoneally daily. Histologic examination and expression analysis of pro-inflammatory genes (Interleukin-1 and 17 (IL-1 and IL-17), Nuclear Factor Kappa B Cells (NF-κB) and Tumor Necrosis Factor-α (TNF-α)), myelin repair, and also regeneration genes (Myelin basic protein (MBP), Oligodendrocyte Transcription Factor 2 (OLIG2) and Platelet-Derived Growth Factor Receptor α (PDGFR-α)) were carried out. Gene studies, Hematoxylin and Eosin (H&E), and Luxol fast blue stain were performed in the lumbar region of the spinal cord. RESULTS: The EAE clinical scores in the carvacrol-treated group were lower than in untreated rats (P < 0.001). The expression of two genes, IL-17 and MBP, was confirmed using fluorescence immunohistochemistry (FIHC). A significant decrease was observed in NF-κB and IL-17, and IL-1 gene expression. The MBP and OLIG2 gene expression was increased in the carvacrol-treated group (p < 0.001). In EAE, PDGFR-α expression increased about four times. However, carvacrol administration did not affect PDGFR-α and TNF-α gene expression. In this treatment, H&E staining of spinal cord regions showed a significant decrease in inflammatory cell infiltration. Moreover, immunostaining analysis demonstrated a considerable increase in MBP and a reduction in IL-17 secretion. CONCLUSION: The results showed that carvacrol administration reduces the entry of inflammatory cells into the CNS by stimulating myelination-related processes employing increasing the expression of genes involved in myelin repair and reducing the expression of inflammatory genes. Our findings confirm that carvacrol improves the clinical and pathological symptoms of EAE through its therapeutic and modification properties as a potential adjunctive therapy and needs to be studied more.

Biosynthesis of titanium dioxide nanoparticles using Hypericum perforatum and Origanum vulgare extracts and their main components, hypericin and carvacrol as promising antibacterial agents.

OBJECTIVE: To evaluate the anti-bacterial activities of titanium dioxide (TiO) nanoparticles of Origanum (O.) vulgare and Hypericum (H.) perforatum extracts, carvacrol and hypericin against Staphylococcus (S.) aureus. METHODS: In this study, TiOnanoparticles of O. vulgare and H. perforatum extracts, carvacrol and hypericin, were prepared and their antibacterial effects were evaluated against Staphylococcus (S.) aureus. In this study, scanning electron microscope, fourier transform infrared spectrometer, atomic force microscopy, dynamic light scattering and zeta potential were used to investigate the structure of synthesized drugs. RESULTS: Anti-bacterial activity of synthesized NPs was tested by minimum inhibitory concentration (MIC), minimum bactericidal concentration and disc diffusion method. MICs of TiO-NPs synthesized using O. vulgare, H. perforatum, carvacrol and hypericin and TiO were obtained 250, 62.5, 250, and 250, and 500 µg/mL, respectively. The MBCs for all of these were obtained 1000 µg/mL. CONCLUSION: Green-synthesized of TiO nanoparticles provides a promising approach to the use of O. vulgare and H. perforatum, carvacrol and hypericin as novel agents and safer antibacterial compounds, especially anti-S. aureus compounds.

Effect of fluoxetine treatment on neurotoxicity induced by lysolecithin in male rats.

Demyelination disorder is an unusual pathologic event, which occurs in the central nervous system (CNS). Multiple sclerosis (MS) is an inflammatory demyelinating disease that affects the CNS, and it is the leading cause of disability in young adults. Lysolecithin (LPC) is one of the best toxin-induced demyelination models. In this study, a suitable model is created, and the effect of fluoxetine treatment is examined on this model. In this case, it was assumed that daily fluoxetine treatment had increased the endogenous remyelination in the LPC model. This study was focused on investigating the influence of the fluoxetine dose of 5 or 10 mg/kg per day for 1 and 4 weeks on LPC-induced neurotoxicity in the corpus callosum region. It was performed as a demyelinating model in male Wistar rats. After 3 days, fluoxetine was injected intraperitoneally (5 or 10 mg/kg per day) for 1 and 4 weeks in each group. After completing the treatment course, the corpus callosum was removed to examine the gene expression and histological analysis was performed. The results of the histopathological study of hematoxylin and eosin staining of the corpus callosum showed that in 1 and 4-week treatment groups, fluoxetine has reduced the level of inflammation at the LPC injection site (5 and 10 mg/kg per day). Fluoxetine treatment in the luxol fast blue (LFB) staining of the corpus callosum has been led to an increase in myelination capacity in all doses and times. The results of the genetic study showed that the fluoxetine has significantly reduced the expression level of tumor necrosis factor-α, nuclear factor κß, and induced nitric oxide synthase in comparison with the untreated LPC group. Also, the fluoxetine treatment has enhanced the expression level of the forkhead box P3 (FOXP3) gene in comparison with the untreated group. Fluoxetine has increased the expression level of myelination and neurotrophic genes such as myelin basic protein (MBP), oligodendrocyte transcription factor 2 (OLIG2), and brain-derived neurotrophic factor (BDNF). The outcomes demonstrated that fluoxetine reduces inflammation and strengthens the endogenous myelination in the LPC-induced demyelination model; however, supplementary studies are required for specifying the details of its mechanisms.

Applying Vasopressin-Pre-Conditioned Human Adipose Mesenchymal Stem Cells Improves Heart Condition after Transplantation into Infarcted Myocardium.

Transplantation of H-AdMSCs may improve heart function after MI. AVP is a neurohypophyseal hormone that reduces cardiovascular damage. This study investigated the role of AVP preconditioning in the survival of MSCs and their effect on myocardial repair in the MI rats. H-AMSCs were isolated and incubated for 3 days. The expression of oxytocin and vasopressin receptors was evaluated by Real-time-PCR. Forty male Wistar rats were divided into 4 groups: control, sham, ASC and AVP-ASC. Ischemia was established by ligation of LAD coronary artery. Electrocardiography, fibrosis, angiogenesis, and apoptosis in myocardium were determined after 7 days. Results showed that preconditioned MSCs significantly increased cardiac function when compared with group that received non-preconditioned MSCs. This was associated with significantly reduced fibrosis, increased vascular density, and decreased resident myocyte apoptosis. Results indicate that AVP preconditioned MSCs can be consider a novel approach to management of MI.

Protective effects of honey, Apis mellifera meda skorikov, on ischemia-reperfusion induced muscle injury / Efectos protectores de la miel, Apis mellifera meda skorikov, sobre la lesión muscular inducida por isquemia-reperfusión

Honey is a natural antioxidant that its protective effects have been proven against ischemia-reperfusion (IR) injury. The aim of this study was to evaluate the ameliorative effect of Persian Honey, Apis mellifera meda skorikov, on gastrocnemius muscle IR injury. Eighty adult male Sprague-Dawley rats weighing 250-300 g were used. They were divided into ten groups (N=8 per group). The ischemia was conducted with a silk suture 6-0 using the slipknot technique. All groups were rendered in ischemic for 3 h, and reperfused for various times of 3 days (3-day reperfusion), 7 days (7-day reperfusion), 14 days (14-day reperfusion), and 28 days (28-day reperfusion). Half of the groups had experimental honey (5 %) treatment immediately after ischemia. After reperfusion, the rats, based on the grouping, were killed with high doses of anesthetic, and the gastrocnemius muscles were removed and fixed. After the tissue processing, the evaluation of edema and mast cells infiltration was performed with hematoxylin-eosin and toluidine blue staining, respectively. TNF-α was detected with immunohistochemistry method. The amount of TNF-α as an index of acute inflammatory except the 3rd day significantly decreased on the other day of reperfusion (7th, 147th and 287th days). The mast cells infiltration was significantly decreased on 77th and 147th days. The tissue edema was decreased significantly in honey administrated group in the comparison with placebo groups. Honey administration can reduce damage caused by ischemia-reperfusion in the rat gastrocnemius muscle.

La miel es un antioxidante natural; sus efectos protectores han sido probados contra la lesión por isquemiareperfusión (IR). El objetivo de este estudio fue evaluar el efecto de mejora de la miel persa Apis mellifera meda skorikov, en la lesión por IR del músculo gastrocnemio. Se utilizaron 80 ratas Sprague-Dawley macho adultas con un peso entre 250 y 300 g divididas en diez grupos (N = 8 por grupo). La isquemia se realizó con una sutura de seda 6-0 utilizando la técnica slipknot permaneciendo isquémicos durante 3 h. La reperfusión se realizó durante varios tiempos de 3 días, 7 días (reperfusión de 7 días), 14 días (reperfusión de 14 días) y 28 días (28 días reperfusión). La mitad de los grupos recibió tratamiento experimental con miel (5 %) inmediatamente después de la isquemia. Después de la reperfusión, las ratas, fueron sacrificadas con altas dosis de anestésico, y los músculos gastrocnemios fueron removidos y fijados. Después de procesar el tejido, se realizó la evaluación del edema y la infiltración de mastocitos se realizó con tinción de hematoxilina-eosina y azul de toluidina, respectivamente. TNF-α se detectó con el método de inmunohistoquímica. La cantidad de TNF-α como índice de inflamación inflamatoria aguda, excepto en el tercer día, disminuyó significativamente al día siguiente de la reperfusión (7, 14 y 28 días). La infiltración de mastocitos disminuyó significativamente a los 7 y 14 días. El edema tisular disminuyó significativamente en el grupo administrado con miel en comparación con los grupos placebo. El tratamiento con miel puede reducir el daño causado por la isquemia-reperfusión en el músculo gastrocnemio de la rata.

Antinociceptive effects of green synthesized copper nanoparticles alone or in combination with morphine.

OBJECTIVE: The aim of this study was to evaluate the antinociceptive effect of biosynthetic copper nanoparticles from aqueous extract of Capparis spinosa fruit. METHODS: In this study, green synthesis of copper nanoparticles (CuNPs) was performed using C. spinosa extract according to the method described previously. The synthesized CuNPs were characterized using the UV-vis spectroscopy, Fourier transforms of infrared (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray (EDX). The antinociceptive effect of CuNPs was evaluated by tail-flick, hot-plate, and rotarod tests following the oral administration of mice with CuNPs at the concentrations of 25, 50, and 75 mg/kg for two weeks. RESULTS: The obtained maximum peak at the wavelength of 414 nm demonstrated the biosynthesis of the copper nanoparticles. SEM approved the particle size of CuNPs between 17 and 41 nm. The statistical analyses of the data of hot plate and tail-flick tests showed the potent analgesic effect of biosynthetic CuNPs. In this regard, the antinociceptive effect of at the doses of 75 mg/kg and 25 mg/kg plus morphine was significantly higher in comparison with the control group receiving morphine alone (P < 0.05). No significant (p > 0.05) difference was observed after the administration of CuNPs at the doses of 25, 50, and 75 mg/kg in the sensory-motor test. CONCLUSION: The present investigation demonstrated the analgesic effects of CuNPs especially in combination with morphine. These findings can provide a new strategy for producing new antinociceptive medications in the future.

Sustained release of silibinin-loaded chitosan nanoparticle induced apoptosis in glioma cells.

In this study, a chitosan nanoparticle formulation was synthesized for loading silibinin as a sustained-release drug system to evaluate its effects on apoptosis in C6 glioma cells. This synthesized nanoparticle was analyzed by measurement methods including Fourier transform infrared (FTIR), field emission-scanning electron microscopy (FE-SEM), dynamic light scattering (DLS), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). The formation and amorphization of nanoparticle were confirmed by FTIR and XRD analysis, respectively. The mean diameter of silibinin-loaded chitosan nanoparticles (SCNP) was 50 ± 7 and 188.6 ± 0.17 nm by using FE-SEM and DLS, respectively. In addition, the positive zeta potential of nanoparticles was +11.5. Rhodamine-conjugated SCNP analysis showed the internalization of silibinin to C6 glioma cells. The cytotoxicity assay indicated that the nanoformulation of silibinin was toxic to C6 glioma cells. Although SCNP significantly increased the expression of the both apoptotic genes in C6 cells, Bax and caspase3, it did not have any significant effect on the level of the antiapoptotic gene, Bcl2. In contrast, SCNP did not have any toxic effect on H9C2 cells. In conclusion, the results of the current study indicated that SCNP can be considered as a sustained-release drug system for future cell-based therapeutic strategies.

Cancer stem cells and nanotechnological approaches for eradication.

Cancer stem cells (CSCs) are currently known as the main cause of tumor recurrence. After chemotherapy is completed, CSCs proliferate and then differentiate to generate new tumor tissues. Similar to normal stem cells, this non-uniformly distributed cell population in the tumor tissue has self-renewal capacity and is responsible for survival of the tumor and difference in its genetic and metabolic characteristics. Followed by gene instability in CSCs, new phenotypic markers are aberrantly expressed in CSCs subpopulation. Hence, some of the surface markers and metabolic pathways that are upregulated in CSCs may be applied as specific targets for development of diagnostic and therapeutic approaches. In this review article, the distinctive properties of CSCs including signal pathways implicated in self-renewal and surface markers were discussed. Moreover, targeting CSCs based on their specific properties using nanodrugs was reviewed.

The efficacy of Crocus sativus (Saffron) versus placebo and Fluoxetine in treating depression: a systematic review and meta-analysis.

Background: Depression represents a serious public health concern, imposing a high burden, both in epidemiological and clinical terms. Crocus sativus (Saffron) is a herbal remedy that has anti-cancer, anti-oxidant, anti-inflammatory and anti-platelet properties. However, the exact mechanisms of Saffron in treating depression are not yet clear. This study was conducted to evaluate the effectiveness of Saffron versus placebo and Fluoxetine in the treatment of depressed patients. Methods: Different bibliographic thesauri, namely the Cochrane Library, Scopus, PubMed/MEDLINE, Centre for Reviews and Dissemination (CRD), EMBASE, and ISI/Web of Science (WoS) were searched up to May 2018. Effect sizes were computed as Standardized Mean Differences (SMD) with their 95% confidence interval (CI). To evaluate the heterogeneity among the studies, I2 test was carried out. Results: Eight studies were included. The SMD was -0.86 (95% CI: -1.73 to 0.00) concerning the comparison of Saffron with placebo. The SMD was found to be 0.11 (95% CI: -0.20 to 0.43) concerning the comparison of Saffron with Fluoxetine. In both sensitivity analyses, the results did not statistically change, confirming the stability of the findings. Conclusion: The findings of this study showed that Saffron administration was well comparable with Fluoxetine and placebo.

Curcumin as a double-edged sword for stem cells: dose, time and cell type-specific responses to curcumin.

BACKGROUND: The beneficial effects of curcumin which includes its antioxidant, anti-inflammatory and cancer chemo-preventive properties have been identified. Little information is available regarding the optimal dose and treatment periods of curcumin on the proliferation rate of different sources of stem cells. METHODS: In this study, the effect of various concentrations of curcumin on the survival and proliferation of two types of outstanding stem cells which includes bone marrow stem cells (BMSCs) and adult rat neural stem/progenitor cells (NS/PCs) at different time points was investigated. BMSCs were isolated from bilateral femora and tibias of adult Wistar rats. NS/PCs were obtained from subventricular zone of adult Wistar rat brain. The curcumin (0.1, 0.5, 1, 5 and 10 µM/L) was added into a culture medium for 48 or 72 h. Fluorescent density of 5-bromo-2'-deoxyuridine (Brdu)-positive cells was considered as proliferation index. In addition, cell viability was assessed by MTT assay. RESULTS: Treatment of BMSCs with curcumin after 48 h, increased cell survival and proliferation in a dose-dependent manner. However, it had no effect on NSCs proliferation except a toxic effect in the concentration of 10 µM of curcumin. After a 72 h treatment period, BMSCs and NS/PCs survived and proliferated with low doses of curcumin. However, high doses of curcumin administered for 72 h showed toxic effects on both stem cells. CONCLUSIONS: These findings suggest that curcumin survival and proliferative effects depend on its concentration, treatment period and the type of stem cells. Appropriate application of these results may be helpful in the outcome of combination therapy of stem cells and curcumin.

Regulation of connexin 43 and microRNA expression via ß2-adrenoceptor signaling in 1321N1 astrocytoma cells.

Connexin 43 (Cx43) is the main gap junction protein in astrocytes and exerts the same effects on growth inhibition in astrocytoma and glioma as microRNA-146a (miR-146a) in glioma. ß2-adrenergic receptor (AR) signaling modulates Cx43 expression in myocytes via components downstream of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). However, it remains to be elucidated how expression of Cx43 is modulated in astrocytes. In the present study, 1321N1 astrocytoma cells were treated with ß2-AR signaling agents in order to evaluate the expression of Cx43 and miRNAs. RNA and protein were extracted from the cells for use in reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results revealed that clenbuterol increased miR-146a level and upregulated Cx43 expression via cAMP/PKA at the mRNA and protein level. Pre-inhibition of adenyl cyclase decreased expression of Cx43 and miR-146a. PKA activation and overexpression of miR-146a in A-1321N1 cells increased the expression of Cx43. ß2-AR stimulation and 6Bnz, a PKA activator, suppressed oncomiRs miR-155 and miR-27a, while 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, an Epac activator, increased their levels. The current findings demonstrated that ß2-AR signaling has growth inhibitory effects via modulation of the cAMP/PKA pathway in A-1321N1 cells through increasing the expression level of Cx43 and miR-146a as well as decreasing miR-155 and miR-27a levels. Thus, stimulation of the ß2-AR and PKA signaling pathway may be a useful approach for astrocytoma therapy.

cAMP-Epac Pathway Stimulation Modulate Connexin-43 and MicroRNA-21 Expression in Glioma Cells.

INTRODUCTION: Malignant astrocytic gliomas are the most common and lethal brain malignancies due to their refractory to the current therapies. Nowadays, molecular targeted therapy has attracted great attention in treatment of glioma. Connexin 43 (Cx43) and micro ribonucleic acid-21(miR-21) are among molecules that are involved in glioma development and progression. These molecules showed potential to be as target molecules with regard to glioma. Some studies have reported that cyclic adenosine monophosphate (cAMP) signaling could be effective on Cx43 and miR-21 in tissues other than in brain. We investigate possible relationship between ß-adrenergic receptor and its newly described downstream, exchange protein directly activated by cAMP (Epac) signaling pathway and expression of Cx43 and miR-21 in low (1321N1) and high grade (U87MG) glioma cell lines. METHODS: We treated cells with ß-adrenergic agonist and Epac activator with and without adenyl cyclase inhibitor. Cx43 and miR-21 expression were measured with real-time PCR. RESULTS: Our data showed that in 1321N1 cells, ß-adrenergic-Epac pathway stimulation up and down-regulated Cx43 and miR-21 expression respectively. Whereas, in U87MG cells these interventions had no effect on Cx43 and miR-21 expression. DISCUSSION: These findings demonstrate that low grade astrocytoma cells have better response to our pharmacological interventions.

Fluoxetin upregulates connexin 43 expression in astrocyte.

INTRODUCTION: Recent studies have shown that astrocytes play major roles in normal and disease condition of the central nervous system including multiple sclerosis (MS). Molecular target therapy studies in MS have revealed that connexin-43 (Cx43) and Aquaporin-4 (AQP4) contents of astrocytes undergo expression alteration. Fluoxetine had some effects in MS patients unrelated to its known antidepressant effects. Some of fluoxetine effects were attributed to its capability of cAMP signaling pathway stimulation. This study aimed to investigate possible acute effects of fluoxetine on Cx43 and AQP4 expression in astrocyte. METHODS: Astrocytoma cells were treated for 24 hours with fluoxetine (10 and 20 µg/ml) with or without adenyl cyclase (AC) and protein kinase A (PKA) inhibition. Cx43 expression at both mRNA and protein levels and AQP4 expression at mRNA level were evaluated. RESULTS: Acquired results showed that fluoxetine with and without AC and PKA inhibition resulted in Cx43 up-regulation both in mRNA and protein levels, whereas AQP4 expression have not changed. DISCUSSION: In conclusion, data showed that fluoxetine alone and in the absence of serotonin acutely up-regulated Cx43 expression in astrocytes that can be assumed in molecular target therapy of MS patients. It seems that cAMP involvement in fluoxetine effects need more researches.

Selective ß2 adrenergic agonist increases Cx43 and miR-451 expression via cAMP-Epac.

It has been demonstrated that connexin 43 (Cx43) and microRNAs have significant roles in glioma. Cyclic adenosine monophosphate (cAMP) is suggested to be a regulator of connexins and microRNAs. However, it remains elusive whether cAMP and exchange protein directly activated by cAMP (Epac2), have a regulatory effect on Cx43 and microRNA-451 (miR-451) in astrocytoma cells. We treated 1321N1 astrocytoma cells with a selective ß2 adrenergic agonist and a selective Epac activator with and without adenyl cyclase and protein kinase A inhibition. Cx43 and miR-451 expression were measured. Next, we evaluated the effect of miR-451 overexpression on Cx43 expression. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated that cAMP-Epac2 increased Cx43 and miR-451 expression. However, the alteration of miR-451 expression required a higher dose of drugs. Overexpression of miR-451 had no significant effect on Cx43 expression. The MTT assay showed that cAMP-Epac stimulation and miR-451 overexpression had a synergic inhibitory effect on cell proliferation. These findings may expand our understanding of the molecular biology of glioma and provide new potential therapeutic targets.

A new and safe method for stereotactically harvesting neural stem/progenitor cells from the adult rat subventricular zone.

BACKGROUND: Adult neural stem/progenitor cells (NS/PCs) are one of the outstanding cell sources for therapeutic purposes in the central nervous system diseases. Autologous transplantation of NS/PCs still is a matter of controversy due to the safety issue as well as efficiency of harvesting these cells from the live mammalian brain subventricular zone (SVZ). NEW METHOD: In this new and safe method, a 16-guage semi-automatic biopsy needle was used stereotactically to remove a piece of SVZ. Then, the proliferation and differentiation capacity of obtained cells were assessed. In addition, the safety of the biopsy procedure was analyzed employing the Morris water maze, modified neurologic severity score, passive avoidance and open field tests. RESULTS: Despite being very small in size, the SVZ specimen could generate a large number of progeny with the ability to differentiate into neuronal and glial cells. The biopsy procedure introduced in this study did not have any impact on the behavioral and neurological processes. COMPARISON WITH EXISTING METHOD(S): existing SVZ biopsy methods were uncontrollable techniques which harvested brain tissue by aspiration using a syringe not a semi-automatic biopsy needle. Also, previous methods were not evaluated in terms of behavior and cognition. CONCLUSIONS: This study revealed a considerable safety and efficacy for the stereotactical removal of the adult rat SVZ to harvest NS/PCs for autologous transplantation.