The expression of fibrosis-related genes is elevated in doxorubicin-induced senescent human dermal fibroblasts, but their secretome does not trigger a paracrine fibrotic response in non-senescent cells.

Tissue fibrosis is associated with the aging process of most of our organs, and organ aging correlates with the chronic accumulation of senescent cells. Fibrosis occurs when fibroblasts proliferate and deposit pathological amounts of extracellular matrix (ECM), leading to progressive tissue scarring and organ dysfunction. Fibroblasts play a key role in fibrosis, especially in the skin where fibroblasts are the most abundant cell type in the dermis and are mainly responsible for the synthesis of ECM. This study aims to investigate how senescent fibroblasts and their secretome influence dermal fibrosis. Here we used human dermal fibroblasts (HDFs) treated with doxorubicin (doxo) to induce senescence. The senescent phenotype of these stress-induced premature senescent (SIPS) cells was confirmed with several markers. The expression of pro-fibrotic genes was quantified and finally, the impact of their secretome on the fibrotic response of non-senescent fibroblasts was assessed. Doxorubicin treatment, induced senescence in fibroblasts which has been confirmed with elevated senescence-associated ß- galactosidase (SA-ß-gal) activity, absence of BrdU incorporation, upregulation of p21, and loss of Lamin b1. Expression levels of the pro-fibrotic genes ACTA2 and FN1 increased in SIPS cells, but in contrast to studies using lung fibroblasts the secretome of these cells failed to induce a paracrine fibrotic response in non-senescent cells. In general, these results suggest that these senescent cells are potentially profibrotic, and their accumulation can trigger fibrosis in organs.

Embryonic cerebrospinal fluid formation and regulation.

The vertebrate brain is organized, from its embryonic origin and throughout adult life, around a dynamic and complex fluid, the cerebrospinal fluid (CSF). There is growing interest in the composition, dynamics and function of the CSF in brain development research. It has been demonstrated in higher vertebrates that CSF has key functions in delivering diffusible signals and nutrients to the developing brain, contributing to the proliferation, differentiation and survival of neural progenitor cells, and to the patterning of the brain. It has also been shown that the composition and the homeostasis of CSF are tightly regulated following the closure of the anterior neuropore, just before the initiation of primary neurogenesis in the neural tissue surrounding brain cavities, before the formation of functional choroid plexus. In this review we draw together existing literature about the composition and formation of embryonic cerebrospinal fluid in birds and mammals, from the closure of the anterior neuropore to the formation of functional fetal choroid plexus, including mechanisms regulating its composition and homeostasis. The significance of CSF regulation within embryonic brain is also discussed from an evolutionary perspective.

Cytotoxicity of mancozeb on Sertoli-germ cell co-culture system: Role of MAPK signaling pathway.

Mancozeb (MZB) is a worldwide fungicide for the management of fungal diseases in agriculture and industrial contexts. Human exposure occurs by consuming contaminated plants, drinking water, and occupational exposure. There are reports on MZB's reprotoxicity such as testicular structure damage, sperm abnormalities, and decrease in sperm parameters (number, viability, and motility), but its molecular mechanism on apoptosis in testis remains limited. To investigate the molecular mechanisms involved in male reprotoxicity induced by MZB, we used primary cultures of mouse Sertoli-germ cells. Cells were exposed to MZB (1.5, 2.5, and 3.5 µM) for 3 h to evaluate viability by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, reactive oxygen species (ROS) generation, and oxidative stress parameters (lipid peroxidation). Cell death and mitogen-activated protein kinase (MAPK) signaling were measured in these cells using flow cytometry and western blotting. In addition, some groups were exposed to N-acetylcysteine (NAC, 5 mM) in the form of co-treatment with MZB. Mancozeb reduced viability and increased the level of intracellular ROS, p38 and c-Jun N-terminal kinases (JNK) MAPK proteins phosphorylation, and apoptotic cell death, which could be blocked by NAC as an inhibitor of oxidative stress. The present study indicated for the first time the toxic manifestations of MZB on the Sertoli-germ cell co-culture. Redox imbalance and p38 and JNK signaling pathway activation might play critical roles in MZB-induced apoptosis in the male reproductive system.

Regeneration of Bone Defects in a Rabbit Femoral Osteonecrosis Model Using 3D-Printed Poly (Epsilon-Caprolactone)/Nanoparticulate Willemite Composite Scaffolds.

Steroid-associated osteonecrosis (SAON) is a chronic disease that leads to the destruction and collapse of bone near the joint that is subjected to weight bearing, ultimately resulting in a loss of hip and knee function. Zn2+ ions, as an essential trace element, have functional roles in improving the immunophysiological cellular environment, accelerating bone regeneration, and inhibiting biofilm formation. In this study, we reconstruct SAON lesions with a three-dimensional (3D)-a printed composite made of poly (epsilon-caprolactone) (PCL) and nanoparticulate Willemite (npW). Rabbit bone marrow stem cells were used to evaluate the cytocompatibility and osteogenic differentiation capability of the PCL/npW composite scaffolds. The 2-month bone regeneration was assessed by a Micro-computed tomography (micro-CT) scan and the expression of bone regeneration proteins by Western blot. Compared with the neat PCL group, PCL/npW scaffolds exhibited significantly increased cytocompatibility and osteogenic activity. This finding reveals a new concept for the design of a 3D-printed PCL/npW composite-based bone substitute for the early treatment of osteonecrosis defects.

Epidermal neural crest stem cell-derived glia enhance neurotrophic elements in an ex vivo model of spinal cord injury.

Growing evidence that cell-based therapies can improve recovery outcome in spinal cord injury (SCI) models substantiates their application for treatment of human with SCI. To address the effectiveness of these stem cells, potential candidates should be evaluated in proper SCI platform that allows direct real-time monitoring. In this study, the role of epidermal neural crest stem cells (EPI-NCSCs) was elucidated in an ex vivo model of SCI, and valproic acid (VPA) was administered to ameliorate the inhospitable context of injury for grafted EPI-NCSCs. Here the contusion was induced in organotypic spinal cord slice culture at day seven in vitro using a weight drop device and one hour post injury the GFP- expressing EPI-NCSCs were grafted followed by VPA administration. The evaluation of treated slices seven days after injury revealed that grafted stem cells survived on the injured slices and expressed GFAP, whereas they did not express any detectable levels of the neural progenitor marker doublecortin (DCX), which was expressed prior to transplantation. Immunoblotting data demonstrated that the expression of GFAP, BDNF, neurotrophin-3 (NT3), and Bcl2 increased significantly in stem cell treated slices. This study illustrated that the fate of transplanted stem cells has been directed to the glial lineage in the ex vivo context of injury and EPI-NCSCs may ameliorate the SCI condition through releasing neurotrophic factors directly and/or via inducing resident spinal cord cells.

Mancozeb induces testicular dysfunction through oxidative stress and apoptosis: Protective role of N-acetylcysteine antioxidant.

Mancozeb (MZB) is one of the fungicides used in pest control programs that might affect human health including reproductive system. The aim of this study was to demonstrate the mechanisms through which MZB induces testicular tissue damage and the probable protective effect of N-acetylcysteine (NAC), a modified amino acid, with antioxidant property, against MZB toxicity in an animal model. Male albino mice ( n = 8) were exposed to different doses of MZB (250 and 500 mg/kg/day) by oral gavage without or with NAC (200 mg/kg, twice/week) for 40 days. Sub-chronic MZB dose-dependently decreased sperm motility and count. Exposure to MZB increased lipid peroxidation and protein carbonyl, while it reduced antioxidant enzymes activities, total antioxidant capacity, and glutathione content. The histopathological examination clearly showed deleterious changes in the testicular structure. At the molecular levels, the results of quantitative real time-poly chain reaction (qRT-PCR) showed that MZB upregulated oxidative stress markers inducible nitric oxide synthase (iNOS) and NADPH oxidase 4 (NOX4) and downregulated expression of the glutathione peroxidase 1 (Gpx1) gene as one of the most important antioxidant enzymes. MZB also induced apoptosis dose-dependently in the testes as determined by the terminal dUTP nick-end labeling assay and immunoblotting. NAC administration decreased the mRNA levels of both iNOS and NOX4 with a concomitant increase in Gpx1 expression. It also significantly decreased MZB-induced oxidative stress and apoptosis. Collectively, the present study showed MZB-induced oxidative damage in testes leading to apoptosis. It revealed that antioxidants such as NAC can mitigate oxidant injury induced by the dithiocarbamate pesticides in the reproductive system.

Suppressing the molecular signaling pathways involved in inflammation and cancer in breast cancer cell lines MDA-MB-231 and MCF-7 by miR-590.

Breast cancer is the most frequent cancer among women worldwide. Tumor immunology suggests relationships between the immune system, chronic inflammation, and cancer. The immune system may either prevent or promote carcinogenesis. Here, we evaluated molecular signaling pathways common in inflammation and cancer and detected the microRNAs which play pivotal roles in mediating these pathways. Using bioinformatics assays, signaling pathways common in inflammation and cancer, and microRNAs mediating these pathways were identified. MiR-590 was selected and cloned into the pLenti-III-eGFP vector and transfected into the breast cancer cell lines. The expression level of microRNA and the candidate genes was evaluated by real-time quantitative reverse transcription polymerase chain reaction, and the apoptosis level in transfected cells was measured by Annexin V-7AAD assay. The cell migration was tested by real-time quantitative reverse transcription polymerase chain reaction for MMP2/MMP9. The expression levels of miR-590 and the selected genes (i.e. JAK2, PI3K, MAPK1, and CREB) were measured 72 h after transfection. While miR-590 showed an over-expression, the genes were significantly down-regulated. A significant increase was observed in apoptosis level in both cell lines and MMP2/MMP9 was significantly decreased in MDA-MB-231 cells. MiR-590 was selected as a microRNA which triggers and down-regulates critical genes of signaling pathways similar in cancer and inflammation. Following the miR-590 treatment, JAK2, PI3K, MAPK1, and CREB were down-regulated and the apoptosis level was increased in breast cancer cell lines. Apparently, some microRNAs can be good candidates for novel treatments of cancer. Although miR-590 showed good results in this study, further studies are required to investigate the role of miR-590 in breast cancer therapy.

Efficient programming of human eye conjunctiva-derived induced pluripotent stem (ECiPS) cells into definitive endoderm-like cells.

Due to pluripotency of induced pluripotent stem (iPS) cells, and the lack of immunological incompatibility and ethical issues, iPS cells have been considered as an invaluable cell source for future cell replacement therapy. This study was aimed first at establishment of novel iPS cells, ECiPS, which directly reprogrammed from human Eye Conjunctiva-derived Mesenchymal Stem Cells (EC-MSCs); second, comparing the inductive effects of Wnt3a/Activin A biomolecules to IDE1 small molecule in derivation of definitive endoderm (DE) from the ECiPS cells. To that end, first, the EC-MSCs were transduced by SOKM-expressing lentiviruses and characterized for endogenous expression of embryonic markers Then the established ECiPS cells were induced to DE formation by Wnt3a/Activin A or IDE1. Quantification of GSC, Sox17 and Foxa2 expression, as DE-specific markers, in both mRNA and protein levels revealed that induction of ECiPS cells by either Wnt3a/Activin A or IDE1 could enhance the expression level of the genes; however the levels of increase were higher in Wnt3a/Activin A induced ECiPS-EBs than IDE1 induced cells. Furthermore, the flow cytometry analyses showed no synergistic effect between Activin A and Wnt3a to derive DE-like cells from ECiPS cells. The comparative findings suggest that although both Wnt3a/Activin A signaling and IDE1 molecule could be used for differentiation of iPS into DE cells, the DE-inducing effect of Wnt3a/Activin A was statistically higher than IDE1.

Toxic effects of magnesium oxide nanoparticles on early developmental and larval stages of zebrafish (Danio rerio).

Magnesium oxide nanoparticles (MgONPs) are used in medicine, manufacturing and food industries. Because of their extensive application in our daily lives, environmental exposure to these nanoparticles is inevitable. The present study examined the effects of MgONPs on zebrafish (Danio rerio) early developmental stages. The results showed that, at different concentrations, MgONPs induced cellular apoptosis and intracellular reactive oxygen species. The hatching rate and survival of embryos decreased in a dose dependent manner. The 96-h LC50 value of MgONPs on zebrafish survival was 428 mg/l and the 48-h EC50 value of MgONPs on zebrafish embryo hatching rate was 175 mg/l. Moreover different types of malformation were observed in exposed embryos. The results demonstrate the toxic effects of MgONPs on zebrafish embryos and emphasize the need for further studies.

Functionalisation and surface modification of electrospun polylactic acid scaffold for tissue engineering.

Repair or replacement of damaged tissues using tissue engineering technology is considered to be a fine solution for enhanced treatment of different diseases such as skin diseases. Although the nanofibers made of synthetic degradable polymers, such as polylactic acid (PLA), have been widely used in the medical field, they do not favour cellular adhesion and proliferation. To enhance cell adherence on scaffold and improve biocompatibility, the surface of PLA scaffold was modified by gelatin in our experiments. For electrospinning, PLA and gelatin were dissolved in hexafluoroisopropanol (HFIP) solvent at varying compositions (PLA:gelatin at 3:7 and 7:3). The properties of the blending nanofiber scaffold were investigated by Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). Modified PLA/gelatin 7/3 scaffold is more suitable for fibroblasts attachment and viability than the PLA or gelatin nanofiber alone. Thus fibroblast cultured on PLA/gelatin scaffold could be an alternative way to improve skin wound healing.

Fabrication and application of salicin-polycaprolactone 3D-printed scaffold in the healing of femur bone defects.

Polycaprolactone (PCL) is a suitable material for bone repair due to good biocompatibility and mechanical properties. However, low bioactivity and hydrophobicity pose major challenges for its biomedical applications. To overcome these limitations, PCL-based scaffolds loaded with bioactive agents have been developed. Salicin (Sal) is an anti-inflammatory and analgesic herbal glycoside with osteogenic potential. In the present study, we aimed to produce a Sal-laden PCL (PCL-Sal) scaffold for bone healing applications. Three-dimensional scaffolds were produced and their biocompatibility, and physical-chemical characteristics were determined. The osteogenic potential of the PCL (PCL) and PCL-Sal scaffolds was evaluated using bone marrow mesenchymal stem cells (BMSCs). Scaffolds were implanted into a 5 mm bone defect created in the femur of adult rats, and the new bone fraction was determined using micro-computed tomography scanning at one-month follow-up. PCL-Sal scaffold had a structure, porosity, and fiber diameter suitable for bone construction. It also possessed a higher rate of hydrophilicity and bioactivity compared to the PCL, providing a suitable surface for the proliferation and bone differentiation of BMSCs. Furthermore, PCL-Sal scaffolds showed a higher capacity to scavenge free radicals compared to PCL. The improved bone healing potential of the PCL-Sal scaffold was also confirmed according toin vivoimplantation results. Our findings revealed that the Sal-laden implant could be considered for bone repair due to desirable characteristics of Sal such as hydrophilicity, surface modification for cell attachment, and antioxidant properties.

MicroRNA 17-92 expressed by a transposone-based vector changes expression level of cell-cycle-related genes.

The miR-17-92 cluster is composed of seven miRNAs (microRNAs; miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1). Previous studies have indicated that this cluster is involved in cell proliferation and their overexpression has been seen in several types of cancer. We have assessed the overexpression effects of miR-17-92 on the expression of several genes associated with cell-cycle regulation. The human miR-17-92 gene was cloned into a transposone-based vector, piggyBac and transfected into HEK-293T [HEK-293 cells (human embryonic kidney cells) expressing the large T-antigen of SV40 (simian virus 40)] cell line. Gene expression analysis indicated that up-regulation of this cluster causes significant changes in the expression of several cell-cycle related genes, including CDK2 (cyclin-dependent kinase 2), cyclin-D2, c-Myc and CREB (cAMP-response-element-binding protein). Other methods of transcripts assessment confirmed miR-17-92 overexpression enhances cell proliferation.

Melittin Prevents Metastasis of Epidermal Growth Factor-Induced MDA-MB-231 Cells through The Inhibition of The SDF-1α/CXCR4 Signaling Pathway.

Objective: Melittin is one of the natural components of bee venom (Apis mellifera), and its anticancer and antimetastatic properties have been well established, but the underlying mechanism remains elusive. The MDA-MB-231 is a triplenegative cell line that is highly aggressive and invasive. Besides, many critical proteins are involved in tumor invasion and metastasis. In this study, we investigated whether melittin inhibits the migration and metastasis of epidermal growth factor (EGF)-induced MDA-MB-231 cells via the suppression of SDF-1α/CXCR4 and Rac1-mediated signaling pathways. Materials and Methods: In this experimental study, cells were treated with melittin (0.5-4 µg/ml), and the toxicity of melittin was assessed by the MTT assay. Afterward, the migration assay was conducted to measure the degree of the migration of EGF-induced cells. The western blot technique was performed to analyze the rate of Rac1, p-Rac1, SDF- 1α, and CXCR4 expression in different groups. Results: The results demonstrated that melittin markedly suppressed the migration of EGF-induced cells and decreased the expression of p-Rac1, CXCR4, and SDF-1α proteins. Conclusion: The results of the present study suggested that the anti-tumor properties of melittin could be through the blocking of the SDF-1α/CXCR4 signaling pathway, which is beneficial for the reduction of tumor migration and invasion.

Ovarian Toxicity Induced by Aluminum Chloride: Alteration of Cyp19a1, Pcna, Puma, and Map1lc3b genes Expression.

Aluminum (Al) is an abundant metal with wide application in our daily lives including medicine, industry, cosmetics, and packaging. After entrance to the body, aluminum binds to transferrin and reaches different tissues. Al is a metalloestrogen that can lead to oxidative stress (OxS) and endocrine disruption. No detailed study can be found addressing the effect of Al on the ovary and granulosa cells (GCs). In this study, the focus is on the treated ovaries and GCs of NMRI mice exposed to low, middle, and high doses of aluminum chloride (AlCl3) via in vitro and in vivo assays. The steroidogenic, proliferative, apoptotic, and autophagic-related genes were examined. Up-regulated expression of steroidogenic and proliferative genes was detected. The observed apoptotic and autophagic genes had variable expression. Interrupted ovarian structure, disrupted folliculogenesis, presence of Call-Exner bodies, overexpression of steroidogenic gene, and unbalanced apoptosis/autophagy and proliferation resembled features of granulosa cell tumor (GCT).

Selenium enhances the expression of miR-9, miR-124 and miR-29a during neural differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: Selenium (Se) is a trace element that plays important role in antioxidant defense in the brain. Sodium selenite (Na2SeO3) is an inorganic salt of Se which has an antioxidant function. In the present study, we investigated the effect of Sodium selenite on the expression of important neuronal microRNAs during neural differentiation of bone marrow-derived stem cells (BMSCs). METHODS: Mesenchymal stem cells were collected from rat bone marrow and cultured in the Dulbecco's Modified Eagle Medium (DMEM) medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was conducted to determine the toxicity of Na2SeO3. For neural induction, BMSCs were divided into control, Na2SeO3 containing (10 ng/mL) and Na2SeO3 free groups and cultured in DMEM medium supplemented with Isobutyl-l-methylxanthine (IBMX), Fibroblast growth factor 2 (FGF2), B27, Retinoic acid, and brain derived neurotrophic factor (BDNF) for 14 days. At the end of the differentiation, immunostaining against Microtubule associated protein 2 (Map-2) and Choline acetyltransferase (ChAT) proteins was performed. Also, the total RNA is extracted from control and neural differentiated cells using a special kit, and the expression of miR-9, miR-124, and miR-29a was analyzed using real-time polymerase chain reaction (RT-PCR). RESULTS: Increasing Na2SeO3 concentrations had increasing toxicity; therefore, the concentration of 10 ng/mL was used as a supplement during neural differentiation. Examination of the expression of Map-2 and ChAT proteins showed that Na2SeO3 increased the expression of them and consequently the neuronal differentiation of BMSCs. Na2SeO3 also significantly increased the expression of miR-9, miR-124, and miR-29a in BMSCs undergoing neuronal differentiation. CONCLUSIONS: Our results suggest that the protective effect of selenium on neural differentiation of stem cells may be mediated through neuron specific microRNAs. This result further highlights the importance of selenium supplementation in preventing neuronal diseases.

Profound changes in cerebrospinal fluid proteome and metabolic profile are associated with congenital hydrocephalus.

The aetiology of congenital hydrocephalus (cHC) has yet to be resolved. cHC manifests late in rodent gestation, and by 18-22 weeks in human fetuses, coinciding with the start of the major phase of cerebral cortex development. Previously we found that cerebrospinal fluid (CSF) accumulation is associated with compositional changes, folate metabolic impairment and consequential arrest in cortical development. Here, we report a proteomics study on hydrocephalic and normal rat CSF using LC-MSMS and a metabolic pathway analysis to determine the major changes in metabolic and signalling pathways. Non-targeted analysis revealed a proteome transformation across embryonic days 17-20, with the largest changes between day 19 and 20. This provides evidence for a physiological shift in CSF composition and identifies some of the molecular mechanisms unleashed during the onset of cHC. Top molecular regulators that may control the shift in the CSF metabolic signature are also predicted, with potential key biomarkers proposed for early detection of these changes that might be used to develop targeted early therapies for this condition. This study confirms previous findings of a folate metabolic imbalance as well as providing more in depth metabolic analysis and understanding of cHC CSF.

Enhanced Neural Differentiation of Epidermal Neural Crest Stem Cell by Synergistic Effect of Lithium carbonate and Crocin on BDNF and GDNF Expression as Neurotrophic Factors.

Neurodegenerative diseases are incurable and debilitating conditions that result in progressive degeneration of nerve cells. Due to the complexity of conditions in neurodegenerative diseases, combination therapy, including cell and drug therapy is important as a new therapeutic strategy. Epidermal neural crest stem cells (EPI-NCSCs) are among the best choices in cell therapy for various neurological diseases. In this study, the effect of Lithium carbonate and Crocin, considering their effects on cellular signaling pathways and neuroprotective properties were investigated on the expression of neurotrophic factors BDNF and GDNF in EPI-NCSCs. EPI-NCSCs were isolated from the hair follicle and treated with different concentrations of drugs [Lithium, Crocin, and lithium + Crocin] for 72h. Then, trial concentrations were selected by MTT assay. The cells were treated with selected concentrations (Lithium 1 mM, Crocin 1.5 mM, and for co-treatment Lithium 1 mM and Crocin 1 mM) for 7 days. The Real-Time PCR results indicated an increasing in expression of BDNF and GDNF in treated cells as compared with control (* p < 0.05, ** p < 0.01 and *** p < 0.001). The results in this study confirmed and supported the neuroprotective/neurogenesis effects of Lithium and Crocin. It also showed that the proposed protocol could be used to increase EPI-NCSCs differentiation potential into neural cells in cell therapy and combination therapy of neurodegenerative diseases.

The association between testicular toxicity induced by Li2Co3 and protective effect of Ganoderma lucidum: Alteration of Bax & c-Kit genes expression.

Ganoderma lucidum has received a lot of attention recently due to its medicinal potential activities. The aim of this designed experiment was to evaluate the beneficial effects of Ganoderma lucidum extract against lithium carbonate induced testicular toxicity and related lesions in mice testis. For this purpose, lithium carbonate at a dose of 30 mg/kg, followed by 75, 150 mg/kg Ganoderma lucidum extract orally were administered for 35 days. The results were obtained from Ganoderma lucidum extract analysis prove contained a large amount of polysaccharides, triterpenoids and poly phenols based on spectrophotometric assay. Also, DPPH assay for Ganoderma lucidum extract showed high level of radical scavenging activity. The hematoxylin & eosin cross section from lithium carbonate treated group exhibited significant alterations in seminiferous tubules. Moreover, lithium carbonate induced oxidative stress via lipid peroxidation and generate MDA (P < 0.001). In addition, lithium carbonate initiated germ cells apoptosis via increase Bax expression (p < 0.001) and reduce germ cells differentiation through down-regulation of c-Kit expression (p < 0.05). Results from CASA showed that sperm parameters like count, motility and viability significantly decreased in lithium treated group (p < 0.001). It is clear that lithium carbonate induce severe damage on male reproductive system and histopathological damages via generation oxidative stress but supplementation with Ganoderma lucidum extract exhibited prevention effects and repaired induced damages.

Melittin inhibits the expression of key genes involved in tumor microenvironment formation by suppressing HIF-1α signaling in breast cancer cells.

HIF-1α has critical roles in the formation of tumor microenvironment by regulating genes involved in angiogenesis and anaerobic respiration. TME fuels tumors' growth and metastasis and presents therapy with several challenges. Therefore, we aimed to investigate if Melittin disrupts HIF-1α signaling pathway in breast adenocarcinoma cell line MDA-MB-231. Breast adenocarcinoma cell line MDA-MB-231 was cultured in the presence of different doses of Melittin, and MTT assay was carried out to measure Melittin's cytotoxic effects. Cells were exposed to 5% O2 to mimic hypoxic conditions and Melittin. Western blot was used to measure HIF-1α protein levels. Gene expression analysis was performed using real-time PCR to measure relative mRNA abundance of genes involved in tumor microenvironment formation. Our results revealed that Melittin effectively inhibits HIF-1α at transcriptional and translational/post-translational level. HIF-1α protein and mRNA level were significantly decreased in Melittin-treated groups. It is found that inhibition of HIF-1α by Melittin is through downregulation of NFκB gene expression. Furthermore, gene expression analysis showed a downregulation in VEGFA and LDHA expression due to inhibition of HIF-1α protein by Melittin. In addition, cell toxicity assay showed that Melittin inhibits the growth of MDA-MB-231 cell line through activation of extrinsic and intrinsic apoptotic pathways by upregulating TNFA and BAX expression. Melittin suppresses the expression of genes responsible for formation of TME physiological hallmarks by suppressing HIF-1α signaling pathway. Our results suggest that Melittin can modulate tumor microenvironment by inhibition of VEGFA and LDHA.

Investigating the synergic effects of valproic acid and crocin on BDNF and GDNF expression in epidermal neural crest stem cells.

Following nerve tissue damage, various events, such as inflammatory responses, microglial activation, endoplasmic reticulum stress, and apoptosis, can occur, which all lead to cell death, prevent axonal growth, and cause axonal circumvolution. So far, several researchers have tended to adopt strategies to reduce the harmful conditions associated with neurological disorders, and stem­cell­based therapy is one of those promising strategies. Epidermal neural crest stem cells (EPI­NCSCs) are a type of stem cell that has widely been employed for the treatment of various neurological disorders. It has been suggested that these stem cells perform their supportive actions primarily through the release of different neurotrophic factors. Hence, in this study, the neuroprotective impacts of valproic acid (VPA) and crocin were evaluated on the mRNA expression levels of brain­derived neurotrophic factor (BDNF) and glial­cell­derived neurotrophic factor (GDNF) in EPI­NCSCs. In this research, we isolated EPI­NCSCs from the hair follicles of the rat whisker pad. Then, the cells were treated with different concentrations of VPA and crocin for 72 h. Subsequently, an MTT assay was performed to define the suitable concentrations of drugs. Finally, real­time PCR was performed to evaluate the mRNA expression levels of BDNF and GDNF in these stem cells. The results of the MTT assay showed that the treatment of EPI­NCSCs with 1 mM VPA and 1.5 mM crocin, and the co­treatment with 1 mM VPA and 500 µM crocin, led to the survival and proliferation of these stem cells. Moreover, the real­time PCR results revealed that both VPA and crocin, both individually and in combination, can significantly increase the expression levels of BDNF and GDNF in EPI­NCSCs. According to the findings of this study, both VPA and crocin, alone and in combination, are potential candidates for enhancing the capacity of EPI­NCSCs to differentiate into neural lineages. Therefore, the co­treatment of EPI­NCSCs with these drugs can be employed for the treatment of various neurological disorders, such as spinal cord injury.