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.

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.

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.

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.

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.

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.

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.

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.

Anticancer Potential of Aguerin B, a Sesquiterpene Lactone Isolated from Centaurea behen in Metastatic Breast Cancer Cells.

BACKGROUND: Breast carcinoma is a malignant disease that represents the most common non-skin malignancy and a chief reason of cancer death in women. Large interest is growing in the use of natural products for cancer treatment, especially with goal of suppression angiogenesis, tumor cell growth, motility, as well as invasion and metastasis with low/no toxicity. It is evident from recent patents on the anticancer properties of sesquiterpene lactones such as parthenolide. OBJECTIVE: In this study, using MDA-MB-231 cells of a human breast adenocarcinoma, the effects of aguerin B, as a natural sesquiterpene lactone, has been evaluated, in terms of the expression of metastatic-related genes (Pak-1, Rac-1 and HIF-1α). METHODS: Cytotoxicity of aguerin B was tested toward MDA-MB-231 breast tumor cells using MTT. Scratch assay was accomplished to evaluate the tumor cell invasion. To understand the underlying molecular basis, the mRNA expressions were evaluated by real time PCR. RESULTS: It was found that aguerin B significantly inhibited human breast cancer cell growth in vitro (IC50 = 2µg/mL) and this effect was accompanied with a persuasive suppression on metastasis. Our results showed that aguerin B in IC50 concentration down-regulated Rac-1, Pak-1, Hif-1α and Zeb-1 transcriptional levels. CONCLUSION: Taken together, this study demonstrated that aguerin B possessed potential anti-metastatic effect, suggesting that it may consider as a potential multi target bio compound for treatment of breast metastatic carcinoma.

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.

Apoptotic Effects of Melittin on 4T1 Breast Cancer Cell Line is associated with Up Regulation of Mfn1 and Drp1 mRNA Expression.

BACKGROUND AND PURPOSE: Melittin, as the main ingredient of honeybee venom, that has shown anticancer properties. The present study aimed at investigating the cytotoxic impacts of melittin on 4T1 breast cancer cells. METHODS: Hemolytic activity of different concentrations (0.125, 0.25, 0.5, 1, 2, 4, 8µg/ml) of melittin was assayed and then cytotoxicity of selected concentrations of melittin (2, 4, 8, 16, 32, and 64µg/ml), 2 and 4µg/ml of cisplatin and 0.513, 0.295 and 0.123µg/ml of doxorubicin was evaluated on 4T1 cells using MTT assay. We used Morphological evaluation and flow cytometric analysis was used. Real time PCR was also used to determine mRNA expression of Mfn1 and Drp1 genes. RESULTS: All compounds showed anti-proliferative effects on the tumor cell line with different potencies. Melittin had higher cytotoxicity against 4T1 breast cancer cells (IC50= 32µg/ml-72h) and higher hemolytic activity (HD50= 1µg/ml), as compared to cisplatin and doxorubicin. Mellitin at 16 and 32µg/ml showed apoptotic effects on 4T1 cells according to the flow cytometric analysis. The Real time PCR analysis of Drp1 and Mfn1 expression in cells treated with 16µg/ml of melittin revealed an up-regulation in Drp1 and Mfn1 genes mRNA expression in comparison with control group. Treatment with 32µg/ml of melittin was also associated with a rise in mRNA expression of Drp1 and Mfn1 as compared to the control group. CONCLUSION: The results of this study showed that melittin has anticancer effects on 4T1 cell lines in a dose and time dependent manner and can be a good candidate for further research on breast cancer treatment.

Down-regulation of TGF-ß, VEGF, and bFGF in vascular endothelial cells of chicken induced by a brittle star (Ophiocoma erinaceus) extract.

Great attention has been focused on the discovery of anti-angiogenic natural and synthetic compounds to be finally used as or at least a part of the treatment of tumors. The marine ecosystems provide diversity in natural chemicals with the potential of being exploited as medicines in the treatment of diseases. Several studies have investigated Ophiuroids as a source of anti-tumor and anti-metastatic organisms. Here, we described the inhibitory effects of an ethanolic crude extract of brittle star (Ophiocoma erinaceus) on angiogenesis and the expression level of TGF-ß, VEGF, and bFGF in chicken chorioallantoic membrane (CAM) as an experimental model. To do this 45 embryonated eggs were randomly divided into six groups including the control group, sham, three experimental groups and positive. The number and the length of vessels were calculated using ImageJ® software. The relative mRNA levels of the genes in different groups were evaluated by qRT-PCR method. Our study was suggestive of an anti-angiogenesis effect of brittle star ethanolic crude extract in a CAM model. The extract also showed a pharmacological effect of down-regulation of mRNA related to VEGF, TGF-ß, and bFGF genes on chicken vascular endothelial cells. It was also showed that the observed inhibitory effect is with a dose-dependent manner in which the highest inhibitory effect belonged to the highest used dose. We indicated the anti-angiogenesis properties of the Persian Gulf brittle star. Further studies are needed in other aspects of the brittle star extract in the treatment of angiogenesis, hyperplasia, and cancers.

In Utero Exposure to Gestational Diabetes Alters DNA Methylation and Gene Expression of CDKN2A/B in Langerhans Islets of Rat Offspring.

OBJECTIVE: DNA methylation, a major epigenetic reprogramming mechanism, contributes to the increased prevalence of type 2 diabetes mellitus (T2DM). Based on genome-wide association studies, polymorphisms in CDKN2A/B are associated with T2DM. Our previous studies showed that gestational diabetes mellitus (GDM) causes apoptosis in ß-cells, leading to a reduction in their number in pancreatic tissue of GDM-exposed adult rat offspring. The aim of this study was to examine the impact of intrauterine exposure to GDM on DNA methylation, mRNA transcription, as well as protein expression of these factors in the pancreatic islets of Wistar rat offspring. Our hypothesis was that the morphological changes seen in our previous study might have been caused by aberrant methylation and expression of CDKN2A/B. MATERIALS AND METHODS: In this experimental study, we delineated DNA methylation patterns, mRNA transcription and protein expression level of CDKN2A/B in the pancreatic islets of 15-week-old rat offspring of streptozotocin-induced GDM dams. We performed bisulfite sequencing to determine the DNA methylation patterns of CpGs in candidate promoter regions of CDKN2A/B. Furthermore, we compared the levels of mRNA transcripts as well as the cell cycle inhibitory proteins P15 and P16 in two groups by qPCR and western blotting, respectively. RESULTS: Our results demonstrated that hypomethylation of CpG sites in the vicinity of CDKN2A and CDKN2B genes is positively related to increased levels of CDKN2A/B mRNA and protein in islets of Langerhans in the GDM offspring. The average percentage of CDKN2A promoter methylation was significantly lower in GDM group compared to the controls (P<0.01). CONCLUSION: We postulate that GDM is likely to exert its adverse effects on pancreatic ß-cells of offspring through hypomethylation of the CDKN2A/B promoter. Abnormal methylation of these genes may have a link with ß-cell dysfunction and diabetes. These data potentially lead to a novel approach to the treatment of T2DM.

The Effects of Embryonic Cerebrospinal Fluid on The Viability and Neuronal Differentiation of Adipose Tissue-Derived Stem Cells in Wistar Rats.

OBJECTIVE: The embryonic cerebrospinal fluid (e-CSF) contains various growth factors and morphogens. Recent studies showed that e-CSF plays significant roles in embryonic brain development. Adipose tissue-derived stem cells (ADSCs) have a mesodermal origin that can be differentiated into mesodermal and ectodermal lineages. This study aimed to evaluate the effects of e-CSF on the proliferation, viability, and neural differentiation of ADSCs in rats. MATERIALS AND METHODS: In this experimental study, adipose tissue was dissected out from the inguinal region of adult male rats. Then, ADSCs were isolated by enzymatic digestion from adipose tissues and mesenchymal cells were confirmed using the flow cytometry analysis that measured the cell surface markers including CD90, CD44, CD73, CD105, CD34, CD45, and CD11b. The multi-potential characteristics of ADSCs were assessed by osteogenic and adipogenic potentials of these cells. Under suitable in vitro conditions, ADSCs were cultured in DMEM supplemented with and without additional 10% e-CSF. These fluids were collected from Wistar rats at the E17, E18, and E19 gestational ages. Cellular proliferation and viability were determined using the MTT assay. Immunocytochemistry was used to study the expression of ß-III tubulin in ADSCs. The neurite outgrowth of cultured cells was assessed using the ImageJ software. RESULT: The results of the present study demonstrated that the viability of ADSCs in cell culture conditioned with E17 and E18 e-CSF were significantly increased in comparison with controls. Cultured cells treated with e-CSF from E18 and E19 established neuronal-like cells bearing long process, whereas no process was observed in the control groups or cultured cells treated with E17 e-CSF. CONCLUSION: This study showed that e-CSF has the ability to induce neuronal differentiation and viability in ADSCs. Our data support a significant role of e-CSF as a therapeutic strategy for the treatment of neurodegenerative diseases.

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.

The synergistic cytotoxic effects of doxorubicin and Viola odorata extract on human breast cancer cell line T47-D.

BACKGROUND: Breast cancer accounts for one-third of cancer cases in women. Doxorubicin (Dox) is one of the chemotherapeutical compounds widely used to treat breast cancer. Chemical drugs have several side effects and their continuous administration leads to drug resistance in patients. To decrease such side effects in cancer treatment, combination therapy as well as application of natural and herbal compounds has been taken into consideration. The aim of this study was to investigate the cytotoxic effect of Viola odorata (Vo) extract on T47-D human breast cancer cells, alone and in combination with Dox. MATERIALS AND METHODS: The cytotoxic effects of V. odorata and Dox were studied by morphological examination and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flowcytometric analysis was performed to determine the type of cell death. Moreover, scratch healing assay was conducted to investigate antimigration effect of V. odorata. RESULTS: The results of MTT assay showed that V. odorata and Dox-induced cell death in T47-D cells in a dose- and time-dependent manner. Morphological analysis revealed that V. odorata and Dox-induced features of apoptotic cell death in T47-D cells. These results were confirmed by flow cytometry analysis. Scratch healing assay revealed that migration rate was reduced in the V. odorata- treated cells. CONCLUSIONS: Our findings suggest that components of V. odorata exert antitumor effects on human breast cancer and could be administered with lower doses of antitumor agent Dox, in combination therapy, to decrease its side effects.

Effects of maternal thyroid hormone deficiency on differentiation of mesenchymal stem cells in CSF-exposed neonatal Wistar rats.

Cerebrospinal fluid (CSF) contains growth and neurotrophic factors which regulate proliferation, differentiation, and neurogenesis. Thyroid hormones play a crucial role in the development of the nervous system and hypothyroidism during development of embryos leads to defects in the nervous system. This study aimed to survey the effects of rat neonatal CSF collected from induced hypothyroid mothers on differentiation of bone marrow mesenchymal stem cells (BM-MSCs). We hypothesized that hypothyroidism affected levels of growth factor in CSF. To induce hypothyroidism, pregnant Wistar rats received methimazole at the third day of gestation. BM-MSCs were obtained from rat femurs and tibias and cultured in medium. CSF was collected from the cisterna magna of newborn rats, and cells were subsequently exposed to CSF with concentrations of 5,7, and 10 /100 (v/v) for 72 h. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and real time polymerase chain reaction (RT-PCR) were used to quantify the cell viability and analyze the expression of neural markers, respectively. Our morphological studies showed that treatment with hypothyroidism CSF (HTH-CSF) resulted in a significant decrease in neurite growth and proliferation as compared to normal CSF (N-CSF). RT-PCR analysis also showed a significant decrease in expression of neural markers (i.e., Nestin, Neurod-1, NeuN) in cells treated with HTH-CSF as compared with the N-CSF group. The most effective concentration of CSF for BM-MSC differentiation was 5% (V/V). Our results showed a significant decrease in differentiation of BM-MSCs in the presence of neonatal CSF of hypothyroid mothers compared with neonatal CSF of healthy mothers. Thus, thyroid hormones are essential in neural development and hypothyroid defects can affect development of the neonatal brain.