Association of Follicular Fluid Antioxidants Activity with Aging and In Vitro Fertilization Outcome: A Cross-Sectional Study.

BACKGROUND: This research was aimed at assessing the relationship between the follicular fluid (FF) antioxidants activity, aging and in vitro fertilization (IVF) outcome. MATERIALS AND METHODS: The present cross-sectional study was carried out on 65 women undergoing IVF/intracytoplasmic sperm injection (IVF/ICSI) cycles due to unexplained infertility. Ovarian stimulation was performed using the long gonadotropin-releasing hormone (GnRH) agonist protocol. After ovum pickup, FF was collected and processed to measure the level of superoxide dismutase (SOD), catalase (CAT) activity, total antioxidant capacity (TAC) and glutathione (GSH). Day 3 after ICSI, fresh embryos were transferred and later, possible pregnancy was assessed. Patients participating in this study were divided into four groups on the basis of age and pregnancy outcome. RESULTS: SOD activity was not significantly different between the groups (P=0.218). GSH in the group whose participants were aged ≤35 years and were pregnant was higher than that in other groups. CAT activity in groups with younger participants was higher compared to the other groups. The mean TAC was higher in groups with pregnant participants compared to the non-pregnant women. Correlation analysis showed that: GSH level had a significant negative correlation with age (P<0.001, R -0.55) and a significant positive correlation with pregnancy (P=0.015, R=0.30). CAT level also had a significant negative correlation with age (P<0.001, R=-0.42) and the level of TAC had a significant positive correlation with pregnancy (P<0.001, R=0.59). CONCLUSION: According to our results, the levels of TAC, GSH and CAT in younger and pregnant women were higher compared with those undergoing ICSI cycles. Given the correlation of FF antioxidant activity with age and pregnancy, it is necessary to carry out more research on these compounds and the maintenance of pregnancy.

Negative effect of varicocele on sperm mitochondrial dysfunction: A cross-sectional study.

Background: Varicocele is an abnormal dilation and enlargement of the scrotal venous pampiniform plexus that impairs normal blood drainage and finally leads to infertility if not treated. Objective: This study aimed to figure out the impact of mitochondria status through the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) assessment and its correlation with semen parameters to illuminate the impact of sperm mitochondria healthiness on normal sperm functionality. Materials and Methods: This analytical cross-sectional study was conducted with 100 men including 50 cases in the normozoospermic group (normal) and 50 in an infertile group with the non-varicocelectomy operation (varicocele) referring to Infertility Research and Treatment Center, ACECR Khuzestan, Iran. Routine semen analysis was performed according to World Health Organization guidelines, DNA fragmentation index, the MMP assay, ATP content, and apoptosis were carried out for all samples. Results: The results showed that the concentration, progressive motility, normal morphology, MMP, and ATP contents of sperm in varicocele were significantly lower than the normal group. In addition, the sperm DNA fragmentation index was significantly higher in the varicocele group in comparison with the normal group. Conclusion: Reduction in MMP and ATP contents, besides the loss of sperm parameters quality and increase in sperm DNA fragmentation, were seriously implicating sperm mitochondria dysfunctionality in varicocele men.

Antibacterial and antioxidant double-layered nanofibrous mat promotes wound healing in diabetic rats.

Diabetic wounds are problematic to heal owing to microbial infections as well as decreased proliferation and high concentrations of reactive oxygen species. In this study, a double-layered nanofibrous mat containing grape seed extract (GSE) and silver sulfadiazine (SSD) was fabricated. A synthetic biodegradable polymer, e.g., polycaprolactone (PCL), and a natural material (i.e., collagen) were employed as wound dressing substances. The results showed that GSE possesses antioxidant activity which can be helpful in reducing free radicals. The platform exhibited antibacterial activity against gram-positive and -negative bacteria. The double-layered nanofibrous mat containing GSE and SSD not only was not toxic but also amplified the cell proliferation compared to a pure mat, showing the effect of plant extract. After induction of a round wound, the animals were divided into three groups, namely (1) normal group (receiving + GSE/-GSE nanofiber), (2) diabetic group (receiving + GSE/-GSE nanofiber), and (3) control group (receiving gauze). In vivo evaluation demonstrated no significant differences in the healing process of normal rats. Surprisingly, fully repaired skin was observed on day 14 in the double-layered nanofibrous mat containing GSE in the normal and diabetic groups whereas the wound of diabetic rats treated with pure mat was not completely healed. The macroscopic and microscopic results after 14 days showed the following order in wound repair: Normal/ + GES > Diabetic/ + GSE > Normal/-GES > Diabetic/-GSE > control (with gauze) (p < 0.05). Accordingly, the double-layered nanofibrous mat containing GSE and SSD used in the present study could be considered as a suitable wound dressing in order to shorten healing time and prevent infection during the wound healing process.

Induction of apoptosis and suppression of Ras gene expression in MCF human breast cancer cells.

Breast cancer is the leading invasive cancer in women globally. This study aimed at evaluating the anti-apoptotic activity of p-Coumaric acid (PCA) on MCF-7 breast cancer cell line. Experiments were conducted in which the MCF-7 cell line was treated with PCA. which showed decreased cell viability, increased lactate dehydrogenase activity, and caspase-3 activation. The results were evaluated with real-time polymerase chain reaction which revealed that PCA reduced the amount of H-Ras and K-Ras transcript in MCF-7 breast cancer cells. In the presence of PCA there was a significant increase in the levels of mRNA gene Bax and late apoptotic cells which was dose dependent. It also retarded the relative expression of antiapoptotic gene, Bcl2 in treated cells. The results suggest that PCA exhibits anti-cancer properties against MCF-7 cells. PCA inhibited the growth of MCF7 cell. The optimum concentration of PCA was 75-150 mM. PCA can inhibit the growth of MCF-7 cells by reducing Ras expression and inducing cell apoptosis. Our results suggest that PCA could prove valuable in the search for possible inhibitors of Ras oncogene functionality and gain further support for its potential utilization in the treatment of patients with breast cancer. PCA is safe and could complement current treatments employed for the disease.

CpG Island Methylation of the Rap1Gap Gene in Medullary Thyroid Cancer.

BACKGROUND: Medullary thyroid cancer (MTC) is a rare type of neuroendocrine tumor. This study aimed to investigate the gene and protein expression of RAP1GAP and DNA methylation patterns of its CpG74a , CpG74b , and CpG24 in an Iranian population with MTC. METHODS: In this case-control study, we selected 55 individuals who underwent thyroidectomy in Erfan hospital, Tehran, between 2018 and 2020. Samples were divided into normal thyroid tissues (control; n=20), benign nodule (n=20), and MTC (n=15). DNA methylation patterns were investigated using MSP (methylation-specific PCR). The protein level and mRNA expression of RAP1GAP were also evaluated using western blotting and real-time PCR, respectively. RESULTS: The hyper-methylation rates of CpG24 and CpG74a in the MTC samples were considerably higher than the controls (83% versus 15% and 74% versus 17%, respectively; P<0.001). The methylation/unmethylation ratio of CpG74a , and CpG24 was considerably higher than the controls (P<0.001). The methylation/unmethylation ratio of CpG24 in the benign nodules was also considerably greater than the controls (P<0.001). The mRNA expression and the protein level of RAP1GAP in the MTC group were considerably lower than the controls (P=0.005 and P=0.035, respectively). In the MTC group, aberrant methylation of CpG74a and CpG24 was significantly correlated with decreasing expression of the Rap1Gap gene (R2 : 0.23; P=0.032 and R2 : 0.56; P=0.001, respectively). CONCLUSION: Hyper-methylation in CpG24 and CpG74a and decreasing expression of RAP1GAP can be considered as diagnostic biomarkers for MTC.

Autophagy Involves in Differentiation of Insulin-Secreting Cells from Adipose Derived Stem Cells.

OBJECTIVE: Destruction of pancreatic beta-cells induces an insulin deficiency and causes type 1 diabetes. The role of autophagy in inducing insulin-secreting cells (ISCs) from adipose-derived mesenchymal stem cells (AMSCs) was investigated in the current study. MATERIALS AND METHODS: In this experimental study, the isolated AMSCs were characterization and exposed to a cocktail differentiation medium (CDM) in the absence or presence of 3-methyladenine (3MA), an autophagy inhibitor. The differentiation of ISCs was confirmed by the evaluation of the expression of beta-cell-specific genes including pancreatic and duodenal homeobox 1 (PDX1), musculoaponeurotic fibrosarcoma oncogene homolog A (MAF-A), Nk class of homeodomain-encoding genes 6.1 and 2.2 (NKX6-1 and NKX2.2), Glucose transporter 2 (GLUT-2) and INSLIN. Using Newport Green (NG), insulin-positive cells were identified. Insulin secretion in response to various glucose concentrations was measured. Autophagy was evaluated by Acridine orange (AO) staining. Also, expression of autophagy-associated genes, including autophagy-related gene 5 (ATG-5), autophagy-related gene 7 (ATG-7), BECLIN-1, and mammalian target of rapamycin (mTOR), was evaluated by Real-time polymerase chain reaction (PCR) method. RESULTS: We observed a significant increase of beta-cell specific genes expression in the CDM-treated cells (P<0.01 or P<0.001), whereas the expression of these genes was down-regulated in 3MA-exposed cells. Expression of INSULIN and GLUT-2 genes (P<0.01 and P<0.05, respectively), insulin secretion in response to glucose (P<0.01), and percentage of NG-positive cells (P<0.05) in the 3MA-exposed cells were considerably lower than the cells treated with CDM. The percentage of AO-positive cells (P<0.01) and the expression of autophagy-related genes (P<0.001) was significantly enhanced in the CDM group. These events were significantly prevented by the 3MA. CONCLUSION: Our data showed that autophagy is necessary for beta-cell differentiation, and preventing autophagy by 3MA causes the reduction of beta-cell differentiation and insulin secretion.

Using ovarian and brain cell culture from the Mullet, Liza klunzingeri, to assess the inhibitory effects of benzo[a]pyrene on aromatase activity.

We assessed the toxic effects of benzo[a]pyrene (BaP) on cell viability, aromatase (Aro) activity and steroid production using ovarian and brain cell cultures obtained from Mullet, Liza klunzingeri. The brain and ovary were minced and digested, and the cells were suspended in Leibovitz's L-15 medium supplemented with 15% and 20% fetal bovine serum. The cell suspensions were seeded on 25-cm2 cell-culture flasks at 1 × 106 cells/mL and incubated at 25 °C for 2 weeks. A BaP concentration of 10-5 mol/L was accepted as the half-maximal inhibitory concentration. Ovarian and brain cells were exposed to different concentrations of BaP [0 (control), 10-6 , 2 × 10-6 , 3 × 10-6 mol/L] and incubated at 30 °C. At different sampling times (0, 12, 24 and 48 h) 40 ng/105 cells of 1,4,6-androstatriene-3,17-dione (ATD) was added to each well. Aro activity, 17ß-estradiol (E2) and ATD production were determined. The sensitivity of the cultivated ovarian and brain cells to BaP increased dose dependently. BaP was a potent inhibitor of Aro activity at 2 × 10-6 and 3 × 10-6 mol/L, both in the cultivated brain and ovarian cells at different sampling times, with 10-6 mol/L BaP found to be the least potent Aro inhibitor. E2 production decreased from cultivated ovarian and brain cells treated by different concentrations of BaP. In conclusion, BaP is able to change the activity of Aro and disrupt the biosynthesis of estrogens, and thus affects reproduction in fish.

Incorporation of Silver Sulfadiazine into An Electrospun Composite of Polycaprolactone as An Antibacterial Scaffold for Wound Healing in Rats.

OBJECTIVE: Fabrication of an antibiotic-loaded scaffold with controlled release properties for wound dressing is one of tissue engineering challenges. The aim of this study was to evaluate the wound-healing effectiveness of 500-µm thick polycaprolactone (PCL) nanofibrous mat containing silver sulfadiazine (SSD) as an antibacterial agent. MATERIALS AND METHODS: In this experimental study, an electrospun membrane of PCL nanofibrous mat containing 0.3% weight SSD with 500 µm thickness, was prepared. Morphological and thermomechanical characteristics of nanofibers were evaluated. Drug content and drug release properties as well as the surface hydrophobicity of the nanofibrous membrane were determined. Antimicrobial properties and cellular viability of the scaffold were also examined. A full thickness wound of 400 mm2 was created in rats, to evaluate the wound-healing effects of PCL/SSD blend in comparison with PCL and vaseline gas used as the control group. RESULTS: SSD at a concentration of 0.3% improved physicochemical properties of PCL. This concentration of SSD did not inhibit the attachment of human dermal fibroblasts (HDFs) to nanofibers in vitro, but showed antibacterial activity against Gram-positive Staphylococcus aureus (ST) and Gram-negative Pseudomonas aeruginosa (PS). Overall, results showed that SSD improves characteristics of PCL nanofibrous film and improves wound-healing process in one-week earlier compared to control. CONCLUSION: Cytotoxicity of SSD in fabricated nanofibrous mat is a critical challenge in designing an effective wound dressing that neutralizes cellular toxicity and improves antimicrobial activity. The PCL/SSD nanofibrous membrane with 500- µm thickness and 0.3% (w/v) SSD showed applicable characteristics as a wound dressing and it accelerated wound healing process in vivo.

Isolating human dermal fibroblasts using serial explant culture.

BACKGROUND: The purpose of this study was to introduce an applicable culture technique to isolate human dermal fibroblasts (HDFs); which could also contribute to research, clinical practices, as well as tissue engineering. METHODS: Samples from the human skin were dissected and cultured via serial explant technique. Subsequently, the isolated fibroblasts were assessed for their protein markers and genetic variations via immunofluorescence (IF) and karyotyping; respectively. Following the employment of this technique, a small piece of explant completely disappeared; while no dermis remained after 10 days. RESULTS: The quantity of HDFs harvested through this culture technique was reported at a normal level. The results of immunostaining also indicated that the isolated fibroblasts had expressed vimentin and fibronectin; whereas no cells had shown cytokeratin and epidermal marker. Moreover, karyotyping results for the fibroblasts isolated by the given technique revealed no chromosomal diversity after passage 20. CONCLUSIONS: It was concluded that serial explant culture was an efficient technique for isolating HDFs from a small piece of skin in short-time periods; which could also preserve their normal morphology and molecular characteristics.

Exposure of liver cell culture from the orange-spotted grouper, Epinephelus coioides, to benzo[a]pyrene and light results in oxidative damage as measured by antioxidant enzymes.

Among the various toxicants discharged into aquatic environments, benzo (a) pyrene (BaP) has been shown to effect on the antioxidant system of fish and the evaluation of its impact on biota is of considerable concern. The aim of the present study was to use the primary hepatocyte culture obtained from the orange-spotted grouper, Epinephelus coioides, to evaluate the adverse effects of benzo (a) pyrene (BaP) on cell viability and liver antioxidant system. BaP was selected for its high ability to produce reactive oxygen species (ROS) and oxidative stress. The liver was minced by a scalpel and digested in the PBS solution with 0.1% collagenase IV at room temperature for 20 min. Then, the cell suspension was transferred to a plate contained an equal amount of Leibovitz's L-15 medium with 20% fetal bovine serum (FBS), 100 IU mL-1 of penicillin and 100 µg mL-1 streptomycin. 5 mL of cell suspension were plated into sterile 25 cm2 tissue culture flasks at the density of 1.5 × 106 cell/ml L-15 and incubated at 30 °C for two weeks. The medium was renewed after 24-48 h. The number of the liver cells was adjusted to 4 × 106 after two weeks. 10-4 mol l-1 was verified by MTT assay as the IC50 of BaP. Then, hepatocytes were exposed to three concentrations of BaP (10-5, 2 × 10-5, 3 × 10-5 mol L-1) and incubated for 24 h. Samples were collected after 6, 12 and 24 h and the amounts of SOD, CAT, GPx, LPO, LDH, AST, ALT, ALP and total protein were analyzed. The results showed that, 10-5 mol L-1 of BaP was not significantly toxic to cultivated hepatocytes, however, the sensitivity of cells to BaP increased in a dose-related pattern. The activity of the antioxidant enzymes (SOD, CAT and GPx) and liver enzymes (ALT, AST, ALP, LDH) significantly increased, though the amount of LPO, total antioxidant power and total protein decreased dose-dependently in BaP-exposed cells. In conclusion, according to the finding of the present study, BaP has a high potential to induce the oxidative stress in primary liver cell culture of E. coioides.

Application of polycaprolactone, chitosan, and collagen composite as a nanofibrous mat loaded with silver sulfadiazine and growth factors for wound dressing.

Fabrication of nanofibrous biomaterials composed of natural and synthetic materials that incorporated with antibiotic and growth factors with controlled release manner is an attractive topic in wound healing. The purpose of this study was to prepare optimal composite of materials as biomimetic nanofibrous mats for application in wound healing. The mat was prepared of polycaprolactone (PCL) in the bottom, chitosan/poly ethylene oxide (Cs/PEO) in the middle, and PCL/collagen (PCL/Coll) in the top layer. A panel of standard characterization tests of nanofibrous mat was performed and its compatibilities in strength and integration were confirmed. Middle layer was loaded with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and silver sulfadiazine (SSD) was incorporated in the bottom layer as an anti-infection factor. Then, on the dorsum of rats, a 400-mm2 wound was created and surrounded by a silicone ring to control the usual tissue contractions. Nanofibrous mats with or without growth factors were applied as wound dressings and at day 14, the healing process was evaluated. At day 14, the treated group by designed mat showed faster epithelialization and angiogenesis. Silicone ring in the test group was desirable in wound closure compared to the control group. Reformation of skin tissue was manifested in a shorter time. This composite nanofibrous mat could be introduced as a dynamic and effective candidate for wound dressing.

Redefining the signaling pathways from pluripotency to pancreas development: In vitro ß-cell differentiation.

Pancreatic ß-cells are destroyed by the immune system, in type 1 diabetes (T1D) and are impaired by glucose insensitivity in type 2 diabetes (T2D). Islet-cells transplantation is a promising therapeutic approach based on in vitro differentiation of pluripotent stem cells (PSCs) to insulin-producing cells (IPCs). According to evolutionary stages in ß-cell development, there are several distinct checkpoints; each one has a unique characteristic, including definitive endoderm (DE), primitive gut (PG), posterior foregut (PF), pancreatic epithelium (PE), endocrine precursor (EP), and immature ß-cells up to functional ß-cells. A better understanding of the gene regulatory networks (GRN) and associated transcription factors in each specific developmental stage, guarantees the achievement of the next successful checkpoints and ensures an efficient ß-cell differentiation procedure. The new findings in signaling pathways, related to the development of the pancreas are discussed here, including Wnt, Activin/Nodal, FGF, BMP, retinoic acid (RA), sonic hedgehog (Shh), Notch, and downstream regulators, required for ß-cell commitment. We also summarized different approaches in the IPCs protocol to conceptually define a standardized system, leading to the creation of a reproducible method for ß-cell differentiation. To normalize blood glucose level in diabetic mice, the replacement therapy in the early differentiation stage, such as EP stages was associated with better outcome when compared with the fully differentiated ß-cells' graft.

Mitochondrial Effects of Teucrium Polium and Prosopis Farcta Extracts in Colorectal Cancer Cells

Background: Teucrium Polium and Prosopis Farcta have been traditionally employed in cancer treatment. In this study we evaluated the effects of methanolic extracts of these two plants in HT-29 cells. Methods: IC50s of extracts were obtained via MTT assay and the levels of ROS production, cell death, collapse of mitochondrial membrane potential and Sirt3 enzyme activity were determined. Results: After 48 hours exposure, IC50s for Teucrium and Prosopis extracts were 3 and 2µg/ml, respectively. Extracts induced higher ROS production after 6 hours than after 12 hours. Mitochondrial membrane potential collapse and cell death rate were also increased; Teucrium caused greater cell death than Prosopis. Extracts from both plants increased Sirt3 activity in its normal form, but only Teucrium extract caused a significant increase in activity of Sirt3 enzyme isolated from cancer cells. Conclusion: Teucrium and Prosopis extracts exert anticancer activity via mitochondrial alterations, as exemplified by increased ROS levels, Sirt3 activity and cell death in HT-29 colorectal cancer cells.

Using a liver cell culture from Epinephelus coioides as a model to evaluate the nonylphenol-induced oxidative stress.

The present study aimed to use primary liver cell culture derived from the orange-spotted grouper, Epinephelus coioides, to assess the toxic effects of nonylphenol (NP) on the hepatocyte viability and the liver antioxidant system. E. coioides was selected due to its commercial importance. NP was used in this study because of its high potential of producing oxidative stress due to increased reactive oxygen species (ROS). A liver of E. coioides was digested with PBS containing 0.1% collagenase IV. The digested cells were moved to Leibovitz L-15 culture medium with 20% fetal bovine serum (FBS), 100IUmL-1 penicillin, 100µgmL-1 streptomycin. Aliquots of cell suspension were seeded as a monolayer into sterile 25cm2 tissue culture flasks and incubated at 30°C for 14days. The medium, containing non-attached cells, was removed after 24 to 48h and a new medium was added. The IC50 of 10-4molL-1 was determined for nonylphenol using MTT assay. Cells were then incubated with L-15 medium containing 10-5, 2×10-5, 3×10-5molL-1 of NP and samples were taken after 6, 12 and 24h of incubation for analysis of LPO, SOD, CAT, GPx, LDH, AST, ALT, and ALP. Based on the results, the lowest concentration of NP was not markedly cytotoxic to primary hepatocytes and the cell sensitivity to NP increased dose-dependently. The activities of SOD, CAT and GPx decreased significantly, while activities of LPO, LDH, AST, ALT and ALP, increased significantly in a dose-related pattern in NP-treated cells. In conclusion, this study revealed that NP could induce the oxidative stress in cultivated hepatocytes of E. coioides during a short-term exposure. NP toxicity is mainly due to the induction of the reactive oxygen species (ROS), which lead to cell membrane disruption, damage of cellular metabolism, and interference with cellular macromolecules.

A dermal equivalent developed from adipose-derived stem cells and electrospun polycaprolactone matrix: an in vitro and in vivo study.

Polycaprolactone (PCL) is used as a material of choice for surgical sutures, wound dressings, contraceptives, fixation devices and dentistry in paramedical sciences. In addition, adipose-derived stem cells (ASCs) have been shown to be effective in the treatment of acute and chronic wounds. This study aimed to evaluate the effect of electrospun PCL fibers on keratinocyte differentiation of ASCs and wound healing. PCL solution was electrospun and characterized. Isolated and characterized ASCs were differentiated into keratinocyte-like cells on a tissue culture plate (TCP) and PCL matrices and compared. PCL nano-/microfibers cultured with ASCs (test group) or alone (control) were implanted as a dermal substitute for wound healing. There were significant increases in the proliferation rate and expression level of cytokeratin 14, filaggrin and involucrin in cells cultured on PCL matrices compared to TCP (p < 0.05). After histological and immunological evaluation of the reconstituted skin, a thick epidermal layer with several skin appendages was evidently observed in the ASC/PCL group, whereas no real and mature epidermis was formed, especially in the central area of the healing wound in the pure PCL group on day 14. Pure PCL, if possessing suitable properties including good adhesiveness, high proliferative capability, inductive elasticity and stiffness for migration and differentiation, could drive the keratinocyte differentiation of ASCs and act as an efficient dermal equivalent to promote wound healing.

Skin-derived precursors possess the ability of differentiation into the epidermal progeny and accelerate burn wound healing.

Skin-derived precursors (SKPs) are remnants of the embryonic neural crest stem cells that reside in the dermis until adulthood. Although they possess a wide range of differentiation potentials, their differentiation into keratinocyte-like cells and their roles in skin wound healing are obscure. The present study aimed to investigate the differentiation of SKPs into keratinocyte-like cells and evaluate their role in healing of third degree burn wounds. To this aim, SKPs were differentiated into keratinocyte-like cells on tissue culture plate and collagen-chitosan scaffold prepared by freeze-drying. Their differentiation capability was detected by real-time RT-PCR and immunofluorescence. Thereafter, they were cultured on scaffold and implanted in a rat model of burn wound. Histopathological and immunohistochemical analyses were employed to examine the reconstituted skin. The research findings revealed that SKPs were able to differentiate along the epidermal lineage and this ability can be enhanced on a suitable scaffold. Additionally, the results indicated that SKPs apparently promoted wound healing process and accelerate its transition from proliferating stage to maturational phase, especially if they were differentiated into keratinocyte-like cells. Regarding the results, it is concluded that SKPs are able to differentiate into keratinocyte-like cells, particularly when they are cultured on collagen-chitosan scaffold. Moreover, they can regenerate epidermal and dermal layers including thick collagen bundles, possibly through differentiation into keratinocyte-like cells.

Impact of Cell Density on Differentiation Efficiency of Rat Adipose-derived Stem Cells into Schwann-like Cells.

BACKGROUND AND OBJECTIVES: Schwann-like (SC-like) cells induced from adipose-derived stem cells (ASCs) may be one of the ideal alternative cell sources for obtaining Schwann cells (SCs). They can be used for treating peripheral nerve injuries. Co-culture with SCs or exposure to glial growth factors are commonly used for differentiation of ASCs to SC-like cells. However, the effect of initial cell density as an inductive factor on the differentiation potential of ASCs into the SC-like cells has not been yet investigated. METHODS AND RESULTS: ASCs were harvested from rat and characterized. The cells were seeded into the culture flasks at three different initial cell densities i.e. 2×10³, 4×10³ and 8×10³ cells/cm² an overnight and differentiated toward SC-like cells using glial growth factors. After two weeks, the differentiation rate of ASCs to SC-like cells at different densities was assessed by immunofluorescence, fluorescence-activated cell sorting analysis and real time RT-PCR. Expression of the typical SCs markers, S-100 proteins and glial fibrillary acidic protein (GFAP) protein, was observed in all cell densities groups although the number of S100-positive and GFAP-positive cells, and the expression of p75NTR mRNA, another SC marker, were significantly higher at the density of 8×10³ cells/cm² when compared with the other cell densities groups (p<0.001). CONCLUSIONS: The results suggest that the higher differentiation rate of ASCs to SC-like cells can be obtained at initial cell density of 8×10³ cells/cm², possibly via increased cell-cell interaction and cell density-dependent influence of glial growth factors.

Potentiation of indomethacin-induced anti-inflammatory response by pioglitazone in carrageenan-induced acute inflammation in rats: Role of PPARγ receptors.

This study aimed to assess the interaction between anti-inflammatory effects of pioglitazone (peroxysome proliferator activated receptor-gamma (PPARγ) agonist, PGL), and indomethacin (cyclooxygenase (COX) inhibitor, IND) and to evaluate the possible underlying mechanisms. Paw edema induced by carrageenan was used to induce inflammation. Different doses of IND (0.3-10mg/kg) and PGL (1-20mg/kg) alone or in combination were administered intraperitoneally to rats. Paw tissue levels of PPARγ, COX-2, and prostaglandin E2 and serum levels of TNF-α and IL-10 were also estimated. Doses of IND and PGL showed a statistically significant anti-inflammatory effect. Combination of a non-effective dose of IND (0.3mg/kg) with increasing doses of PGL (1-10mg/kg) resulted in potentiated anti-inflammation and vise versa. IND, PGL and the combination were able to reduce the COX-2, PGE2 contents and TNF-α level. Moreover, all these treatments caused elevation in PPARγ levels and IL-10 levels. However, when the rats were pre-treated with GW-9662 (a selective PPARγ antagonist), all the anti-inflammation and alterations in the biochemical factors were antagonized. These results showed that PGL markedly enhanced the anti-inflammatory activity of IND and this effect mediated partly at least, through PPARγ. Possible mechanisms of the interaction were that PGL stimulates the PPARγ and inhibits COX-2 by those cytokines that trigger the PPARγ and also inhibit COX-2. This study suggests that combination therapy with pioglitazone and indomethacin may provide an alternative for the clinical control of inflammation especially in patients with diabetes.

Human Sperm Quality and Metal Toxicants: Protective Effects of some Flavonoids on Male Reproductive Function.

BACKGROUND: Metals can cause male infertility through affection of spermatogenesis and sperm quality. Strong evidences confirm that male infertility in metal-exposed humans is mediated via various mechanisms such as production of reactive oxygen species (ROS). Flavonoids have antioxidant and metal chelating properties which make them suitable candidates for neutralizing adverse effects of metals on semen quality. In the current study, we have evaluated the effects of five types of flavonoids (rutin, naringin, kaempferol, quercetin, and catechin) on recovery of sperm motility and prevention of membrane oxidative damage from aluminum chloride (AlCl3), cadmium chloride (CdCl2), and lead chloride (PbCl4). MATERIALS AND METHODS: In this experimental study, motility and lipid peroxidation of metalexposed sperm was investigated in the presence of different concentrations of five kinds of flavonoids. Malondialdehyde (MDA) production was assessed as a lipid peroxidation marker. RESULTS: Aluminum chloride (AlCl3), cadmium chloride (CdCl2), and lead chloride (PbCl4) diminished sperm motility. Treatment of metal-exposed sperm with rutin, naringin, and kaempferol attenuated the negative effects of the metals on sperm motility. Quercetin and catechin decreased the motility of metal-exposed sperm. CONCLUSION: Based on the MDA production results, only AlCl3 significantly induced lipid peroxidation. Treatment with rutin, naringin, and kaempferol significantly decreased MDA production.