Ameliorative effect of Stevia rebaudiana Bertoni on sperm parameters, in vitro fertilization, and early embryo development in a streptozotocin-induced mouse model of diabetes.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.

Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation.

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

The expressions of NLRP1, NLRP3, and AIM2 inflammasome complexes in the contusive spinal cord injury rat model and their responses to hormonal therapy.

Spinal cord injury (SCI) is a devastating condition with a growing incidence in developing countries. The activity of inflammasome complexes initiates neuroinflammation, which is a key player in SCI pathogenesis. Here, NLRP1, NLRP3, and absent in melanoma 2 (AIM2) inflammasome complexes were assessed in the contusive (T6) SCI rats for their expression profiles and their response to hormonal therapy (10 mg/kg melatonin or 25 µg/kg 17ß-estradiol [E2] every 12 h until 72 h). Two phases was considered in this study: the dominant time of inflammasome activation, which was 72 h post-SCI and the response from each complex to hormonal therapy at this time. Gene and protein expressions of NLRP1, NLRP3, AIM2, ASC, and caspase-1 were evaluated by real-time PCR (for gene analysis), western blot, and immunohistochemistry (IHC), and biochemical presence of IL-18 and IL-1ß in spinal cord tissue homogenates was analyzed by enzyme-linked immunosorbent assay (ELISA). The whole inflammasome complexes showed high expressions in the SCI group, while after hormonal therapy, these alterations were counteracted, which were more conspicuous for the NLRP1 and NLRP3. Melatonin had no predilection over E2 for such effect. Finally, the expression profile of signaling related to the synthesis (TLR4/NF-κB) and activation (NADPH oxidase 2 [NOX2]/TXNIP) of inflammasome complexes was surveyed, and there were low activities for the two pathways in SCI rats that underwent hormone therapy. From the findings, it is concluded that both melatonin and E2 are efficient to target inflammasome activation in the SCI rats.

Ovarian vein thrombosis after coronavirus disease (COVID-19) infection in a pregnant woman: case report.

Corona virus outbreak started in December 2019, and the disease has been defined by the World Health Organization as a public health emergency. Coronavirus is a source of deep venous thrombosis (DVT) due to complications such as over-coagulation, blood stasis, and endothelial damage. In this study, we report a 26-year-old pregnant woman with coronavirus who was hospitalized with a right ovarian vein thrombosis at Besat Hospital in Sanandaj. Risk classification for deep vein thrombosis (DVT) disease is of crucial importance for the forecast of coronavirus.

Macrophage polarity in cancer: A review.

Macrophages are the most abundant cells within the tumor stroma displaying noticeable plasticity, which allows them to perform several functions within the tumor microenvironment. Tumor-associated macrophages commonly refer to an alternative M2 phenotype, exhibiting anti-inflammatory and pro-tumoral effects. M2 cells are highly versatile and multi-tasking cells that directly influence multiple steps in tumor development, including cancer cell survival, proliferation, stemness, and invasiveness along with angiogenesis and immunosuppression. M2 cells perform these functions through critical interactions with cells related to tumor progression, including Th2 cells, cancer-associated fibroblasts, cancer cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells. M2 cells also have negative cross-talks with tumor suppressor cells, including cytotoxic T cells and natural killer cells. Programed death-1 (PD-1) is one of the key receptors expressed in M2 cells that, upon interaction with its ligand PD-L1, plays cardinal roles for induction of immune evasion in cancer cells. In addition, M2 cells can neutralize the effects of the pro-inflammatory and anti-tumor M1 phenotype. Classically activated M1 cells express high levels of major histocompatibility complex molecules, and the cells are strong killers of cancer cells. Therefore, orchestrating M2 reprogramming toward an M1 phenotype would offer a promising approach for reversing the fate of tumor and promoting cancer regression. Macrophage switching toward an anti-inflammatory M1 phenotype could be used as an adjuvant with other approaches, including radiotherapy and immune checkpoint blockades, such as anti-PD-L1/PD-1 strategies.

Post-treatment of melatonin with CCl4 better reduces fibrogenic and oxidative changes in liver than melatonin co-treatment.

Therapeutic effects of melatonin (MEL) in targeting CCl4 -induced liver fibrosis has been widely known, but there is no study comparing oxidative and fibrogenic changes in co- and post-treatment of MEL with CCl4 , which was further aimed in this experiment. Male SD rats were injected with CCl4 (1 mL/kg/i.p./daily) dissolved 1:1 in olive oil for 1 month. Some animals received MEL (20 mg/kg/i.p./daily) diluted in 1 mL PBS in combination with CCl4 (co-treatment), and some rats were treated with MEL, beginning with injection of the last dose of CCl4 for one month (post-treatment). The groups were control, CCl4 , CCl4 -co vehicle, CCl4 -post vehicle, post-CCl4 , MEL co-treatment, and MEL post-treatment. MEL post-treatment group showed significantly lower lipid deposition, serum malondialdehyde (MDA), serum alanine aminotransferase (ALT), and liver hydroxyproline. This group also had low expressions of Bax and transforming growth factor-ß1 (TGF-ß1). MEL post-treatment group revealed higher sera levels of albumin, superoxide dismutase (SOD) and glutathione peroxidase (GPx). Expression levels of metalloproteinase-13 (MMP-13) and Bcl2 was also higher in this group (P ≤ 0.05 vs co-treatment). Results of the present study indicated that MEL post-treatment is more powerful in reduction of CCl4 -induced liver fibrosis through reduction of oxidative stress and maintenance of matrix balance.

Investigating the effect of Nigella sativa on the testicular function of first-generation offspring of mice treated with titanium oxide nanoparticles.

Objective: Nanoparticles include primary particles with at least one of their dimensions being less than 100 nm. The goal of this research was to determine the possible protective role of Nigella sativa (NS) against toxic effects mediated by titanium oxide nanoparticle (TNP). Materials and Methods: 30 adult mice (10 males and 20 females) were used. After mating, the pregnant female mice were randomly divided into 4 study groups (n=5 mice in each group). From the 13th day of gestation until delivery, the mice were given TNP and NS. After delivery, 10 newborn male mice were selected from each group and kept under standard conditions until puberty according to the previous grouping (4 groups). The epididymis of each mouse was removed and the sperm was collected for the evaluation of in vitro fertilization and testis for histopathology and spermatogenesis of in vitro fertilization of first-generation mice. Results: No significant difference was observed between the NS group and the control group (p>0.05). In the TNP, a degree of epithelial lysis and a significant decrease in sperm motility was observed (p<0.05) compared with the control group. In the TNP and NS group, NS had an ameliorating effect on TNP-induced testicular germ cell damage (p<0.05). Conclusion: In the present study, it was found that NS had no destructive effect on the germinal epithelium. However, NS had an ameliorating effect on TNP-induced testicular germ cell damage in mice.

A case report of greater saphenous vein thrombosis in a patient with coronavirus (COVID-19) infection.

In December 2019, the World Health Organization (WHO) announced a series of pneumonia cases caused by an unknown origin, discovered in Wuhan, China. A dangerous virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a disease named acute respiratory syndrome, which was later popularly called coronavirus infection (COVID-19). Patients with acute COVID-19 are at high risk of thrombosis in various blood vessels due to hypercoagulability, blood stasis, and endothelial damage. In this study, we present a case report of a patient with COVID-19, who was hospitalized in one of the hospitals in Sanandaj, Iran. There were symptoms of fever, chills, muscle aches, cough, and tachycardia. Laboratory tests showed high levels of CRP, ESR, Ferritin CLIA, LDH and D-Dimer in this patient. Doppler ultrasound of the patient also revealed an abnormal finding, thrombosis in the right greater saphenous vein. This suggests that COVID-19 may lead to other negative effects through damage to blood vessels.

Increased pulmonary embolism in patients with COVID-19: a case series and literature review.

There is some recent evidence that the coronavirus disease 2019 (COVID-19) increases the risk of venous thromboembolism by creating a prothrombotic state. COVID-19 and pulmonary embolism (PE) are both associated with tachypnoea, hypoxemia, dyspnoea, and increased D-dimer. Diagnosis of pulmonary embolism in a patient with COVID-19 compared to an individual without it, using the conventional clinical and biochemical evidence is challenging and somehow impossible. In this study, we reported four male cases affected by COVID-19 and admitted to hospitals in Sanandaj, Iran. The patients were all older adults (ranging between 56 and 95 years of age). Fever, chills, muscle pain, and cough were evident in all the cases. Red blood cell levels were low, and pulmonary embolism was clearly detected on spiral computed tomographic (CT) angiography of the pulmonary circulation of all patients. These cases demonstrated that COVID-19 may lead to pulmonary embolism by causing blood coagulation problems. As COVID-19 continues to cause considerable mortality, more information is emerging which reveals its complicated pathogenicity. In the meantime, venous thromboembolism remains an uncommon finding in patients with COVID-19. It is essential that health care providers perform the necessary diagnostic evaluations and provide appropriate treatment for patients.

Metformin Therapy Attenuates Pro-inflammatory Microglia by Inhibiting NF-κB in Cuprizone Demyelinating Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic disorder characterized by reactive gliosis, inflammation, and demyelination. Microglia plays a crucial role in the pathogenesis of MS and has the dynamic plasticity to polarize between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes. Metformin, a glucose-lowering drug, attenuates inflammatory responses by activating adenosine monophosphate protein kinase (AMPK) which suppresses nuclear factor kappa B (NF-κB). In this study, we indirectly investigated whether metformin therapy would regulate microglia activity in the cuprizone (CPZ)-induced demyelination mouse model of MS via measuring the markers associated with pro- and anti-inflammatory microglia. Evaluation of myelin by luxol fast blue staining revealed that metformin treatment (CPZ + Met) diminished demyelination, in comparison to CPZ mice. In addition, metformin therapy significantly alleviated reactive microgliosis and astrogliosis in the corpus callosum, as measured by Iba-1 and GFAP staining. Moreover, metformin treatment significantly downregulated the expression of pro-inflammatory associated genes (iNOS, H2-Aa, and TNF-α) in the corpus callosum, whereas expression of anti-inflammatory markers (Arg1, Mrc1, and IL10) was not promoted, compared to CPZ mice. Furthermore, protein levels of iNOS (pro-inflammatory marker) were significantly decreased in the metformin group, while those of Trem2 (anti-inflammatory marker) were increased. In addition, metformin significantly increased AMPK activation in CPZ mice. Finally, metformin administration significantly reduced the activation level of NF-κB in CPZ mice. In summary, our data revealed that metformin attenuated pro-inflammatory microglia markers through suppressing NF-κB activity. The positive effects of metformin on microglia and remyelination suggest that it could be used as a promising candidate to lessen the incidence of inflammatory neurodegenerative diseases such as MS.

Acute Ischemic and Hemorrhagic Stroke and COVID-19: Case Series.

The coronavirus COVID-19 pandemic is the defining global health crisis of our time and the greatest challenge we have faced since the World Wars; it can attack several systems in the body and has high complications and mortality. COVID-19 can cause venous and arterial thromboembolism due to immobility, high inflammation, extensive intravascular blood coagulation, and hypoxia. In this study, we report 5 cases of adults with COVID-19, hospitalized in Tohid Hospital, Sanandaj, Iran. Three patients were male and two were female. The youngest patient was 20 years old and the oldest was 55 years old. All patients had at least one family member with coronavirus. Fever, chills, muscular pain, cough, and tachypnea were present in all patients. Red blood cell (RBC) was observed in all patients at a low level. Computed tomography (CT) scans of all patients showed abnormal findings in different areas of the brain. These cases indicate that COVID-19 may damage blood vessels in the brain and lead to stroke.

The effect of adding Rosmarinic and Ascorbic acids to vitrification media on fertilization rate of the mice oocyte: An experimental study.

BACKGROUND: Oocytes vitrification is a pivotal step for the widespread and safekeeping of animal genetic resources. Oocytes endure notable morphological and functional damage during cryopreservation. Oxidative stress is one of the adverse effects that vitrification imparts on oocytes. OBJECTIVE: In the present study, we investigated the antioxidant effect of Rosmarinic and Ascorbic acids on the quality and fertilizing ability of frozen-thawed mice oocyte. MATERIALS AND METHODS: In this experimental study, germinal vesicle oocytes obtained from two-months-old (30-40gr) NMRI mice were randomly divided into four groups. The basic cryoprotectants were 7.5% (v/v) ethylene glycol+7.5% (v/v) Propanediol as an equilibration media. Vitrification medium contained 15% (v/v) ethylene glycol+15% (v/v) propanediol, and 0.5 M sucrose. In the first group (Control), nothing was added to vitrification mediums, whereas, in the second and third groups, 0.5 mmol/L of Ascorbic acid and 105 µmol/L of Rosmarinic acid were added into vitrification medium, respectively. The cumulative concentration of Rosmarinic and Ascorbic acids were added to group 4. Mouse oocytes were vitrified and preserved for one month. The thawed oocytes were transferred into the α-MEM medium (Alpha Minimum Essential Medium) and maintained in this medium for 24 hr, to be matured and reach the metaphase II stage. RESULTS: The addition of Rosmarinic and Ascorbic acids to the vitrification solution improved the survival, maturation of Germinal vesicles, fertilization rate, and finally development to 4-cell stage. Maturation rates to 4-cell stage for Ascorbic acid, Rosmarinic acid, and both of them together were 80%, 80.76%, and 86.61%, respectively. CONCLUSION: These results indicate that the addition of a cumulative concentration of 0.5 mmol/L Ascorbic acid and 105 µmol/L of Rosmarinic acid to the cryopreservation solution for the mouse immature oocytes would be of significant value (p < 0.01).

Combined Effect of Retinoic Acid and Basic Fibroblast Growth Factor on Maturation of Mouse Oocyte and Subsequent Fertilization and Development.

BACKGROUND: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. MATERIALS AND METHODS: In this experimental study, germinal vesicle (GV) oocytes obtained from two-months-old NMRI mice were randomly divided into control, sham and three experimental groups. The basic culture medium was α-MEM supplemented with 10% fetal bovine serum (FBS), 50 mg/l streptomycin, 60 mg/l penicillin and 10 ng/ ml epidermal growth factors. Each of the experimental groups received one of the following treatments: RA (2 µM), bFGF (20 ng/ml) or combination of RA and bFGF with the indicated concentrations. After 24 hours, capacitated spermatozoa were added to in vitro matured oocytes. Five hours later, the oocytes were cultured in fresh droplets of M2 medium for 24 hours and assessed for cleavage to the two-cells stage. RESULTS: As compared with the control group, the rate of maturation was significantly increased in the RA (P<0.001) and bFGF+RA (P<0.02) groups with 58 ± 10 and 57 ± 3.46, respectively. The rate of maturation was significant in the RA (P<0.02) and bFGF+RA (P<0.03) groups, in comparison with the bFGF group. The bFGF+RA group had higher rate (83 ± 1.52) of two-cells development, than control (33 ± 1, P<0.001). CONCLUSION: Our findings showed beneficial effects of 2 µM RA and 20 ng/ml bFGF combination on mouse oocyte IVM.

In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells.

OBJECTIVE: Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy. MATERIALS AND METHODS: This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA. RESULTS: The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticß-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells. CONCLUSION: Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical ap- plications.

Intravenous transplantation of very small embryonic like stem cells in treatment of diabetes mellitus.

BACKGROUND: Diabetes Mellitus (DM), simply known as diabetes, refers to a group of metabolic diseases in which there are high blood sugar levels over a prolonged period. In this study, the feasibility and safety of intravenous transplantation of Very Small Embryonic Like stem cells (VSELs) were investigated for diabetes repair, and finally the migration and distribution of these cells in hosts were observed. METHODS: Mouse bone marrow VSELs were isolated by Fluorescent Activating Cell Sorting (FACS) method by using fluorescent antibodies against CD45, CXCR4 and Sca1 markers. Sorted cells were analyzed for expression of oct4 and SSEA1 markers with immunocytochemistry staining method. To determine multilineage differentiation, sorted cells were differentiated to Schwann, osteocyte and beta cells. Ten days after the establishment of a mouse model of pancreas necrosis, DiI-labeled VSELs were injected into these mice via tail vein. Pancreases were harvested 4 weeks after transplantation and the sections of these tissues were observed under fluorescent microscope. RESULTS: It was proved that CD45-, CXCR4+, and Sca1+ sorted cells express oct4 and SSEA1. Our results revealed that intravenously implanted VSELs could migrate into the pancreas of hosts and survive in the diabetic pancreas. In treated groups, blood glucose decreased significantly for at least two month and the weights of mice increased gradually. CONCLUSION: This study provides a strategy for using VSELs for curing diabetes and other regenerative diseases, and the strategy is considered an alternative for other stem cell types.

In vitro Therapeutic Effects of Low Level Laser at mRNA Level on the Release of Skin Growth Factors from Fibroblasts in Diabetic Mice.

BACKGROUND: Numerous in vitro reports suggest that Low Level Laser Therapy (LLLT) affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light (which is more useful in clinic because of its higher penetration) on secretion of Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF), as important growth factors in wound healing. METHODS: Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT (energy density of 1 J/cm (2)) using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time poly-merase chain reaction. RESULTS: Diabetic irradiated group showed significant increase in FGF (p = 0.017) expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. CONCLUSION: These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts.