Melatonin pretreated blastocysts along with calcitonin administration improved implantation by upregulation of heparin binding-epidermal growth factor expression in murine endometrium

Objective: Implantation failure is an obstacle in assisted reproduction techniques (ART). Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor (HB-EGF) expression in murine endometrium. Materials and Methods: In this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in GTM medium with or without 10-9 M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal (IP) injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P<0.05 was considered statistically significant. Results: Melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein (P<0.001) in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels (P<0.001). Compared with the control group (2.6 ± 0.5), the average number of implantation sites in the melatonin group (4.6 ± 0.5, P<0.05) and calcitonin group (7 ± 1, P<0.001) significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group (8.6 ± 0.5, P<0.001). Calcitonin significantly enhanced calcitonin receptor mRNA (P<0.001) and protein (P<0.05) in the uteri of naturally pregnant and pseudo-pregnant mice. Conclusion: Melatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudopregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice

Melatonin pretreated blastocysts along with calcitonin administration improved implantation by upregulation of heparin binding-epidermal growth factor expression in murine endometrium

Objective: Implantation failure is an obstacle in assisted reproduction techniques (ART). Calcitonin is a molecules involved in uterine receptivity and embryo implantation. Melatonin can promote embryo quality and improve implantation. This study examines the effect of pretreatment of blastocysts with melatonin and calcitonin on heparin binding-epidermal growth factor (HB-EGF) expression in murine endometrium. Materials and Methods: In this experimental study, we collected 2-cell embryos from the oviducts of 1.5 day pregnant NMRI mice. Embryos were cultured to the blastocyst in GTM medium with or without 10-9 M melatonin. Pregnant and pseudo-pregnant mice received intraperitoneal (IP) injections of 2 IU calcitonin. After 24 hours, we transferred the cultured blastocysts into the uteri of pseudo-pregnant mice. Two days later, implantation sites were counted and we assessed the levels of HB-EGF mRNA and protein in the uteri of naturally pregnant and pseudo-pregnant mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Statistical analysis was performed with one-way ANOVA followed by the Tukey post hoc test. P&lt;0.05 was considered statistically significant. Results: Melatonin pretreatment of blastocysts along with calcitonin administration significantly increased HB-EGF mRNA and protein (P&lt;0.001) in the endometrium of pseudo-pregnant mice. Administration of calcitonin in naturally pregnant mice significantly increased HB-EGF mRNA and protein levels (P&lt;0.001). Compared with the control group (2.6 ± 0.5), the average number of implantation sites in the melatonin group (4.6 ± 0.5, P&lt;0.05) and calcitonin group (7 ± 1, P&lt;0.001) significantly increased. There was a significant increase in implantation sites in the combined melatonin and calcitonin group (8.6 ± 0.5, P&lt;0.001). Calcitonin significantly enhanced calcitonin receptor mRNA (P&lt;0.001) and protein (P&lt;0.05) in the uteri of naturally pregnant and pseudo-pregnant mice. Conclusion: Melatonin pretreated blastocysts along with calcitonin increased HB-EGF expression in the uteri of pseudopregnant mice. Calcitonin administration upregulated HB-EGF in uteri of naturally pregnant mice

Preconditioning of mesenchymal stem cells with non-toxic concentration of hydrogen peroxide against oxidative stress induced cell death: The role of hypoxia-inducible factor-1

Purpose: To investigate the protective effect of preconditioning with non-toxic dose of hydrogenperoxide (H2O2) as a possible cell signaling molecule, against cell death induced by toxicconcentration of H2O2 or by serum deprivation in human Wharton’s jelly-derived mesenchymalstem cells (HWJ-MSCs) and underlying mechanisms.Methods: HWJ-MSCs were isolated and identified using flow cytometry. After finding non-toxicconcentration of H2O2, cells preconditioning was performed by H2O2 (20 μM) for 12 h and celltolerance against serum deprivation or toxic levels of H2O2 was assayed by MTT test. Effect ofpreconditioning on mRNA and protein expression of Akt-1, Bcl-2 and Bax were examined usingreverse transcription polymerase chain reaction (RT-PCR) and western blotting respectively. Roleof hypoxia-inducible factor (HIF)-1α was explored in presence HIF-1α inhibitor.Results: Preconditioning with 20 μM H2O2 for 12 h was non-toxic and decreased cell deathinduced by oxidative stress and serum deprivation in MSC cultures. However, the increasedtolerance reversed in the presence of inhibitor of HIF-1α. By regards to RT-PCR and westernblotting data, although expression of Akt-1, Bcl-2 and Bax was not change considerably butphosphorylated Akt-1 (pAkt-1) was up regulated after treatment with 20 μM H2O2 compared tocontrol group. Moreover after exposure to 100 μM H2O2, western blotting analysis showed thatcell pretreatment with 20 μM H2O2, decremented Bax/Bcl2 ratio and up-regulated HIF-1α andpAkt-1 compared to the control group.Conclusion: Increased tolerance of H2O2-pretreated cells led to the suggestion that transplantationof H2O2 preconditioned MSCs may improve therapeutic potential of stem cells in cell therapyprocedures

Teucrium polium extract enhances the anti-angiogenesis effect of tranilast on human umbilical vein endothelial cells

Purpose: Angiogenesis plays an important role in numerous pathophysiological events like cancer. As a result of this, tranilast as an anti-fibrotic drug induces the promising antitumor activities through the inhibition of angiogenesis. Further, Teucrium polium (TP) is a herbal medicine (family Lamaceae) with antitumor properties. This study was conducted to investigate the combination effects of tranilast and T. polium on human umbilical vein endothelial cells (HUVECs) viability and apoptotic genes expression. Methods: The HUVECs line was treated using different doses of tranilast and T. polium alone or their combination. The cell cytotoxicity was evaluated using MTT and LDH assays; apoptosis was examined using acridine orange/ethidium bromide staining, nitric oxide (NO) production was evaluated using Griess reaction and the expression of BAX and BCL-2 genes were detected using real-time RT-PCR. One-way analysis of variance (ANOVA) test was used to compare the data in different groups. Results: The survival rate of HUVECs was significantly reduced (p<0.05) in a dose dependent manner by tranilast and T. polium. However, T. polium and tranilast combination significantly (p<0.001) reduced cell viability and increased apoptotic cells as compared to each drug alone. Also, HUVECs treated with Tranilast / T. polium combination showed a reduced level of NO as regards to cells exposed only to Tranilast or T. polium (p<0.05). Furthermore, a significant increase in BAX and a decrease in BCL-2 mRNA expression were observed in combination group (p<0.001). Conclusion: T. polium synergistically increased the antiangiogenic effect of tranilast on in vitro angiogenic model of HUVECs

A Laboratory Study on Human Jaw Osteoblastic Stem Cells Culturing

Abstract: Introduction: The goal of this study was to evaluate different methods of cultivating human bone cells. Method: Five periosteal and bone specimens obtained from the human jaw were divided into small pieces in the laboratory. After the addition of trypsin and collagenase enzymes and releasing of cells, three primary periostal, endosteal, and bony chips cells were prepared. After passing the required time for the growth of specimens, lamella were prepared and stained with alkaline phosphatase (ALP) in order to determine ALP positive cells. Results: Mean time of cellular growth was 23 days. Conclusion: Human bone cells have the capability of being cultured under special sterile laboratory conditions and three dimensional culturing of them can be used for reconstruction of maxillofacial region defects. Keywords: Stem Cell Culture, Osteoblast, Human ja

Enhancing the butyrylcholinesterase activity in hek-293 cell line by dual-promoter vector decorated on lipofectamine

Purpose: Human butyrylcholinesterase (BChE) serves as a bio scavenger to counteract organophosphate poisoning. It is also a potential drug candidate in several therapeutic fields. Therefore, in the present study, we constructed a new dual-promoter plasmid consisting of Cytomegalovirus (CMV) and human elongation factor 1α (EF-1α) promoters and transfected that into HEK-293 cells using Lipofectamine to enhance the BChE secretion. Methods: The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellman�s method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector. Results: Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellman�s method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity. Conclusion: The designed construction with CMV and EF-1α promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Largescale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials. © 2020 Mirzaie et al

Hydroalcoholic extract and seed of Foeniculum vulgare improve folliculogenesis and total antioxidant capacity level in F1 female mice offspring

Background: Foeniculum vulgare (fennel) is traditionally suggested for the fertility improvement in Iranian lore due to its antioxidant and phytoestrogen compounds. The present study aimed to investigate the effects of fennel seed and its hydroalcoholic extract on the serum total antioxidant capacity (TAC) and folliculogenesis in offspring exposed to either of the treatments in utero and 56 days after birth (PND 56). Methods: Pregnant NMRI mice were randomly divided into 5 groups of 7: Extract-treated groups received 500 and 1000 mg/kg/day fennel extract (FE), seed-treated groups received 500 and 1000 mg/kg/day fennel seed (FS), and the control group (CTL) received no treatment. The treatments started from pregnancy day 1 and continued until PND 56. Body and right ovary weights and ovary dimensions were recorded. Hematoxylin and eosin stained ovary sections were prepared to calculate the proportion of different follicles. The level of TAC in the serums was also measured by fluorescence recovery after photo bleaching. Results: A marked rise in the body and ovary weights of treated mice was observed compared to the CTL group. The mean number of primordial, primary, pre-antral, and pre-ovulatory follicles as well as corpus luteum size in the treated offspring was significantly higher compared to those of CTL offspring. The atretic follicle number was nonsignificantly lower in either of the treatment groups compared with that in the CTL. However, treatment of animals with 500 mg/kg FE showed a more pronounced effect. Animals in FS500, FE500 and FE1000 groups had a significantly higher level of serum TAC compared to the CTL group. Conclusions: Fennel extract and seed administration in pregnancy and lactation period improve offspring's folliculogenesis. Higher level of TAC in the serum of offspring might have positively altered the folliculogenesis milieu. © 2020 The Author(s)