sincalide and Hypoxia

Thin endometrium remains a severe clinical challenge with no effective therapy to date. We aimed at exploring the role and molecular mechanism of human umbilical cord mesenchymal stem cell- (hucMSC-) derived exosomes (hucMSC-Ex) in repairing hypoxic injury of endometrial epithelial cells (EECs).. Exosomes were harvested from the conditioned medium of hucMSC and characterized using western blot, transmission electron microscopy (TEM), flow cytometry, and nanoparticle tracking analysis (NTA). EECs were subjected to hypoxic conditions before cocultured with hucMSC-Ex. Cell viability, apoptosis, and migration were determined with CCK-8, flow cytometry, and wound healing assay, respectively. Apoptosis/EMT-related proteins were detected by western blot. The miRNA profiling was determined by RNA sequencing. The expression of miR-663a and CDKN2A was measured by qRT-PCR. MiR-663a in EECs was overexpressed by transfecting with miR-663a mimics.. Mesenchymal stem cells (MSCs) markers CD73, CD90, and CD106 were positively expressed in hucMSCs. Exosome isolated from hucMSC expressed CD63 and TSG101, and were 100-150 nm in diameter. HucMSC-Ex promoted cell proliferation inhibited by hypoxia. And hucMSC-Ex also inhibited hypoxia-induced apoptosis, migration, and EMT of EECs by upregulating the expression of Bcl-2 and E-cadherin and downregulating Bax and N-cadherin levels. Further, bioinformatics research found that hucMSC-Ex coculture can significantly upregulate the expression of miR-663a and decrease the expression of CDKN2A in hypoxia-induced EECs. Furthermore, miR-663a overexpression inhibited CDKN2A expression and increased the expression of Bcl-2 and E-cadherin in hypoxia-induced EECs.. HucMSC-Ex promoted cell proliferation, inhibited cell apoptosis, migration, and EMT in hypoxia-induced EECs, thereby alleviating hypoxia-induced EECs injury, which may be related to its regulation of miR-663a/CDKN2A expression. Our study indicated that hucMSC-Ex might benefit for repairing thin endometrium.

Topics: bcl-2-Associated X Protein; Cadherins; Culture Media, Conditioned; Cyclin-Dependent Kinase Inhibitor p16; Endometrium; Epithelial Cells; Exosomes; Female; Humans; Hypoxia; Mesenchymal Stem Cells; MicroRNAs; Sincalide; Umbilical Cord

This study aimed to investigate the effect and mechanism of miR-26a-5p on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R).After construction of an H/R model in rat cardiomyocyte H9c2 cells, miR-26a-5p in the cells was interfered with (cells transfected with miR-26a-5p inhibitor) or overexpressed (cells transfected with a miR-26a-5p mimics). The viability and the apoptosis rate of cells in each group were detected using CCK-8 and flow cytometry; the relationship between miR-26a-5p and WNT5A was verified by a dual-luciferase reporter assay; the expression of miR-26a-5p, WNT5A, cleavedcaspase3 and Wnt/β-catenin signaling pathway-related proteins in each group was detected using qRT-PCR or Western blot; LDH release, SOD, and GSH-PX activities in each group were detected by kit.In the H/R group, the expression level of miR-26a-5p was significantly decreased, whereas the expression level of WNT5A was significantly increased. The activity of the Wnt/β-catenin signaling pathway was up-regulated; the level of LDH released was significantly increased; and activities of SOD and GSH-PX were significantly decreased. The aforementioned changes resulted in decreased cell activity and increased apoptosis rate. The overexpression of miR-26a-5p could reduce the expression level of WNT5A, the activity of the Wnt/β-catenin signaling pathway, and the apoptosis rate and restore the cell viability.These results suggest that miR-26a-5p can target WNT5A and thus, inhibit the Wnt/β-catenin signaling pathway activity, inhibiting H/R-induced cardiomyocyte injury and apoptosis.

Topics: Acute Disease; Animals; Apoptosis; Cell Survival; Flow Cytometry; Glutathione Peroxidase; Hypoxia; MicroRNAs; Models, Animal; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Protective Factors; Rats; Sincalide; Superoxide Dismutase; Transfection; Up-Regulation; Wnt Signaling Pathway; Wnt-5a Protein

We sought to determine the expression levels of hypoxia-inducible factor-1α (HIF-1α) in the bone marrow chronic myelogenous leukemia (CML) patients. We also tried to determine the roles HIF-1α in the proliferation of CML cells by small interfering RNA (siRNA) knockdown. Real-time PCR was performed to determine the expression levels of HIF-1α in the bone marrows of CML patients and healthy volunteers. HIF-1α knockdown by siRNA in K562 cells was confirmed by RT-PCR. Proliferation and colony formation of the treated cells were determined by CCK8 after HIF-1α knockdown. RT-PCR and western blotting were performed to detect mRNA and protein levels of p21 and p53 in K562 cells. HIF-1α mRNA expression in the bone marrow of CML patients was significantly higher than that in the control, which was statistically significant (P < 0.05). HIF-1α knockdown dramatically reduced the proliferation of K562 cells, which was also statistically significant (P < 0.05). HIF-1α knockdown markedly reduced the colony formation ability of K562 cells, which was also statistically significant (P < 0.05). The mRNA and protein expression of p21 were significantly reduced in K562 cell after HIF-1α knockdown with affecting the mRNA and protein levels of p53. HIF-α promotes chronic CML cell proliferation by up-regulating p21 expression.

Topics: Apoptosis; Bone Marrow; Cell Line, Tumor; Cell Proliferation; Cholecystokinin; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Peptide Fragments; RNA, Small Interfering; Tumor Suppressor Protein p53; Up-Regulation

The aim of the study was to investigate whether repetitive/temporal hypoxia up-regulated P-glycoprotein (P-gp) in cultured rat brain microvascular endothelial cells (rBMECs). Cultured rBMECs were used as in vitro blood brain barrier (BBB) model. Cells reached confluence were subjected to temporal hypoxic exposure. Under free-glucose cultured medium, the cells were covered by sterile paraffin oil for 15 min, inducing temporal hypoxic exposure. The hypoxic-exposure was carried out once every day up to 8 days, leading to the repetitive/temporal hypoxia in rBMECs. The cell viability was tested using CCK-8 kit, function and levels of P-gp in the cells were measured using rhodamine 123 uptake and western blot, respectively. It was found that 8-temporal hypoxic exposure induced 1.6-fold increase of P-gp level in cells, accompanied by decrease of cellular accumulation of rhodamine 123. Cellular accumulation of phenobarbital was also decreased. These findings indicated that repetitive/temporal hypoxia may be one of the factors resulting in P-gp overexpression in refractory epilepsy.

Topics: Animals; Animals, Newborn; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Brain; Cell Survival; Cells, Cultured; Cyclosporine; Endothelial Cells; Endothelium, Vascular; Enzyme Inhibitors; Gene Expression Regulation; Hypoxia; Rats; Rats, Sprague-Dawley; Rhodamine 123; Sincalide; Time Factors

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for cholecystokinin octapeptide sulfate (CCK-8S) has been developed using N-terminal specific antibody for CCK-8S. In this assay CCK-8S coupled with poly-L-Glu (CCK-poly-Glu), which is adsorbed on a solid phase, competes with CCK-8S for the binding sites of rabbit anti-CCK antibody, and the complex of the immobilized antibody and CCK-poly-Glu is measured using goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is less than 24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2-6.2% and the interassay coefficient of variation is 5.9-8.5%. This assay is sensitive enough to detect 9 pg of CCK-8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6-hydroxy-dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.

Topics: Animals; Animals, Newborn; Antibody Specificity; Brain; Brain Chemistry; Enzyme-Linked Immunosorbent Assay; Hydroxydopamines; Hypoxia; Immune Sera; Kinetics; Oxidopamine; Peptide Fragments; Radioimmunoassay; Rats; Rats, Inbred Strains; Sincalide; Tissue Distribution