scholarly journals Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling

2021 ◽  
Vol 22 (21) ◽  
pp. 12060
Author(s):  
Simon Lin ◽  
Subramanya Pandruvada ◽  
Hong Yu
Keyword(s):  
Alkaline Phosphatase ◽  
Vesicle Trafficking ◽  
High Dose ◽  
Mrna Levels ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Smad Signaling ◽  
Murine Bone Marrow ◽  
Sphingosine 1 Phosphate ◽  
Associated Proteins

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 μM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 μM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

2021 ◽  
pp. 1-5
Author(s):  
Kari Hanson ◽  
Carly Isder ◽  
Kristen Shogren ◽  
Anthony L. Mikula ◽  
Lichun Lu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
High Dose ◽  
Inhibitory Effects ◽  
Alizarin Red ◽  
Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

scholarly journals Epigallocatechin-3-Gallate Promotes Osteo-/Odontogenic Differentiation of Stem Cells from the Apical Papilla through Activating the BMP–Smad Signaling Pathway

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1580
Author(s):  
Zeni Liu ◽  
Yuxiu Lin ◽  
Xiaolin Fang ◽  
Jingwen Yang ◽  
Zhi Chen
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Signaling Pathway ◽  
Matrix Protein ◽  
Type I ◽  
Alizarin Red ◽  
Smad Signaling ◽  
Odontogenic Differentiation ◽  
Smad Signaling Pathway ◽  
Apical Papilla

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16–20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP–Smad signaling pathway.

scholarly journals Macromolecule Biomaterials Promote the Osteogenic Differentiation Capacity of Equine Adipose -Derived Mesenchymal Stem Cells

2020 ◽  
Author(s):  
Mohamed I. Elashry ◽  
Nadine Baulig ◽  
Alena-Svenja Wagner ◽  
Michele C. Klymiuk ◽  
Benjamin Kruppke ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Fluid Shear Stress ◽  
Basal Medium ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Alp Activity

Abstract Background: Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. In the present study, the effect of biomaterial bone substitutes on equine adipose derived MSCs morphology, viability, adherence, migration and osteogenic differentiation were investigated. Methods: MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and B30Str biomaterials in osteogenic differentiate medium either under static or mechanical fluid shear stress (FSS) culture conditions. The data were generated by histological means, life cell imaging, cell viability, adherence and migration assays. Osteogenic differentiation was detected by semi-quantification of alkaline phosphatase (ALP) activity, matrix mineralization using Alizarin Red S (ARS) staining and quantification of the osteogenic markers; runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression using RT-qPCR. All data were statistically analyzed using ANOVA. Results: The data revealed that combined mechanical stress with MC but not B30 enhanced MSCs viability and promoted their migration. Combined osteogenic medium with MC, B30 and B30Str increased ALP activity compared to cultivation in basal medium. Osteogenic induction with MC, B30 and B30Str resulted in diffused matrix mineralization by means of ARS. FSS increased the viability in the presence of the osteogenic medium with MC but not B30 or B30Str. FSS enhanced osteogenic differentiation in the presence of B30Str. Upregulation of Runx2 and ALP expression was detected with osteogenic differentiation together with B30 and B30Str regardless of static or FSS culture. Conclusions: Taken together, the data revealed that FSS in conjunction with biomaterials promoted osteogenic differentiation of MSCs. This combination may be considered as a marked improvement for clinical applications to cure bone defects.

scholarly journals Viburnum opulus L. Juice Phenolic Compounds Influence Osteogenic Differentiation in Human Osteosarcoma Saos-2 Cells

2020 ◽  
Vol 21 (14) ◽  
pp. 4909 ◽  
Author(s):  
Małgorzata Zakłos-Szyda ◽  
Adriana Nowak ◽  
Nina Pietrzyk ◽  
Anna Podsędek
Keyword(s):  
Phenolic Compounds ◽  
Osteogenic Differentiation ◽  
Bone Metabolism ◽  
Activity Measurement ◽  
Mrna Levels ◽  
Mineral Density ◽  
Viburnum Opulus ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Bone Mass Loss

Bone mass loss occurs with a decrease in osteoblast proliferation and differentiation, or the enhancement of bone resorption, which further leads to the impairment of bone mineral density and increase in bone fracture. Recent studies suggest that some phenolic compounds found in food play positive role in bone metabolism. High content of phenolic compounds with potential beneficial effects on bone metabolism have been identified in the Viburnum opulus fruit. The aim of the study was to determine the influence of V. opulus fresh juice (FJ) and juice purified by solid phase extraction (PJ) on osteogenesis processes with osteosarcoma Saos-2 cell lines. V. opulus purified juice revealed stronger potential as an inducer of Saos-2 osteogenic differentiation. Saos-2 cells matrix mineralization was evaluated with alkaline phosphatase (ALP) activity measurement and alizarin red S staining. Gene expression analysis showed the elevation of the mRNA levels of Runt-related transcription factor 2 (RUNX2), ALP, collagen type 1 and osteonectin, whereas the nuclear factor-κB ligand and osteoprotegerin ratio (RANKL/OPG) decreased. Furthermore, V. opulus was able to diminish the secretion of pro-inflammatory cytokines Il6 and TNFα, however had no effect on vascular endothelial growth factor (VEGF). It decreased intracellular oxidative stress and induced DNA repair, but had no effect on the growth inhibition of lactic acid beneficial microorganisms.

scholarly journals Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 2175
Author(s):  
Adonis Z. Wu ◽  
Tzu-Lun Ohn ◽  
Ren-Jay Shei ◽  
Huei-Fang Wu ◽  
Yong-Cyuan Chen ◽  
...  
Keyword(s):  
Chromaffin Cells ◽  
Low Dose ◽  
Single Channel ◽  
Cell System ◽  
K Channel ◽  
Lipid Mediator ◽  
Bkca Channel ◽  
High Dose ◽  
Open Probability ◽  
Sphingosine 1 Phosphate

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca2+-activated K+ channel (BKCa) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca2+ imaging, and computational modeling, we evaluated the effects of S1P on the Ca2+-activated K+ currents (IK(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BKCa channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca2+ concentration (Cai) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BKCa was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed IK(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of IK(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced IK(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BKCa conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited IK(Ca) and the permissive role of S1P on the BKCa activity was also effectively observed in the PC12 cell system. The S1P-mediated IK(Ca) stimulation may result from the elevated Cai, whereas the inhibition of BKCa activity by S1P appears to be direct. By the differentiated tailoring BKCa channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.

Hepatotoxic effects of chloroform extract of Artemisia macivera Linn in rats following intraperitoneal administration of different subchronic doses

2010 ◽  
Vol 30 (1) ◽  
pp. 25-33 ◽  
Author(s):  
SE Atawodi ◽  
AC Ene ◽  
DA Ameh
Keyword(s):  
Alkaline Phosphatase ◽  
Total Protein ◽  
Aspartate Aminotransferase ◽  
Alanine Aminotransferase ◽  
Intraperitoneal Administration ◽  
Chloroform Extract ◽  
Hepatic Tissue ◽  
Albino Rats ◽  
High Dose ◽  
Hepatocellular Injury

The possible hepatotoxic effects of chloroform extract of Artemisia maciverae was evaluated biochemically and histologically using male Swiss albino rats, randomly assigned into four groups of 24 animals each. The groups (control, 50, 100 and 200 mg/kg body weight) were treated for 60 days and then monitored for another 30 days before sacrifice. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin (total and direct), total protein and albumin were assessed colorimetrically, while tissue specimens were subjected to histological examination following standard hematoxyline-eosin staining techniques. After 1 week of treatment, the extract caused statistically significant elevation in levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and bilirubin (total and direct), while there was significant (p < 0.05) decrease in the levels of serum total protein and albumin at the onset of treatment when compared with the control. These abnormalities in the levels of serum biochemical parameters were spontaneously corrected within 2 weeks of treatment. Similarly, histological assessment showed severe hepatic tissue injuries after 1 week, but these organs recovered spontaneously by the second week of treatment. The results indicate that long-term exposure to therapeutic doses of chloroform extract of A maciverae is relatively safe, but high dose exposure may result in hepatocellular injury.

scholarly journals GnRH Receptor Gene Expression in the Developing Rat Hippocampus: Transcriptional Regulation and Potential Roles in Neuronal Plasticity

Endocrinology ◽  
2010 ◽  
Vol 152 (2) ◽  
pp. 568-580 ◽  
Author(s):  
Anne-Laure Schang ◽  
Valérie Ngô-Muller ◽  
Christian Bleux ◽  
Anne Granger ◽  
Marie-Claude Chenut ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Neuronal Plasticity ◽  
Gnrh Receptor ◽  
Lateral Septum ◽  
Rat Hippocampus ◽  
Marker Genes ◽  
Mrna Levels ◽  
Primary Role ◽  
Placental Alkaline Phosphatase ◽  
Human Placental Alkaline Phosphatase

Abstract In the pituitary of mammals, the GnRH receptor (GnRHR) plays a primary role in the control of reproductive function. It is further expressed in the hippocampus, where its function, however, is not well defined. By quantitative RT-PCR analyses, we demonstrate herein that the onset of GnRHR gene (Gnrhr) expression in the rat hippocampus was unexpectedly delayed as compared to the pituitary and only occurred after birth. Using a previously described transgenic mouse model bearing the human placental alkaline phosphatase reporter gene under the control of the rat Gnrhr promoter, we established a positive correlation between the temporal pattern of Gnrhr mRNA levels and promoter activity in the hippocampal formation. The gradual appearance of human placental alkaline phosphatase transgene expression occurred simultaneously in the hippocampus and interconnected structures such as the lateral septum and the amygdala, coinciding with the establishment of hippocampo-septal projections. Analysis of transcription factors together with transient transfection assays in hippocampal neurons indicated that the combinatorial code governing the hippocampus-specific expression of the Gnrhr is distinct from the pituitary, likely involving transactivating factors such as NUR77, cyclic AMP response element binding protein, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene homolog. A silencing transcription factor acting via the -3255/-1135 promoter region of the Gnrhr may be responsible for the transcriptional repression observed around birth. Finally, GnRH directly stimulated via activation of its receptor the expression of several marker genes of neuronal plasticity such as Egr1, synaptophysin, and spinophilin in hippocampal primary cultures, suggesting a role for GnRHR in neuronal plasticity. Further characterization of these mechanisms may help unravel important functions of GnRH/GnRHR signaling in the brain.

scholarly journals Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mariane Beatriz Sordi ◽  
Raissa Borges Curtarelli ◽  
Izabella Thaís da Silva ◽  
Gislaine Fongaro ◽  
Cesar Augusto Magalhães Benfatti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Culture Media ◽  
Deciduous Teeth ◽  
Free Calcium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

scholarly journals Paradoxical Regulation of Biotin Utilization in Brain and Liver and Implications for Inherited Multiple Carboxylase Deficiency

2004 ◽  
Vol 279 (50) ◽  
pp. 52312-52318 ◽  
Author(s):  
Diana Pacheco-Alvarez ◽  
R. Sergio Solórzano-Vargas ◽  
Roy A. Gravel ◽  
Rafael Cervantes-Roldán ◽  
Antonio Velázquez ◽  
...  
Keyword(s):  
High Dose ◽  
Mrna Levels ◽  
Holocarboxylase Synthetase ◽  
Biotin Deficiency ◽  
Multiple Carboxylase Deficiency ◽  
Regulatory Impact ◽  
Normal Metabolism ◽  
Liver And Kidney ◽  
Essential Function ◽  
The Brain

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of five carboxylases in human cells, and mutations of HCS cause multiple carboxylase deficiency (MCD). Although HCS also participates in the regulation of its own mRNA levels, the relevance of this mechanism to normal metabolism or to the MCD phenotype is not known. In this study, we show that mRNA levels of enzymes involved in biotin utilization, including HCS, are down-regulated during biotin deficiency in liver while remaining constitutively expressed in brain. We propose that this mechanism of regulation is aimed at sparing the essential function of biotin in the brain at the expense of organs such as liver and kidney during biotin deprivation. In MCD, it is possible that some of the manifestations of the disease may be associated with down-regulation of biotin utilization in liver because of the impaired activity of HCS and that high dose biotin therapy may in part be important to overcoming the adverse regulatory impact in such organs.

Effect of gallic acid on the reproduction of adolescent male Brandt’s vole (Lasiopodomys brandtii)

2021 ◽  
Author(s):  
Xin Dai ◽  
Xiao-Feng Sun ◽  
Ai-Qin Wang ◽  
Wanhong Wei ◽  
Sheng-Mei Yang
Keyword(s):  
Gallic Acid ◽  
Low Dose ◽  
Regulatory Protein ◽  
Antioxidant Properties ◽  
Adolescent Male ◽  
Seminiferous Tubules ◽  
High Dose ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Lasiopodomys Brandtii

Gallic acid (GA), a phenol that is present in various plants, potentially contains antioxidant properties. This study aimed to investigate the effects of GA on the reproduction of adolescent male Brandt’s voles (Lasiopodomys brandtii (Radde, 1861)). Antioxidant levels and apoptosis in the testis, as well as reproductive physiology, were evaluated in adolescent males treated with GA. The results showed that a low dose of GA enhanced relative epididymis weight and the sperm density in the epididymis, increased the mRNA levels of steroidogenic acute regulatory protein in the testis, and reduced the percentages of abnormal and dead sperm. In addition, a low dose of GA significantly increased the levels of superoxide dismutase, catalase, and glutathione peroxidase, and decreased the level of malondialdehyde in the testis, as well as the mRNA and protein levels of the apoptosis related gene, caspase-3. However, a high dose of GA sharply reduced the average diameter of the seminiferous tubules compared to a low dose. Collectively, these findings demonstrate that GA treatment during puberty affects the reproductive responses of male Brandt’s voles in a dose-dependent manner by regulating antioxidant levels and apoptosis.

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