Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes.

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and âˆ¼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 µM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 µM) and mGPDH (Ki 54.70 µM) activities. siRNAs transfection showed ∼50 % and âˆ¼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Enhanced anti-biofilm and anti-caries potential of arginine combined with calcium glycerophosphate and fluoride.

OBJECTIVE: The aim of this work was to evaluate the antibiofilm and anticaries properties of the association of arginine (Arg) with calcium glycerophosphate (CaGP) and fluoride (F). METHODS: An active attachment, polymicrobial biofilm model obtained from saliva and bovine teeth discs were used. After the initial biofilm growth period, the enamel discs were transferred to culture medium. The treatment solutions were added to the culture media to achieve the desired final concentration. The following groups were used: negative control (Control); F (110 ppm F); CaGP (0.05 %); Arg (0.8 %) and their associations (F + CaGP; Arg + F; Arg + CaGP; Arg +F + CaGP). The following analyses were carried out: bacterial viability (total bacteria, aciduric bacteria and mutans streptococci), pH assessment of the spent culture medium, dry weight quantification, evaluation of surface hardness loss (%SH) and subsurface mineral content. Normality and homoscedasticity were tested (Shapiro-Wilk and Levene's test) and the following tests were applied: two-way ANOVA (acidogenicity), Kruskall-Wallis (microbial viability) and one way ANOVA (dry weight, %SH, mineral content). RESULTS: The association Arg + F + CaGP resulted in the lowest surface hardness loss in tooth enamel (-10.9 ± 2.3 %; p < 0.05). Arg +F + CaGP exhibited highest values of subsurface mineral content (10.1 ± 2.9 gHAP/cm3) in comparison to Control and F (p < 0.05). In comparison to Control and F, Arg +F + CaGP promoted the highest reduction in aciduric bacteria and mutans streptococci (5.7 ± 0.4; 4.4 ± 0.5 logCFU/mL, p < 0.05). CONCLUSIONS: The Arg-F-Ca association demonstrated to be the most effective combination in protecting the loss of surface hardness and subsurface mineral content, in addition to controlling important virulence factors of the cariogenic biofilm. CLINICAL SIGNIFICANCE: Our findings provide evidence that the Arg-F-Ca association showed an additive effect, particularly concerning protection against enamel demineralization. The combination of these compounds may be a strategy for patients at high risk of caries.

A role for sirtuin 1 in FGF23 activation following ß-glycerophosphate treatment.

Phosphate homeostasis is vital for many biological processes and disruptions in circulating levels can be detrimental. While the mechanisms behind FGF23 regulation have been regularly studied, the role of extracellular phosphate sensing and its impact on fibroblast growth factor 23 (FGF23) expression remains unclear. This study aimed to investigate the involvement of reactive oxygen species (ROS), silent information regulator 1 (SIRT1), and Hairy and Enhancer of Split-1 (HES1) in regulating FGF23 in FGF23 expressing MC3T3-E1 cells. MC3T3-E1 cells treated with ß-glycerophosphate (BGP) resulted in increased Fgf23 expression. Inhibition of ROS formation by inhibition of NADPH oxidase, which is essential for ROS production, did not affect this response to BGP, suggesting ROS is not involved in this process. Moreover, treatment with tert-butyl hydroperoxide (TBHP), a ROS-inducing agent, did not increase Fgf23 expression. This suggests that ROS machinery is not involved in FGF23 stimulation as previously suggested. Nonetheless, inhibition of SIRT1 using Ex527 eliminated the Fgf23 response to BGP, indicating its involvement in FGF23 regulation after BGP treatment. Indeed, activation of SIRT1 using SRT1720 increased Fgf23 expression. Moreover, transcription factor Hes1 was upregulated by BGP treatment, which was diminished when cells were treated with Ex527 implying it is also regulated through SIRT1. These findings suggest the existence of an upstream SIRT1-HES1 axis in the regulation of FGF23 by phosphate, though we were unable to find a role for ROS in this process. Further research should provide insights into phosphate homeostasis and potential therapeutic targets for phosphate-related disorders.

Defining the Most Potent Osteoinductive Culture Conditions for MC3T3-E1 Cells Reveals No Implication of Oxidative Stress or Energy Metabolism.

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)'s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and ß-glycerophosphate (ßGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 µg.mL-1/2 mM Asc/ßGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.

Mir-195-5p targets Smad7 regulation of the Wnt/ß-catenin pathway to promote osteogenic differentiation of vascular smooth muscle cells.

In this study, we sought to investigate the mechanisms of action of miR-195-5p in the osteogenic differentiation of vascular smooth muscle cells (VSMCs), and thereby provide novel insights and a reference for the targeted therapy of arterial media calcification. VSMC differentiation was induced using sodium ß-glycerophosphate, and we investigated the effects of transfecting cells with miR-195-5p mimics, vectors overexpressing Smad7, and the Wnt/ß-catenin pathway inhibitor (KYA1797K) on VSMC differentiation by determining cell viability and apoptosis, and the mRNA and protein expression of factors associated with osteogenic differentiation and the Wnt/ß-catenin pathway. The results revealed that miR-195-5p mimics enhanced the osteogenic differentiation of VSMCs induced by ß-glycerophosphate, whereas the overexpression of Smad7 reversed this phenomenon. In addition, KYA1797K was found to promote the effects of Smad7 overexpression. In conclusion, by targeting, Smad7, miR-195-5p promotes the Wnt/ß-catenin pathway. and thus the osteogenic differentiation of VSMCs. These findings will provide a reference for elucidating the mechanisms whereby miR-195-5p regulates osteogenic differentiation.

Differing responses of osteogenic cell lines to ß-glycerophosphate.

Ascorbic acid (Asc), dexamethasone (Dex) and ß-glycerophosphate (ß-Gly) are commonly used to promote osteogenic behaviour by osteoblasts in vitro. According to the literature, several osteosarcoma cells lines appear to respond differently to the latter with regards to proliferation kinetics and osteogenic gene transcription. Unsurprisingly, these differences lead to contrasting data between publications that necessitate preliminary studies to confirm the phenotype of the chosen osteosarcoma cell line in the presence of Asc, Dex and ß-Gly. The present study exposed Saos-2 cells to different combinations of Asc, Dex and ß-Gly for 14 days and compared the response with immortalised human mesenchymal stromal/stem cells (MSCs). Cell numbers, cytotoxicity, mineralised matrix deposition and cell proliferation were analysed to assess osteoblast-like behaviour in the presence of Asc, Dex and ß-Gly. Additionally, gene expression of runt-related transcription factor 2 (RUNX2); osteocalcin (OCN); alkaline phosphatase (ALP); phosphate regulating endopeptidase homolog X-linked (PHEX); marker of proliferation MKI67 and proliferating cell nuclear antigen (PCNA) was performed every two days during the 14-day cultures. It was found that proliferation of Saos-2 cells was significantly decreased by the presence of ß-Gly which contrasted with hMSCs where no change was observed. Furthermore, unlike hMSCs, Saos-2 cells demonstrated an upregulated expression of late osteoblastic markers, OCN and PHEX that suggested ß-Gly could affect later stages of osteogenic differentiation. In summary, it is important to consider that ß-Gly significantly affects key cell processes of Saos-2 when using it as an osteoblast-like cell model.

Effect of the association of microparticles and nano-sized ß-calcium glycerophosphate in conventional toothpaste on enamel remineralization: In situ study.

OBJECTIVES: This in situ study aimed to assess the remineralizing effect of a fluoride toothpaste supplemented with ß-calcium glycerophosphate in both micro (ß-CaGPm) and nano-sized forms (ß-CaGPn). METHODS: This blind and cross-over study was performed in 4 phases, each spanning 3 days. Twelve volunteers utilized palatal appliances containing four bovine enamel blocks with artificial caries lesions. Volunteers were randomly assigned to the following treatment groups: Placebo (no F-ß-CaGPm-ß-CaGPn); 1100 ppm F alone (1100F); 1100F plus 0.5% micrometric ß-CaGP (1100F-0.5%ß-CaGPm); and 1100F plus 0.25%nano-sized ß-CaGP (1100F-0.25%ß-CaGPn). Participants were instructed to brush their natural teeth with the palatal appliances in the mouth for 1 min (3 times/day), ensuring that the enamel blocks were exposed to the natural toothpaste slurries. Following each phase, evaluations were conducted to determine the percentage of surface hardness recovery (%SHR), integrated recovery of subsurface hardness (ΔIHR), profile subsurface lesion through polarized light microscopy (PLM), as well as fluoride (F), calcium (Ca), and phosphorus (P) concentrations within the enamel. Data were analyzed by ANOVA and Student-Newman-Keuls test (p < 0.001). RESULTS: Treatment with 1100F-0.25%ß-CaGPn resulted in %SHR ∼69 % and ∼40 % higher when compared to 1100F and 1100F-0.5%ß-CaGPm (p < 0.001). The reduction in lesion body (ΔIHR; PLM) was ∼40 % higher with 1100F-0.25%ß-CaGPn (p < 0.001) compared to 1100F. The addition of ß-CaGPm and ß-CaGPn did not influence enamel F concentration (p > 0.001). Treatment with 1100F-0.25%ß-CaGPn led to an increase in the concentration of Ca and P in the enamel (p < 0.001). CONCLUSION: The addition of 0.25%ß-CaGPn into 1100F formulation increased the bioavailability of calcium and phosphate, promoting a higher remineralizing effect. CLINICAL SIGNIFICANCE: Toothpaste containing 1100F-0.25%ß-CaGPn showed a potential of higher remineralization to 1100 ppm F and 1100 ppm F micrometric ß-CaGP could be a strategy for patients at caries activity.

The effect of a novel toothpaste in children with white spot lesions.

OBJECTIVE: To investigate the effect of a novel mineral containing toothpaste in comparison to a fluoride toothpaste in children with white spot lesions. METHODS: The clinical study was conducted from 2016 to 2018 at Marmara University Department of Pediatric Dentistry Clinic after approval from the ethics review committee of Yeditepe University, Istanbul, Turkey and comprised children of either gender aged 4-5 years having white spot lesions. They were randomly allocated into two groups. The FT (Fluoridated Toothpaste) group was given a 500ppm fluoridated toothpaste, while the Mineral Containing Toothpaste (MCT) group was given toothpaste containing calcium glycerophosphate, magnesium chloride, and 12% xylitol. The white spot lesions were examined using Laser Fluorescence (LF) at baseline and after a month of usage. The two readings were compared. Stimulated saliva was collected for measuring the salivary potential of hydrogen, buffering capacity, and streptococcus mutans. Data was analysed using SPSS 19. RESULTS: Of the 26 children, 10(38%) were girls and 16(62%) were boys. The overall mean age was 4.77±0.54 years. There were 13(50%) subjects in each of the two groups. Of the 381 measurements done, 198(52%) were in the MCT group and 183(48%) in the FT group. LF scores decreased in both the groups (p=0.001). The remineralising potential was not significantly different (p=0.866), while salivary buffering capacity and potential of hydrogen increased in both the groups but the change was not significant (p>0.05). The number of children positive for streptococcus mutans decreased in both the groups (p>0.05). CONCLUSIONS: The toothpaste containing calcium glycerophosphate, magnesium chloride and 12% xylitol had the remineralization properties needed for the prevention of gwhite spot lesions in children.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by ß-glycerophosphate (ß-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in ß-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In ß-GP-treated VSMCs, BK channel opener NS1619 (20 µM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 µM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 µM) or FoxO1 inhibitor AS1842856 (3 µM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

Calcium glycerophosphate and fluoride affect the pH and inorganic composition of dual-species biofilms of Streptococcus mutans and Candida albicans.

OBJECTIVES: This study evaluated the influence of calcium glycerophosphate (CaGP), combined with or without fluoride (F), on the pH and concentrations of F, Ca, and P of dual-species biofilms of Streptococcus mutans and Candida albicans, with or without exposure to sucrose. METHODS: The biofilms (n = 9) received three treatments (72, 78, and 96 h after the start of their formation) at three CaGP concentrations (0.125, 0.25, or 0.5%), with or without F at 500 ppm (as NaF). Solutions containing 500 and 1100 ppm F and artificial saliva were also tested as controls. Biofilm pH was measured, and the concentrations of F, Ca, P, and CaGP were determined (solid and fluid phases). In a parallel experiment, after the third treatment, the treated biofilms were exposed to a sucrose solution, and the pH of the medium, F, Ca, P, and CaGP was determined. Data were subjected to two-way ANOVA, followed by Fisher's LSD test (p < 0.05). RESULTS: Treatment with CaGP and 500 ppm F led to the highest pH values and F and Ca concentrations in the biofilm biomass, both with and without sucrose exposure. CaGP without F led to higher Ca and P concentrations in the biofilm fluid. CONCLUSIONS: CaGP increased F, Ca, and P concentrations in the biofilm, and its presence promoted an increase in the pH of the medium, even after exposure to sucrose. CLINICAL SIGNIFICANCE: The present results elucidate the mechanism by which CaGP and F act on biofilms, further interfering with dental caries dynamics.

Gamma-Aminobutyric Acid (GABA) Promotes Growth in Zebrafish Larvae by Inducing IGF-1 Expression via GABAA and GABAB Receptors.

Insulin-like growth factor-1 (IGF-1) primarily increases the release of gamma-aminobutyric acid (GABA) in neurons; moreover, it is responsible for the promotion of longitudinal growth in children and adolescents. Therefore, in this study, we investigated whether exogenous GABA supplementation activates IGF-mediated growth performance. Zebrafish larvae treated with GABA at three days post fertilization (dpf) showed a significant increase in the total body length from 6 to 12 dpf through upregulation of growth-stimulating genes, including IGF-1, growth hormone-1 (GH-1), growth hormone receptor-1 (GHR-1), and cholecystokinin A (CCKA). In particular, at 9 dpf, GABA increased total body length from 3.60 ± 0.02 to 3.79 ± 0.03, 3.89 ± 0.02, and 3.92 ± 0.04 mm at concentrations of 6.25, 12.5, and 25 mM, and the effect of GABA at 25 mM was comparable to 4 mM ß-glycerophosphate (GP)-treated larvae (3.98 ± 0.02 mm). Additionally, the highest concentration of GABA (50 mM) -induced death in 50% zebrafish larvae at 12 dpf. GABA also enhanced IGF-1 expression and secretion in preosteoblast MC3T3-E1 cells, concomitant with high levels of the IGF-1 receptor gene (IGF-1R). In zebrafish larvae, the GABA-induced growth rate was remarkably decreased in the presence of an IGF-1R inhibitor, picropodophyllin (PPP), which indicates that GABA-induced IGF-1 enhances growth rate via IGF-1R. Furthermore, we investigated the effect of GABA receptors on growth performance along with IGF-1 activation. Inhibitors of GABAA and GABAB receptors, namely bicuculline and CGP 46381, respectively, considerably inhibited GABA-induced growth rate in zebrafish larvae accompanied by a marked decrease in the expression of growth-stimulating genes, including IGF-1, GH-1, GHR-1, and CCKA, but not with an inhibitor of GABAC receptor, TPMPA. Additionally, IGF-1 and IGF-1R expression was impaired in bicuculline and CGP 46381-treated MC3T3-E1 cells, but not in the cells treated with TPMPA. Furthermore, treatment with bicuculline and CGP 46381 significantly downregulated GABA-induced IGF-1 release in MC3T3-E1 cells. These data indicate that GABA stimulates IGF-1 release via GABAA and GABAB receptors and leads to growth promotion performance via IGF-1R.

Globular adiponectin inhibits osteoblastic differentiation of vascular smooth muscle cells through the PI3K/AKT and Wnt/ß-catenin pathway.

Lipid metabolism is closely related to the improvement of vascular calcification (VC) in chronic kidney disease (CKD). Globular adiponectin (gAd) has been reported to be involved in the development of VC in CKD, but the detailed regulatory role remains unclear. The present study is aimed to investigate the biological function and the underlying regulation mechanism of gAd in the process of VC during CKD. Vascular smooth muscle cells (VSMCs) calcification was determined by Alizarin Red S staining. Protein signaling related with VC was tested by western blotting. The expression and intracellular localization of runt-related transcription factor 2 (Runx2) was detected by immunofluorescence and uraemic rat with VC was established by a two-step nephrectomy. Combined with the results of Alizarin Red S staining, we discovered that ß-glycerophosphate (ß-Gp)-induced the osteoblastic differentiation of VSMCs was significantly reversed by gAd treatment. Along with the VSMCs calcification and the increase of Runx2 in ß-Gp-exposed VSMCs, the activities of protein kinase B (AKT) and Wnt/ß-catenin pathway were enhanced, but that were counteracted by the exposure of gAd in rat and human VSMCs. After administration with agonists of the Wnt (SKL2001) and AKT (SC79), there appeared more osteoblastic differentiation and higher expression of Runx2 in gAd-treated VSMCs, but showing lower impact in the presence of SC79 than that in the presence of SKL2001. In the in vivo experiments, intravenous injection of gAd also significantly inhibited VC and Runx2 level in uraemic rat in a dose-dependent manner, possibly through regulating Wnt/ß-catenin pathway. This study demonstrates that gAd ameliorates osteoblastic differentiation of VSMCs possibly by blocking PI3K/AKT and Wnt/ß-catenin signaling transduction. The findings provide an important foundation for gAd in treating VC in kidney diseases.

κ-opioid receptor stimulation alleviates rat vascular smooth muscle cell calcification via PFKFB3-lactate signaling.

In the present study, the effects and mechanism of action of U50,488H (a selective κ-opioid receptor agonist) on calcification of rat vascular smooth muscle cells (VSMCs) induced by ß-glycerophosphate (ß-GP) were investigated. VSMCs were isolated and cultured in traditional FBS-based media. A calcification model was established in VSMCs under hyperphosphatemia and intracellular calcium contents. Alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and lactate were detected in cell culture supernatants before and after treatment. Alizarin red staining was used to detect the degree of calcification of VSMCs. Expression levels of key molecules of osteogenic markers, fructose-2,6-biphosphatase 3 (PFKFB3), and proline hydroxylase 2 (PHD2), were determined using western blotting. Further, vascular calcification was induced by vitamin D3 plus nicotine in rats and isolated thoracic aortas, calcium concentration was assessed in rat aortic rings in vitro. We demonstrated that U50,488H inhibited VSMC calcification in a concentration-dependent manner. Moreover, U50,488H significantly inhibited osteogenic differentiation and ALP activity in VSMCs pretreated with ß-GP. Further studies confirmed that PFKFB3 expression, LDH level, and lactate content significantly increased during calcification of VSMCs; U50,488H reversed these changes. PHD2 expression showed the opposite trend compared to PFKFB3 expression. nor-BNI or 3-PO abolished U50,488H protective effects. Besides, U50,488H inhibited VSMC calcification in rat aortic rings ex vivo. Collectively, our experiments show that κ-opioid receptor activation inhibits VSMC calcification by reducing PFKFB3 expression and lactate content, providing a potential drug target and strategy for the clinical treatment of vascular calcification.

Novel quinazolinone MJ­33 induces AKT/mTOR­mediated autophagy­associated apoptosis in 5FU­resistant colorectal cancer cells.

Novel quinazolinone compounds have been studied in the field of drug discovery for a long time. Among their broad range of pharmacological effects, certain compounds effectively inhibit cancer cell proliferation. MJ­33 is a quinazolinone derivative with proposed anticancer activities that was synthesized in our laboratory. The present study aimed to evaluate the anticancer activity of MJ­33 in fluorouracil (5FU)­resistant colorectal cancer cells (HT­29/5FUR) and to investigate the underlying molecular mechanisms. The cell viability assay results indicated that HT­29/5FUR cell viability was inhibited by MJ­33 treatment in a concentration­dependent manner compared with the control group. The cellular morphological alterations observed following MJ­33 treatment indicated the occurrence of apoptosis and autophagy, as well as inhibition of cell proliferation in a time­dependent manner compared with the control group. The acridine orange, LysoTracker Red and LC3­green fluorescent protein staining results indicated that MJ­33 treatment significantly induced autophagy compared with the control group. The DAPI/TUNEL dual staining results demonstrated increased nuclear fragmentation and condensation following MJ­33 treatment compared with the control group. The Annexin V apoptosis assay and image cytometry analysis results demonstrated a significant increase in apoptotic cells following MJ­33 treatment compared with the control group. The western blotting results demonstrated markedly decreased Bcl­2, phosphorylated (p)­BAD, pro­caspase­9 and pro­caspase­3 expression levels, and notably increased cytochrome c and apoptotic peptidase activating factor 1 expression levels following MJ­33 treatment compared with the control group. Moreover, the expression levels of autophagy­related proteins, including autophagy related (ATG)­5, ATG­7, ATG­12, ATG­16, p62 and LC3­II, were increased following MJ­33 treatment compared with the control group. Furthermore, MJ­33­treated HT­29/5FUR cells displayed decreased expression levels of p­AKT and p­mTOR compared with control cells. The results suggested that MJ­33­induced apoptosis was mediated by AKT signaling, and subsequently modulated via the mitochondria­dependent signaling pathway. Therefore, the results suggested that suppression of AKT/mTOR activity triggered autophagy in the HT­29/5FUR cell line. In summary, the results indicated that MJ­33 inhibited HT­29/5FUR cell viability, and induced apoptosis and autophagy via the AKT/mTOR signaling pathway. The present study may provide novel insight into the anticancer effects and mechanisms underlying MJ­33 in 5FU­resistant colorectal cancer cells.

Expression of RUNX2 and Osterix in Rat Mesenchymal Stem Cells during Culturing in Osteogenic-Conditioned Medium.

We studied the expression of transcription factors RUNX2 and Osterix after addition of a concentrate of osteogenic-conditioned medium to the culture medium for osteogenic differentiation of mesenchymal stem cells (MSC). The obtained concentrate of osteogenic-conditioned medium containing a complex of bioactive substances with a molecular weight >10 kDa provided MSC differentiation into osteoblasts, which was confirmed by high level of expression of transcription factors RUNX2 and Osterix in comparison with the negative control. The highest expression of transcription factor Osterix was revealed on day 14 of MSC culturing in the presence of osteogenic supplement StemPro (positive control) and the studied concentrate of osteogenic-conditioned medium.

Fermented Oyster Extract Promotes Insulin-Like Growth Factor-1-Mediated Osteogenesis and Growth Rate.

Fermented oyster (Crassostrea gigas) extract (FO) prevents ovariectomy-induced osteoporosis by inhibiting osteoclastogenesis and activating osteogenesis. However, the molecular mechanisms underlying FO-mediated bone formation and growth rate are unclear. In the current study, we found that FO significantly upregulated the expression of growth-promoting genes in zebrafish larvae including insulin-like growth factor 1 (zigf-1), insulin-like growth factor binding protein 3 (zigfbp-3), growth hormone-1 (zgh-1), growth hormone receptor-1 (zghr-1), growth hormone receptor alpha (zghra), glucokinase (zgck), and cholecystokinin (zccka). In addition, zebrafish larvae treated with 100 µg/mL FO increased in total body length (3.89 ± 0.13 mm) at 12 days post fertilization (dpf) compared to untreated larvae (3.69 ± 0.02 mm); this effect was comparable to that of the ß-glycerophosphate-treated zebrafish larvae (4.00 ± 0.02 mm). Furthermore, FO time- and dose-dependently increased the extracellular release of IGF-1 from preosteoblast MC3T3-E1 cells, which was accompanied by high expression of IGF-1. Pharmacological inhibition of IGF-1 receptor (IGF-1R) using picropodophyllin (PPP) significantly reduced FO-mediated vertebrae formation (from 9.19 ± 0.31 to 5.53 ± 0.35) and growth performance (from 3.91 ± 0.02 to 3.69 ± 0.01 mm) in zebrafish larvae at 9 dpf. Similarly, PPP significantly decreased FO-induced calcium deposition in MC3T3-E1 cells by inhibiting GSK-3ß phosphorylation at Ser9. Additionally, DOI hydrochloride, a potent stabilizer of GSK-3ß, reduced FO-induced nuclear translocation of RUNX2. Transient knockdown of IGF-1Rα/ß using specific silencing RNA also resulted in a significant decrease in calcium deposition and reduction in GSK-3ß phosphorylation at Ser9 in MC3T3-E1 cells. Altogether, these results indicate that FO increased phosphorylated GSK-3ß at Ser9 by activating the autocrine IGF-1-mediated IGF-1R signaling pathway, thereby promoting osteogenesis and growth performance. Therefore, FO is a potential nutritional supplement for bone formation and growth.

The protective effects of long non-coding RNA-ANCR on arterial calcification.

INTRODUCTION: Arterial calcification is a major factor for cardiovascular events and is characterized by vascular smooth muscle cells (VSMCs) transformed into osteoblast-like cells. Long non-coding RNAs (lncRNA) were recognized as important regulators of diverse biological processes. Previous studies have demonstrated that lncRNAs could regulate the proliferation and apoptosis of VSMCs. LncRNA-ANCR (Anti-differentiation ncRNA) is an essential mediator governing the differentiation of human osteoblast. However, it is unclear whether ANCR could regulate the osteoblastic differentiation of VSMCs. In this study, we determined the effect of ANCR on VSMCs differentiation and arterial calcification. MATERIALS AND METHODS: Both cellular and mouse model of arterial calcification were, respectively, established to investigate the role of ANCR in the mechanism of arterial calcification. ANCR overexpressing lentivirus were used to investigate the effects of ANCR on the expression of bone proteins and autophagy-related molecules. RESULTS: ANCR could inhibit ß-glycerophosphate (ß-GP)-induced VSMCs osteoblastic differentiation and mineralization due to decreased expressions of Runt-related transcription factor 2, bone morphogenetic protein-2, and formation of mineralized nodule, and attenuate high calcitriol-induced mice model of arterial calcification. Furthermore, ANCR could significantly increase LC3 and autophagy protein 5 expression in ß-GP-stimulated VSMCs, and the effect could be inhibited by 3-methyladenine, a pharmacological inhibitor of autophagy. CONCLUSION: ANCR may inhibit the osteoblastic differentiation of VSMCs and attenuate mice arterial calcification through activating autophagy.

1-O-alkyl glycerophosphate-induced CD36 expression drives oxidative stress in microglial cells.

Microglia, the tissue-resident macrophages in the central nervous system, are important for the initiation and perpetuation of neuroinflammation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-inducible transcription factor and plays an important role in fatty acid metabolism. Our previous study found that 1-O-alkyl glycerophosphate (AGP), a naturally occurring ether analog of lysophosphatidic acid, is a high-affinity, partial agonist of PPARγ. In this study, we investigated the role of AGP in microglial activation and illustrated the underlying molecular mechanism. We found that AGP treatment increased the production of intracellular reactive oxygen species and induced PPARγ activation in microglial cells. Interestingly, AGP also up-regulated the expression levels of the cluster of differentiation 36 (CD36) scavenger receptor, a high-affinity receptor for oxidized low-density lipoproteins. The findings suggest that AGP induces PPARγ activation, enhances CD36 expression and increases the production of intracellular reactive oxygen species (ROS) in microglial cells.

Melatonin Attenuates ß-Glycerophosphate-Induced Calcification of Vascular Smooth Muscle Cells via a Wnt1/ß-Catenin Signaling Pathway.

BACKGROUND: Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. The wingless-type MMTV integration site family member 1 (Wnt1)/ß-catenin pathway is associated with cardiovascular calcification. This study aimed to explore whether melatonin could attenuate VSMC calcification through regulating the Wnt1/ß-catenin signaling pathway. METHODS: The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualized by Alizarin Red Staining. Calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure the expression of runt-related transcription factor 2 (Runx2), α-smooth muscle actin (α-SMA), and cleaved caspase-3. RESULTS: Melatonin markedly ameliorated calcium deposition and ALP activity. Runx2 and cleaved caspase-3 were found to be reduced and α-SMA was found to be increased by melatonin, together with a decrease in apoptosis. Immunofluorescence assay revealed a lower Runx2 protein level in the melatonin group. Melatonin treatment significantly decreased the expression of Wnt1 and ß-catenin. Treatment with lithium chloride or transglutaminase 2 abrogated the protective effects of melatonin. CONCLUSION: Melatonin can attenuate ß-GP-induced VSMC calcification through the suppression of Wnt1/ß-catenin system.

Downregulation of miR-542-3p promotes osteogenic transition of vascular smooth muscle cells in the aging rat by targeting BMP7.

BACKGROUND: Aging is believed to have a close association with cardiovascular diseases, resulting in various pathological alterations in blood vessels, including vascular cell phenotypic shifts. In aging vessels, the microRNA(miRNA)-mediated mechanism regulating the vascular smooth muscle cell (VSMC) phenotype remains unclarified. MiRNA microarray was used to compare the expressions of miRNAs in VSMCs from old rats (oVSMCs) and young rats (yVSMCs). Quantitative reverse transcription real-time PCR (qRT-PCR) and small RNA transfection were used to explore the miR-542-3p expression in oVSMCs and yVSMCs in vitro. Calcification induction of yVSMCs was conducted by the treatment of ß-glycerophosphate (ß-GP). Alizarin red staining was used to detect calcium deposition. Western blot and qRT-PCR were used to investigate the expression of the smooth muscle markers, smooth muscle 22α (SM22α) and calponin, and the osteogenic markers, osteopontin (OPN), and runt-related transcription factor 2 (Runx2). Lentivirus was used to overexpress miR-542-3p and bone morphogenetic protein 7 (BMP7) in yVMSCs. Luciferase reporter assay was conducted to identify the target of miR-542-3p. RESULTS: Compared with yVSMCs, 28 downregulated and 34 upregulated miRNAs were identified in oVSMCs. It was confirmed by qRT-PCR that oVSMC expressed four times lower miR-542-3p than yVSMCs. Overexpressing miR-542-3p in yVSMCs suppressed the osteogenic differentiation induced by ß-GP. Moreover, miR-542-3p targets BMP7 and overexpressing BMP7 in miR-542-3p-expressing yVSMCs reverses miR-542-3p's inhibition of osteogenic differentiation. CONCLUSIONS: miR-542-3p regulates osteogenic differentiation of VSMCs through targeting BMP7, suggesting that the downregulation of miR-542-3p in oVSMCs plays a crucial role in osteogenic transition in the aging rat.