Oleanolic Acid Modulates 25-Hydroxyvitamin D3 1-alpha-hydroxylase in Osteoblasts and Human Mesenchymal Stem Cells

Objectives: 25-Hydroxyvitamin D3 1-alpha-hydroxylase (CYP27B1) catalyzes the hydroxylation of 25-hydroxyvitamin D3 (25(OH)D3) to 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), the bioactive form of vitamin D3. Our previous studies suggested that oleanolic acid (OA), a pentacyclic triterpenoid presents in many food and herbs, can improve circulating 1,25(OH)2D3 in ovariectomized (OVX) mice and increase CYP27B1 expression in human renal proximal tubular cells (HKC-8). However, the role of OA in regulating CYP27B1 in bone is far from clear. The present study is designed to study the effects of OA on CYP27B1 expressions and 1,25(OH)2D3 production in bone cells. Methods: The mRNA and protein expressions of CYP27B1, as well as bone metabolism markers were determined in osteoblast-like UMR-106 cells in response to treatments of parathyroid hormone (PTH) (10–7 M) or OA (10–9 - 10–5 M) for 24 hours. By using excessive 25(OH)D3 (10–6 M) as substrate, cellular production of 1,25(OH)2D3 was measured to determine CYP27B1 activity. To mimic the physiological condition, human mesenchymal stem cells (hMSCs) were pre-treated with 25(OH)D3 (10–7 M) for 12 hours, followed by OA treatment (10–9 - 10–6 M) for another 24 hours, the osteogenic effects of OA on alkaline phosphatase (ALP) activity and CYP27B1 expression were evaluated. Results: PTH (10–7 M, p<0.001) and OA (10–9 M, p<0.05) significantly upregulated mRNA and protein expressions of CYP27B1 in UMR-106 cells. 4-hour treatments of PTH (10–7 M) and OA (10–9 M) also stimulated the 1,25(OH)2D3 production by 46.02 % (p<0.001) and 17.60 % (p<0.01), respectively. Moreover, the mRNA expressions of ALP and osteocalcin (OCN) involved in osteoblast differentiation, were upregulated in response to PTH and OA in UMR-106 cells (p<0.05). The protein expression of CYP27B1 was upregulated by treatment with OA in hMSCs supplemented with 25(OH)D3 (10–8 M, p<0.05 vs. supplement alone). Furthermore, OA (10–8 M) potentiated the effects of 25(OH)D3 on osteogenesis in hMSCs by enhancing ALP activity by 47.77 % (p<0.01). Conclusions: Our results indicated that the bone anabolic effects of OA are associated with its actions to improve local bioactivation of vitamin D3 in osteoblasts and hMSCs, suggesting the involvement of paracrine or autocrine activities of 1,25(OH)2D3 in mediating the actions of OA in bone. Funding Sources: This work is supported by research studentship of Wen-Xuan Yu, The Hong Kong Polytechnic University.

Multifunctional Properties of Binary Polyrhodanine Manganese Ferrite Nanohybrids—From the Energy Converters to Biological Activity

The PRHD@MnFe2O4 binary hybrids have shown a potential for applications in the biomedical field. The polymer cover/shell provides sufficient surface protection of magnetic nanoparticles against adverse effects on the biological systems, e.g., it protects against Fenton’s reactions and the generation of highly toxic radicals. The heating ability of the PRHD@MnFe2O4 was measured as a laser optical density (LOD) dependence either for powders as well as nanohybrid dispersions. Dry hybrids exposed to the action of NIR radiation (808 nm) can effectively convert energy into heat that led to the enormous temperature increase ΔT 170 °C (>190 °C). High concentrated colloidal suspensions (5 mg/mL) can generate ΔT of 42 °C (65 °C). Further optimization of the nanohybrids amount and laser parameters provides the possibility of temperature control within a biologically relevant range. Biological interactions of PRHD@MnFe2O4 hybrids were tested using three specific cell lines: macrophages (RAW 264.7), osteosarcoma cells line (UMR-106), and stromal progenitor cells of adipose tissue (ASCs). It was shown that the cell response was strongly dependent on hybrid concentration. Antimicrobial activity of the proposed composites against Escherichia coli and Staphylococcus aureus was confirmed, showing potential in the exploitation of the fabricated materials in this field.

Analyses of In-vitro antioxidant and anticancer activity of Cissus quadrangularis stem extract in osteoblastic cell line -UMR- 106

Cancer is one of the leading causes of death worldwide with an estimated 7.8 lakh deaths in 2018 in India. Osteosarcoma, a primary tumour of the bone is the second highest cause of bone cancer related death in young people. It is treated with chemotherapy, radiation, and surgery which cause side effects. Certain traditional plants may have antitumour activities. This research aimed to evaluate the invitro antioxidant and apoptosis induced activity of stem extract of cissus quadrangularis (CQ) on osteoblastic cell line (UMR-106). Various extracts of CQ was subjected to phytochemical screening and antioxidant studies. Ethanolic extract of CQ showed maximum phytoconstituents and better antioxidant activity for both DPPH(2,2-diphenyl-1-picryl-hydrazyl-hydrate)and FRAP(Ferric reducing antioxidant potential) assay. The UMR-106 cell line was treated with different concentrations of the extract ranging from 25μg to 400μg for varying time periods. The dose and time optimization of the ethanolic extract was evaluated in the osteoblastic cell line (UMR-106) using MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) which showed IC50 value with 200μg/ml at 24 hours. There was a reduction in the percentage of cells by 50% in the treated group by SRB assay showing the cytotoxic potency of the extract. Flow cytometry analysis indicated that the extract of CQ induced apoptosis in UMR-106 cell line. Ethanolic extract of CQ has antioxidant effects, also inducing G0/G1 arrest and apoptosis in osteoblasts, suggesting that this might represent a novel and effective agent against bone cancer.

Susceptibility to Localized Corrosion of ASTM F745 and UNS S32750 Stainless Steels Influence of pH and Cytotoxicity Evaluation

Background: Body fluids are highly corrosive as they contain chlorides and hydroxides ions, as well as salts, bacteria, proteins and dissolved oxygen. The pH of the body is usually around 7.4, although this value can vary in a range of 4 to 9 after surgery or because of haematomas, inflammations and infections. ASTM F745 (type 316L) stainless steel has been used for load bearing partial and total joint replacements and post trauma reconstructive surgeries. However, long exposure to the aggressive effect of chloride ion present in the human body, may increase the susceptibility to suffer localized corrosion. Although UNS S32750 has greater corrosion resistance to chloride ion, its magnetic characteristics inhibit its use in implantable devices. Nevertheless, this stainless steel could be used in temporary implants and orthodontic appliances such as brackets, wire arches and bands, due to its high resistance to corrosion, the greater mechanical resistance and the high capacity of plastic forming. Objectives: The objective was to evaluate the susceptibility to localized corrosion in simulated body fluid, in the pH range of 4 to 9. Another objective was to evaluate the cytotoxicity of Cr and Ni present in the chemical composition of both stainless steels. Cytocompatibility was also analysed by seeding cells on the surfaces of both stainless steels. Methods: Cyclic polariation test was performed to evaluate the susceptibility to localized corrosion in 0.9 wt% NaCl aqueous solution, at pH between 4 and 9, maintained at 37°C. For cytotoxicity evaluation, neutral red, MTT and collagen assays were performed using UMR-106 cell line. Cytocompatibility was analysed by seeding UMR-106 cells on the surfaces of both stainless steels. Results: F745-SS was more susceptible to suffer localized corrosion than UNS S32750. Although it showed a tendency to develop transpassive reactions at low pH, galvanostatic tests did not reveal the onset of localized corrosion. The results from the cytotoxicity assays indicated that no adverse effects were observed. UMR-106 osteoblastic cells showed high viability, however, a slight reduction in the collagen production was observed. The cytocompatibility was also satisfactory, since the cells seeded on the surfaces had adequate proliferation. Conclusion: F745-SS is more susceptible to suffer localized corrosion than UNS S32750 in the pH range between 4 and 9. UNS S32750 showed an extensive passive region, however, transpassive reactions were observed at lower pH. On the other hand, no cytotoxic effects were promoted by both stainless steels, although a slight reduction in collagen production was observed. Cells seeded on F745-SS and UNS S32750 surfaces had an acceptable proliferation, without evidence of changes in their morphology.

Silencing of long-non-coding RNA ANCR suppresses the migration and invasion of osteosarcoma cells by activating the p38MAPK signalling pathway

Background Osteosarcoma (OS) is a malignancy of the bone that has no clearly identified prognostic factors for diagnosis. In this study, we evaluated the regulatory role of long non-coding RNA (lncRNA) ANCR on the migration and invasion of OS cells as well as the possible mechanism involving the p38MAPK signalling pathway. Methods ANCR expression was determined in OS tissues and OS cell lines (MG-63, S1353, U2OS, and UMR-106) by qRT-PCR. It was observed that ANCR was down-regulated in MG-63 and U2OS cells by 48 h of siRNA-ANCR (si-ANCR) transfection. The proliferation of transfected cells was determined using the CCK-8 and the EdU assays. The migration and invasion of transfected cells were determined by the Transwell assay. The expression of E-cadherin, N-cadherin, and phosphorylated p38MAPK (p-p38MAPK) proteins was determined by Western blot. In addition, combinatorial treatment of cells with si-ANCR + SB203580 (p38MAPK inhibitor) was performed to investigate the association between ANCR and MAPK signalling in OS cells. Results ANCR was up-regulated in OS cells and tissues. ANCR silencing significantly inhibited the proliferation rate, decreased the percentage of migration and invasion cells, down-regulated N-cadherin, and up-regulated E-cadherin and p-p38MAPK in MG-63 and U2OS cells. Inhibition of the p38MAPK signalling pathway (SB203580) in MG-63 and U2OS cells rescued si-ANCR-induced inhibition of cell migration and invasion. Conclusions Silencing of ANCR inhibited the migration and invasion of OS cells through activation of the p38MAPK signalling pathway.

Activation of unliganded FGF receptor by extracellular phosphate potentiates proteolytic protection of FGF23 by its O-glycosylation

Fibroblast growth factor (FGF) 23 produced by bone is a hormone that decreases serum phosphate (Pi). Reflecting its central role in Pi control, serum FGF23 is tightly regulated by serum Pi alterations. FGF23 levels are regulated by the transcriptional event and posttranslational cleavage into inactive fragments before its secretion. For the latter, O-glycosylation of FGF23 by GALNT3 gene product prevents the cleavage, leading to an increase in serum FGF23. However, the molecular basis of Pi sensing in the regulation of serum FGF23 remains elusive. In this study, we showed that high Pi diet enhanced the skeletal expression of Galnt3, but not Fgf23, with expected increases in serum FGF23 and Pi in mice. Galnt3 induction by high Pi was further observed in osteoblastic UMR 106 cells, and this was mediated by activation of the extracellular signal-regulated kinase (ERK) pathway. Through proteomic searches for the upstream sensor for high Pi, we identified one subtype of the FGF receptor (FGFR1c), which was phosphorylated by high Pi in the absence of FGFs. The mode of unliganded FGFR activation by high Pi appeared different from that of FGFR bound to a canonical FGFR ligand (FGF2) when phosphorylation of the FGFR substrate 2α and ERK was monitored. Finally, we showed that an FGFR inhibitor and conditional deletion of Fgfr1 in osteoblasts/osteocytes abrogated high Pi diet-induced increases in serum FGF23 and femoral Galnt3 expression in mice. Thus, these findings uncover an unrecognized facet of unliganded FGFR function and illustrate a Pi-sensing pathway involved in regulation of FGF23 production.

Sustained Klotho delivery reduces serum phosphate in a model of diabetic nephropathy

Diabetic nephropathy (DN) is a primary cause of end-stage renal disease and is becoming more prevalent because of the global rise in type 2 diabetes. A model of DN, the db/db uninephrectomized ( db/db-uni) mouse, is characterized by obesity, as well as compromised renal function. This model also manifests defects in mineral metabolism common in DN, including hyperphosphatemia, which leads to severe endocrine disease. The FGF23 coreceptor, α-Klotho, circulates as a soluble, cleaved form (cKL) and may directly influence phosphate handling. Our study sought to test the effects of cKL on mineral metabolism in db/db-uni mice. Mice were placed into either mild or moderate disease groups on the basis of the albumin-to-creatinine ratio (ACR). Body weights of db/db-uni mice were significantly greater across the study compared with lean controls regardless of disease severity. Adeno-associated cKL administration was associated with increased serum Klotho, intact, bioactive FGF23 (iFGF23), and COOH-terminal fragments of FGF23 ( P < 0.05). Blood urea nitrogen was improved after cKL administration, and cKL corrected hyperphosphatemia in the high- and low-ACR db/db-uni groups. Interestingly, 2 wk after cKL delivery, blood glucose levels were significantly reduced in db/db-uni mice with high ACR ( P < 0.05). Interestingly, several genes associated with stabilizing active iFGF23 were also increased in the osteoblastic UMR-106 cell line with cKL treatment. In summary, delivery of cKL to a model of DN normalized blood phosphate levels regardless of disease severity, supporting the concept that targeting cKL-affected pathways could provide future therapeutic avenues in DN. NEW & NOTEWORTHY In this work, systemic and continuous delivery of the “soluble” or “cleaved” form of the FGF23 coreceptor α-Klotho (cKL) via adeno-associated virus to a rodent model of diabetic nephropathy (DN), the db/db uninephrectomized mouse, normalized blood phosphate levels regardless of disease severity. This work supports the concept that targeting cKL-affected pathways could provide future therapeutic avenues for the severe mineral metabolism defects associated with DN.