Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: OCN attenuates LPS-caused detrimental effects in WT and OCN −/− mice. ( A ) Experimental design for the LPS-induced acute inflammation. The OCN −/− and WT mice were injected intraperitoneally with PBS or OCN (500 ng) followed by LPS (10 mg/kg) treatment. Mice survival rate and serum inflammatory were analyzed in each time point, accordingly. ( B ) Survival curves of OCN −/− and WT mice treated with PBS or OCN in response to LPS stimulation. Sample sizes are presented in brackets ( n = 15). * p < 0.05, as compared to PBS. ( C , D ) ELISA analysis of serum levels of IL-6 ( C ) and TNFα ( D ) in OCN −/− and WT mice with PBS or OCN pretreatment after 6h of LPS administration. Sample sizes are indicated as dots in each column. Data are presented as mean ± SD. * p < 0.05 and ** p < 0.01, as compared to PBS.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: OCN inhibits the production of pro-inflammatory factor in macrophage. ( A , B ) Effects of OCN treatment on the mRNA expression of pro-inflammatory genes, i.e., IL-6 ( A ) and TNFα ( B ), in LPS (500 ng/mL) treated peritoneal macrophages from WT mice. Data are presented as means ± SD ( n = 3 biological replicates). * p < 0.05 and ** p < 0.01, as compared to PBS. ( C , D ) Cytokine levels in a culture medium of peritoneal macrophage from WT and OCN −/− mice. Peritoneal macrophages were treated with LPS (500 ng/mL), together PBS or OCN (10 nM) for 12 h, and the levels of IL-6 ( C ) and TNFα ( D ) in culture medium were measured by ELISA analysis. Data are presented as means ± SD. ( n = 3 biological replicates) * p < 0.05, as compared to PBS. ( E ) The mRNA expression analysis of genes associated with anti-inflammatory in peritoneal macrophages isolated from WT mice. Data are presented as means ± SD ( n = 3 biological replicates). ** p < 0.01 and *** p < 0.001, as compared to PBS.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: GPR37 mediates OCN-induced intracellular responses in macrophages. ( A ) Overview of the experimental design testing intracellular signals in macrophages. Peritoneal macrophages were isolated from WT and GPR37 −/− mice to study the changes of intracellular calcium level (iCa 2+ ), cAMP production, and pERK level in response to OCN treatment. ( B ) Representative response curves of OCN-triggered iCa 2+ changes in macrophages from WT and GPR37 −/− mice. ( C ) OCN-triggered inhibition of cAMP production in macrophages from WT and GPR37 −/− mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS, and n.s. indicates not significant. ( D , E ) Representative immunoblot images ( D ) and relative quantification ( E ) showing that OCN-triggered an increase in the pERK level in macrophages from WT but not in that from GPR37 −/− mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS. ( F ) Representative response curves of OCN-triggered iCa 2+ changes in PTX pretreated macrophage from WT mice. ( G ) OCN-triggered inhibition of cAMP production in PTX pretreated macrophage from WT mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS, and n.s. indicates not significant. ( H , I ) Representative immunoblot images ( H ) and relative quantification ( I ) showing that PTX pretreatment blocked the OCN-triggered increase in the pERK level in macrophages from WT mice. Data are presented as mean ± SD ( n = 3 biological replicates). * p < 0.05, as compared to PBS.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Isolation, Inhibition, Western Blot, Quantitative Proteomics

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: GPR37 mediates the inhibitory effects of OCN on pro-inflammatory factors in macrophage. ( A , B ) Cytokine levels in the culture medium of peritoneal macrophages from WT and GPR37 −/− mice. Cells were treated with LPS (500 ng/mL, 37 °C, 24 h) together with PBS or OCN (10 nM). ELISA analysis was performed to measure the level of IL-6 ( A ) and TNF-α ( B ). Data are presented as mean ± SD ( n = 3 biological replicates), * p < 0.05, as compared to PBS, and n.s. indicates not significant. ( C , D ) Representative immunoblot images ( C ) and quantitative analysis ( D ) of NFκB p65 level in macrophages treated with LPS in the presence of OCN. Data are presented as mean ± SD ( n = 3 biological replicates), * p < 0.05 as compared to PBS, and n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: OCN promotes macrophage phagocytosis via GPR37. ( A ) Representative images of in vitro phagocytosis assay, in which pHrodo zymosan particles were incubated with peritoneal macrophages from WT or OCN −/− mice, treated with PBS or OCN (10 nM, 30 min, 37 °C). Red fluorescence suggests an intracellular update indicating phagocytosis. Scale bars: 20 μm. ( B ) Quantification of macrophage phagocytic activity of zymosan positives cells. Data are presented as mean ± SD ( n = 3 biological replicates), ** p < 0.01, as compared to PBS. ( C ) Representative images of an in vitro phagocytosis assay, in which pHrodo zymosan particles were incubated with peritoneal macrophages from WT or GPR37 −/− mice, treated with PBS or OCN (10 nM, 30 min, 37 °C). Scale bars: 20 μm. ( D ) Quantification of macrophage phagocytic activity of zymosan positives cells. Data are presented as mean ± SD ( n = 3 biological replicates), ** p < 0.01, as compared to PBS, and n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: In Vitro, Phagocytosis Assay, Incubation, Fluorescence, Activity Assay

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: The protective function of OCN against LPS is absent in GPR37 −/− mice. ( A ) Experimental design for LPS-induced acute inflammation in GPR37 −/− and WT mice. After intraperitoneal injection of PBS or OCN (500 ng), followed by LPS (10 mg/kg) treatment, mice survival rate and serum inflammatory cytokine level were analyzed accordingly. ( B ) Survival curves of GPR37 −/− and WT mice treated with PBS or OCN in response to an LPS challenge. Sample sizes are presented in brackets ( n = 15). * p < 0.05, as compared to PBS. ( C , D ) After 6h of LPS injection, with or without OCN pretreatment, serum IL-6 ( C ) and TNFα ( D ) levels were analyzed in GPR37 −/− and WT mice. Sample sizes are indicated as dots in columns. * p < 0.05 and ** p < 0.01, as compared to PBS; n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Injection

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: Adoptive transfer of OCN-treated macrophages confers protection against LPS stimulation. ( A ) Experimental design, to test whether adoptive transfer of macrophages pretreated with OCN, could attenuate LPS-induced acute inflammation. Peritoneal macrophages were isolated from WT or GPR37 −/− mice and then treated with PBS or OCN (10 nM) for 24 h, followed by a washout of OCN and an adoptive transfer of macrophages (I.P.) into GPR37 −/− mice challenged with LPS for 1 h. ( B , C ) ELISA analysis of serum IL-6 ( B ) and TNFα ( C ) levels, in GPR37 −/− mice with adoptive transfer, of macrophages derived from WT or GPR37 −/− mice. Sample sizes are indicated as dots in each column. Data are presented as mean ± SD. * p < 0.05 and ** p < 0.01, as compared to PBS; n.s. indicates not significant.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Adoptive Transfer Assay, Isolation, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Biomedicines

Article Title: Osteocalcin Alleviates Lipopolysaccharide-Induced Acute Inflammation via Activation of GPR37 in Macrophages

doi: 10.3390/biomedicines10051006

Figure Lengend Snippet: A schematic diagram illustrating that the bone-derived hormone OCN, via the activation of GPR37 in macrophages, plays a protective function against LPS challenge through the regulation of macrophage inflammatory reactions and phagocytic function.

Article Snippet: Mouse osteocalcin (OCN) peptides were synthesized in the Beijing Genomics Institute (BGI, Beijing, China).

Techniques: Derivative Assay, Activation Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Measurement of bioactive osteocalcin in humans using a novel immunoassay reveals association with glucose metabolism and β-cell function

doi: 10.1152/ajpendo.00321.2019

Figure Lengend Snippet: Generation of monoclonal antibodies recognizing human uncarboxylated osteocalcin (ucOCN). A: schematic representation of the different forms of OCN used in this study. B: results of the screening strategy used to identify clones specific to the ucOCN epitope or to the OCN C-terminal region. C: recombinant uncarboxylated (ucOCN1–49) and synthetic partially carboxylated (2XGla OCN1–49) or fully carboxylated (cOCN1–49) human osteocalcins were spotted on nitrocellulose membranes and detected by Western blot using anti-ucOCN, anti-C-terminal OCN, and anti-γ-carboxyglutamic acid [Gla (Pan-Gla)] antibodies. Glu, glutamate.

Article Snippet: Recombinant uncarboxylated (ucOCN 1–49 ), synthetic partially carboxylated (2XGla OCN 1–49 ), or synthetic fully carboxylated (cOCN 1–49 ) human osteocalcin (Peptide Institute Inc., Japan) were diluted in PBS 1× containing 0.01% BSA and spotted on nitrocellulose membranes.

Techniques: Bioprocessing, Clone Assay, Recombinant, Western Blot

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Measurement of bioactive osteocalcin in humans using a novel immunoassay reveals association with glucose metabolism and β-cell function

doi: 10.1152/ajpendo.00321.2019

Figure Lengend Snippet: Establishment and validation of the human uncarboxylated osteocalcin (ucOCN) ELISA. A: identification of 5 antibody pairs (capture/detection) to detect ucOCN. The results were normalized to the highest signal (100) from the 8H4/4B6 pair. B: schematic representation of the sandwich ELISA described and tested in this study. Human ucOCN is measured using the 8H4 clone as the capture antibody and biotinylated 4B6 as the detection antibody in combination with horseradish peroxidase (HRP)-conjugated avidin. C: measurement of ucOCN, fully carboxylated OCN (cOCN), and partially carboxylated OCN (2XGla OCN) showing that the ucOCN ELISA specifically recognizes the uncarboxylated form of OCN from 0.037 to 1.8 ng/mL. D: ucOCN detection in media from human embryonic kidney 293 cells overexpressing human OCN-V5 cultured with vitamin K1 (VK1; 20 μM) or vehicle. E: ucOCN measurements in serum from individuals treated with warfarin (n = 6) or untreated controls (Ctrl; n = 15). **P < 0.01; ***P < 0.001.

Article Snippet: Recombinant uncarboxylated (ucOCN 1–49 ), synthetic partially carboxylated (2XGla OCN 1–49 ), or synthetic fully carboxylated (cOCN 1–49 ) human osteocalcin (Peptide Institute Inc., Japan) were diluted in PBS 1× containing 0.01% BSA and spotted on nitrocellulose membranes.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Avidin-Biotin Assay, Cell Culture

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Measurement of bioactive osteocalcin in humans using a novel immunoassay reveals association with glucose metabolism and β-cell function

doi: 10.1152/ajpendo.00321.2019

Figure Lengend Snippet: Association between uncarboxylated osteocalcin (ucOCN) and insulin resistance and diabetes in severely obese subjects with and without type 2 diabetes (T2D; Cohort B). A and B: circulating level of total OCN (tOCN; A) and ucOCN (B) in the nondiabetic (ND) and T2D subpopulations of the cohort. C–E: Pearson’s correlation between ucOCN and glucose (C), glycated hemoglobin (HbA1c; D), and the insulin disposition index (E). **P < 0.01.

Article Snippet: Recombinant uncarboxylated (ucOCN 1–49 ), synthetic partially carboxylated (2XGla OCN 1–49 ), or synthetic fully carboxylated (cOCN 1–49 ) human osteocalcin (Peptide Institute Inc., Japan) were diluted in PBS 1× containing 0.01% BSA and spotted on nitrocellulose membranes.

Techniques:

Journal: Nature Communications

Article Title: Long-lasting renewable antibacterial porous polymeric coatings enable titanium biomaterials to prevent and treat peri-implant infection

doi: 10.1038/s41467-021-23069-0

Figure Lengend Snippet: a CCK-8 assay about the proliferation of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl after cells were cultured for 1, 3 and 7 days ( n = 3; P = 0.673, 0.639 and 0.145 for 1, 3 and 7 days compared with Ti-OH, respectively; Student’s t test). b Morphology of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 1 day (green for F-actin, blue for cell nucleus, scale bar = 20 μm). c ALP activity of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 7 and 14 days (n = 3; P = 0.132 and 0.954 for 7 and 14 days compared with Ti-OH; Student’s t test). d Alizarin Red S staining of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 21 days (red for calcium nodules, scale bar = 1 mm) and semi-quantitative measurement of calcium content ( n = 3; P = 0.147 compared with Ti-OH; Student’s t test). e Western blot results of osteogenic-related proteins (OCN, OPN and RUNX2) expressed in MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 3, 7 and 14 days after osteogenic induction. f RT-qPCR results of expression levels of osteogenic-related genes (OCN, OPN and RUNX2) in MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 3, 7 and 14 days after osteogenic induction ( n = 3; P > 0.05 for all time points of these three genes between groups; Student’s t test). g In vivo biocompatibility. HE staining and CD68 immunofluorescent staining of tissues around Ti-OH and Ti-PAA-NCl embedded in the backs of nude mice for 4 weeks (fluorescent green for CD68, fluorescent blue for cell nucleus, white scale bars in HE images = 100 μm, black scale bars in HE images = 25 μm, scale bars in CD68 immunofluorescent images = 100 μm). All error bars = s.d.

Article Snippet: After that, proteins were blotted onto immobilon-P polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany) and blocked with 5% skim milk (Difco, BD, USA) for 1 h. Western blot was performed using rabbit anti-mouse OCN (Affinity Biosciences, Cincinnati, USA), OPN (Proteintech, Chicago, USA), RUNX2 (Abcam, Cambridge, UK) polyclonal antibodies and mouse-derived GAPDH monoclonal antibody (Emarbio, Beijing, China) overnight at 4 °C.

Techniques: CCK-8 Assay, Cell Culture, Activity Assay, Staining, Western Blot, Quantitative RT-PCR, Expressing, In Vivo