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: The primary antibodies included anti-NF-κB-p65 (Cat#ab16502, Abcam, Cambridge, UK), anti-NF-κB-p-p65 (S536) (Cat# ab76302, Abcam, Cambridge, UK), anti-tERK (Cat#8544, CST, Boston, USA), anti-tERK (Cat#4695, CST, Boston, MA, USA), anti-GPR37 (Cat#14820-1-AP, Proteintech, Wuhan, China), anti-GAPDH (Cat# 10494-1-AP, Proteintech, Wuhan, China).

Techniques: Isolation, Inhibition, 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: 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: The primary antibodies included anti-NF-κB-p65 (Cat#ab16502, Abcam, Cambridge, UK), anti-NF-κB-p-p65 (S536) (Cat# ab76302, Abcam, Cambridge, UK), anti-tERK (Cat#8544, CST, Boston, USA), anti-tERK (Cat#4695, CST, Boston, MA, USA), anti-GPR37 (Cat#14820-1-AP, Proteintech, Wuhan, China), anti-GAPDH (Cat# 10494-1-AP, Proteintech, Wuhan, 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: The primary antibodies included anti-NF-κB-p65 (Cat#ab16502, Abcam, Cambridge, UK), anti-NF-κB-p-p65 (S536) (Cat# ab76302, Abcam, Cambridge, UK), anti-tERK (Cat#8544, CST, Boston, USA), anti-tERK (Cat#4695, CST, Boston, MA, USA), anti-GPR37 (Cat#14820-1-AP, Proteintech, Wuhan, China), anti-GAPDH (Cat# 10494-1-AP, Proteintech, Wuhan, 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: The primary antibodies included anti-NF-κB-p65 (Cat#ab16502, Abcam, Cambridge, UK), anti-NF-κB-p-p65 (S536) (Cat# ab76302, Abcam, Cambridge, UK), anti-tERK (Cat#8544, CST, Boston, USA), anti-tERK (Cat#4695, CST, Boston, MA, USA), anti-GPR37 (Cat#14820-1-AP, Proteintech, Wuhan, China), anti-GAPDH (Cat# 10494-1-AP, Proteintech, Wuhan, 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: The primary antibodies included anti-NF-κB-p65 (Cat#ab16502, Abcam, Cambridge, UK), anti-NF-κB-p-p65 (S536) (Cat# ab76302, Abcam, Cambridge, UK), anti-tERK (Cat#8544, CST, Boston, USA), anti-tERK (Cat#4695, CST, Boston, MA, USA), anti-GPR37 (Cat#14820-1-AP, Proteintech, Wuhan, China), anti-GAPDH (Cat# 10494-1-AP, Proteintech, Wuhan, 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: The primary antibodies included anti-NF-κB-p65 (Cat#ab16502, Abcam, Cambridge, UK), anti-NF-κB-p-p65 (S536) (Cat# ab76302, Abcam, Cambridge, UK), anti-tERK (Cat#8544, CST, Boston, USA), anti-tERK (Cat#4695, CST, Boston, MA, USA), anti-GPR37 (Cat#14820-1-AP, Proteintech, Wuhan, China), anti-GAPDH (Cat# 10494-1-AP, Proteintech, Wuhan, China).

Techniques: Derivative Assay, Activation Assay