Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: GPR37 agonists instantly attenuate bone cancer pain. A) Schematic of the establishment of bone cancer pain (BCP) model. B,C) Von Fery testing to detect cancer induced mechanical allodynia, as assessed by withdrawal threshold (B) and withdrawal frequency (C) in mice i.t. injected with vehicle, NDP1 (200 ng) or ARU (20 µg) on day 11 after LLC inoculation. n = 10 mice/group. D) Acetone response test to determine cold allodynia after the indicated treatment. n = 10 mice/group. E) Change of mechanical allodynia through von Frey testing after i.p. injection with vehicle, NPD1 (25 µg kg −1 ) or ARU (25 mg kg −1 ) on day 11 after tumor inoculation. n = 10 mice/group. All data are displayed as mean ± SEM, and analyzed using repeated‐measures two‐way ANOVA with Bonferroni's post‐hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Injection

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: GPR37 agonists attenuate persistent cancer pain and improve locomotor function. A) Experimental design to perform behavior tests after continuous i.p. injection of vehicle, NPD1 (25 µg kg −1 ) or ARU (25 mg kg −1 ). B,C) Von Frey testing to measure mechanical allodynia through withdrawal threshold (B) and withdrawal frequency (C) on day 7, 10 and 14 after tumor inoculation. D) Cold allodynia via acetone response test in mice with indicated treatment. E) Observation of spontaneous pain as shown by flinching (left) or guarding behaviors (right) in vehicle NPD1 or ARU treated mice on day14 after LLC implantation. F) Change of body weight in mice with indicated treatment. G,H) Open field testing detecting distance traveled and mean speed over a 20 min duration in naïve mice or mice with bone cancer pain treated with vehicle, NPD1 or ARU at day 14 after tumor inoculation with representative traces (G) and quantification (H). n = 8 mice/group for the above panels. All data are expressed as mean ±SEM, and analyzed using repeated‐measures two‐way ANOVA with Bonferroni's post‐hoc test (B, C, D, F), or one‐way ANOVA with Bonferroni's post‐hoc test (E, H), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Injection

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: GPR37 agonists protect against cancer induced bone destruction without affecting tumor burden. A) Experimental diagram for continuous i.p. administration of vehicle, NPD1 (25 µg kg −1 ) or ARU (25 mg kg −1 ). B) Representative images showing bone destruction assessed by X‐ray. Bone destruction score is indicated in each image and arrows show bone lesions with scores over 3. C) Quantification for bone destruction scores. D) Comparison of ratio of bone fracture from tumor bearing femora taken from vehicle, NPD1 or ARU‐treated mice on day 17 after LLC inoculation. E,F) In vivo bioluminescence imaging showing no effects of vehicle, NPD1 or ARU treatment on total flux of LL/2‐Luc2 bearing femur on days 7, 10, and 14 after tumor inoculation (F). Images (E) were acquired at 15 min after i.p. injection of d‐luciferin (30 mg kg −1 ). G) Comparison of ratio of maximum thigh circumference in mice with indicated treatment on day 17 after LLC implantation. n = 8 mice/group for A‐G. Data are expressed as mean ± SEM, and analyzed using repeated‐measures two‐way ANOVA with Bonferroni's post‐hoc test (C, F, G), or two‐sided Fisher's exact test (D), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Comparison, In Vivo, Imaging, Injection

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: GPR37 mediates the pain relief and bone protection from NPD1 treatment. A) Study design to detect the protective effect of i.p. injection of NPD1 (25 µg kg −1 ) in WT or Gpr37 −/− mice. B,C) Mechanical allodynia measured by withdrawal threshold (B) and withdrawal frequency (C) on day 7, 10, and 14 after tumor inoculation. D) Cold allodynia via acetone response testing. E,F) Representative X‐ray images (E) and quantification of bone destruction score (F) in WT or Gpr37 −/− mice applied with vehicle or NPD1 (25 µg kg −1 ) on day 8, 11 and 15 post LLC inoculation. Bone destruction score is indicated in each image and arrows show bone lesions with scores over 3. G) Comparison of ratio of bone fracture in tumor bearing femora on day 17 after LLC implantation. n = 6 mice/group. All data displayed represent the mean ± SEM, and are analyzed using repeated‐measures two‐way ANOVA with Bonferroni's post‐hoc test (B, C, D, F), or two‐sided Fisher's exact test (G), * p < 0.05, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Injection, Comparison

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: NPD1 and ARU impaired the activity of small‐sized DRG neurons in BCP mice. A) Schematic of DRG neurons preparation, drug treatment, and bright field image with a recording micropipette sealed on a small‐diameter neuron (nociceptor). B) Representative current‐evoked action potential (AP) traces (left) and quantification of the firing rate (right) in a small DRG neuron from WT naïve mice, BCP mice after vehicle or NPD1 perfusion (10 ng mL −1 , 2 min). C) Representative traces of rheobases (left) and comparison of the averages of rheobases (right) from naïve mice, vehicle or NPD1 treated group (naïve: n = 7 neurons/5 mice; vehicle: n = 9 neurons/5 mice; NPD1: n = 13 neurons/5 mice). D) Representative current‐evoked AP traces (left) and quantification of the No. of AP (right) in small DRG neurons from WT naïve mice, BCP mice after vehicle or ARU perfusion (10 µM, 2 min). E) Representative traces of rheobases (left) and comparison of the averages of rheobases (right) from naïve mice, vehicle or ARU treated group (naïve: n = 8 neurons/5 mice; vehicle: n = 9 neurons/5 mice; ARU: n = 11 neurons/5 mice). F) Representative current‐evoked AP traces (left) and comparison of the firing rate (fight) in small‐diameter DRG neuron from Gpr37 −/− naïve mice, Gpr37 −/− BCP mice after vehicle or NPD1 perfusion. G) Representative traces of rheobases (left) and comparison of the averages of rheobases (right) from Gpr37 −/− naïve mice, vehicle or NPD1 treated group (naïve: n = 8 neurons/5 mice; vehicle: n = 11 neurons/5 mice; NPD1: n = 11 neurons/5 mice). H) Representative current‐evoked AP traces (left) and comparison of the firing rate (right) in small‐diameter DRG neuron from Gpr37 −/− naïve mice, Gpr37 −/− BCP mice after vehicle or ARU perfusion. I) Representative traces of rheobases (left) and comparison of the averages of rheobases (right) from Gpr37 −/− naïve mice, vehicle or ARU treated group (naïve: n = 7 neurons/5 mice; vehicle: n = 11 neurons/5 mice; ARU: n = 8 neurons/5 mice). Data are presented as mean ± SEM, and analyzed using repeated‐measures two‐way ANOVA with Bonferroni's post‐hoc test (B, D, F, H), or one‐way ANOVA with Bonferroni's post‐hoc test (C, E, G, I); ns, not significant; ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Activity Assay, Comparison

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: GPR37 agonist inhibit osteoclastogenesis through IL‐10. A) Immunostaining showed wide expression of GPR37 in bone marrow cells from murine femur section. Scale bar, 50 µm. B,C) Representative images (B) and quantification (C) of TRAP staining to reveal osteoclast numbers in the tumor‐bearing distal femora from mice continuously treated with vehicle, NPD1 (25 µg kg −1 ) or ARU (25 mg kg −1 ) measured on day 11 after LLC inoculation. n = 5 mice/group. Scale bar, 500 µm. D) Level of serum CTX‐I at baseline or on day 17 after tumor implantation. n = 5 mice/group. E,F) TRAP staining revealing osteoclast numbers after differentiation from RAW264.7 cells stimulated with 35 ng mL −1 RANKL, in the presence of vehicle, NPD1 (100 nM) or ARU (10 µM). Arrows indicate TRAP + multinucleated osteoclasts. E Representative TRAP‐stained images. F quantification (n = 3 biologically independent experimental replicates). Scale bar, 200 µm. G,H) TRAP staining for osteoclasts differentiated from BMDM cells from WT mice or Gpr37 −/− mice, each treated with vehicle, NPD1 (100 nM) or ARU (10 µM). RANKL: 35 ng mL −1 , MCSF: 20 ng mL −1 . G Representative images of TRAP staining. Arrows indicate TRAP + osteoclasts. Scale bar, 100 µm. H Quantification for (G), n = 3 independent cultures. I) Measurement of IL‐10 level in the culture medium of Raw 264.7 cells 24 h after vehicle, NPD1 (100 nM) or ARU (10 µM) co‐incubation with RANKL (35 ng mL −1 ). n = 3 biologically independent experimental replicates. J) TRAP staining showing No. of osteoclasts differentiated form Raw 264.7 cells treated with vehicle, ARU (10 µM), anti‐IL‐10 antibody (1 µg mL −1 ) or ARU + anti‐IL‐10 antibody. Left, representative images and arrows indicate TRAP + osteoclasts.; right, quantification. Scale bar, 200 µm. n = 3 biologically independent experimental replicates. Data indicate the mean ± SEM, and are analyzed using one‐way ANOVA with Bonferroni's post‐hoc test (C, F, H, I, J), or repeated‐measures two‐way ANOVA with Bonferroni's post‐hoc test (D), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Immunostaining, Expressing, Staining, Tumor Implantation, Incubation

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: β‐arrestin 2 mediates IL‐10 production and neuronal modulation of GPR37 activation. A) IL‐10 level in the culture medium of Raw 264.7 cells 24 h after vehicle, ARU (10 µM), Forskolin (1 µM) or ARU + Forskolin co‐incubation with RANKL (35 ng mL −1 ). n = 5 biologically independent experimental replicates. B) Western blot showing decreased expression of β‐arrestin 2 in shRNAs treated Raw 264.7 cells. C) RT‐PCR determining reduced level of β‐arrestin 2 mRNA in shRNAs treated Raw 264.7 cells. n = 3 independent experimental replicates. D) Measurement of IL‐10 level in the culture medium of scramble or Arrb2 ‐shRNA‐treated RAW 264.7 cells administered with Vehicle or ARU (10 µM) and with RANKL (35 ng mL −1 ). n = 5 biologically independent experimental replicates. E‐F) TRAP staining for osteoclasts differentiated from scramble or Arrb2 ‐shRNA‐treated RAW 264.7 cells, each treated with vehicle or ARU (10 µM). RANKL: 35 ng mL −1 . E Representative images of TRAP staining. Arrows indicate TRAP + osteoclasts. Scale bar, 200 µm. F Quantification for (E), n = 3 biologically independent experimental replicates. G) Schematic for the establishment of Arrb2 conditional knockout (CKO) mice via DRG injection. H) IF staining showing β‐arrestin 2 expression in DRG neurons from naïve mice or β‐arrestin 2 CKO mice with BCP. Scale Bar, 50 µm. I) Representative current‐evoked AP traces (left) and quantification of the firing rate (right) in small‐diameter DRG neurons from WT mice or β‐arrestin 2 CKO BCP mice after NPD1 (10 ng mL −1 , 2 min) or ARU perfusion (10 µM, 2 min). J) Representative traces of rheobases (left) and comparison of the averages of rheobases (right) from WT mice or β‐arrestin 2 CKO BCP mice after NPD1 or ARU perfusion. (n = 7–8 neurons/5 mice, each group). Data are expressed as mean ± SEM, and analyzed using one‐way ANOVA with Bonferroni's post‐hoc test (A, C, D, F, J), or repeated‐measures two‐way ANOVA with Bonferroni's post‐hoc test (I), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, shRNA, Staining, Knock-Out, Injection, Comparison

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: Effect of NPD1 and ARU on sEPSCs in spinal dorsal horn from BCP mice. A) Immunostaining showed the expression of GPR37 and NeuN in lamina I‐II of the spinal dorsal horn (SDH) from mice with BCP. Scale bar, 50 µm. B) Chart recording showing spontaneous excitatory postsynaptic currents (sEPSCs) in the absence and presence of NPD1 (10 ng mL −1 , 2 min) obtained from WT BCP mouse. C) The average of sEPSCs frequency (left) and amplitude (right) before (control) and 4–5 min after the beginning of NPD1 perfusion, respectively. D) Chart recording showing sEPSCs in the absence and presence of ARU (10 µM, 2 min) obtained from WT BCP mouse. E) The average of sEPSCs frequency (left) and amplitude (right) before (control) and 4–5 min after the beginning of ARU perfusion, respectively. F) Chart recording showing sEPSCs in the absence and presence of NPD1 (10 ng mL −1 , 2 min) obtained from Gpr37 −/− BCP mouse. G) The average of sEPSCs frequency (left) and amplitude (right) before (control) and 4–5 min after the beginning of NPD‐1 perfusion, respectively. H) Chart recording showing sEPSCs in the absence and presence of ARU (10 µM, 2 min) obtained from Gpr37 −/− BCP mouse. I) The average of sEPSCs frequency (left) and amplitude (right) before (control) and 4–5 min after the beginning of ARU perfusion, respectively. The duration of drug perfusion is indicated by a horizontal bar above the chart recording, while consecutive traces of spontaneous events are shown at an expanded time scale, marked by a short bar below the recording. n = 8–10 neurons from 6 mice for B‐I. Data are presented as mean ± SEM, and analyzed using two‐tailed Student's t‐test; ns, not significant; ** p < 0.01, *** p < 0.001.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Immunostaining, Expressing, Control, Two Tailed Test

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: NPD1 is negatively correlated with cancer pain and CTX‐I level in patients with metastatic cancer. A) Schematic diagram for the design of the clinical study. B) Comparison of pain intensity via number rating score (NRS) in cancer patients with bone metastasis. These patients were divided into NPD1‐low and NPD1‐high groups according to their levels of serum NPD1. n = 40 patients in NPD1‐low group and n = 39 patients in NPD1‐high group. C) Correlation between serum NPD1 level and NRS score in the enrolled patients. n = 79 patients. D) Elisa measurement of plasma CTX‐I level in NPD1‐low (n = 40) and NPD1‐high (n = 39) patients. E) Correlation between plasma NPD1 level and plasma CTX‐I level in the enrolled patients (n = 79). F) Graphical abstract of the current study. GPR37 activation reduces cancer pain and cancer induced bone destruction via peripheral and neuronal mechanism. NPD1 or ARU acting on GPR37 expressed in macrophage could promote the release of IL‐10 via recruitment of β‐arrestin 2 which further inhibit cancer induced ostoclastogenesis. Moreover, GPR37 activation in DRG could suppress No. of action potentials via β‐arrestin 2 pathway, while GPR37 activation in SDH decreases the frequency of sEPSCs, both leading to the inhibition of cancer induced neuronal hyperexcitability. Data in A and C are expressed as median (interquartile range), and analyzed with Mann‐Whitney test (B, D) and Person correlation test (C, E), * p < 0.05.

Article Snippet: The following reagents from the indicated vendors were used in this study: NPD1 (Cayman Chemical, 10 010 390), ARU (Cayman Chemical, 11 817), TX14 (Anaspec, AS‐60248‐1), mouse RANKL protein (R&D systems, 462‐TEC), mouse MCSF (R&D systems, 416‐ML), anti‐IL‐10 antibody (Millipore Sigma, i5145), Forskolin (MCE, HY‐15371).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Activation Assay, Inhibition, MANN-WHITNEY

Journal: Advanced Science

Article Title: GPR37 Activation Alleviates Bone Cancer Pain via the Inhibition of Osteoclastogenesis and Neuronal Hyperexcitability

doi: 10.1002/advs.202417367

Figure Lengend Snippet: NPD1 is negatively correlated with cancer pain and CTX‐I level in patients with metastatic cancer. A) Schematic diagram for the design of the clinical study. B) Comparison of pain intensity via number rating score (NRS) in cancer patients with bone metastasis. These patients were divided into NPD1‐low and NPD1‐high groups according to their levels of serum NPD1. n = 40 patients in NPD1‐low group and n = 39 patients in NPD1‐high group. C) Correlation between serum NPD1 level and NRS score in the enrolled patients. n = 79 patients. D) Elisa measurement of plasma CTX‐I level in NPD1‐low (n = 40) and NPD1‐high (n = 39) patients. E) Correlation between plasma NPD1 level and plasma CTX‐I level in the enrolled patients (n = 79). F) Graphical abstract of the current study. GPR37 activation reduces cancer pain and cancer induced bone destruction via peripheral and neuronal mechanism. NPD1 or ARU acting on GPR37 expressed in macrophage could promote the release of IL‐10 via recruitment of β‐arrestin 2 which further inhibit cancer induced ostoclastogenesis. Moreover, GPR37 activation in DRG could suppress No. of action potentials via β‐arrestin 2 pathway, while GPR37 activation in SDH decreases the frequency of sEPSCs, both leading to the inhibition of cancer induced neuronal hyperexcitability. Data in A and C are expressed as median (interquartile range), and analyzed with Mann‐Whitney test (B, D) and Person correlation test (C, E), * p < 0.05.

Article Snippet: Elisa kits including NPD1 (Biomatik, EKF58060), CTX‐I (CUSABIO, CSB‐E11224 h), IL‐10 (CUSABIO, CSB‐E04594 m) were obtained and applied.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Activation Assay, Inhibition, MANN-WHITNEY

Journal: Metabolites

Article Title: Lipidomics in Understanding Pathophysiology and Pharmacologic Effects in Inflammatory Diseases: Considerations for Drug Development

doi: 10.3390/metabo12040333

Figure Lengend Snippet: Summary of lipidomics studies in inflammatory disease research.

Article Snippet: In a Sprague-Dawley MCAO model, intravenous administration of aspirin-triggered neuroprotectin D1 (AT-NPD1) 3 h post-stroke improved neurologic scores up to 7 days after stroke, reduced radiographic measures of cerebral edema, and decreased histopathologic infarct volume [ ].

Techniques: Clinical Proteomics, Targeted Proteomics, Produced, Activation Assay, Activity Assay, Biomarker Discovery, Membrane, Mouse Assay, Derivative Assay