Journal: Neural Regeneration Research

Article Title: Trans-spinal magnetic stimulation upregulates microglial SOCS3 to attenuate neuroinflammation in chronic constriction injury–induced neuropathic pain

doi: 10.4103/NRR.NRR-D-24-00912

Figure Lengend Snippet: rMS-induced upregulation of SOCS3 in microglia is CaMKKβ/AMPK-dependent in vitro . (A) A schematic diagram of the in vitro study. (B) Western blotting data indicating that the p-CaMKKβ protein level in BV-2 cells increased at 5 minutes after a single administration of rMS and was highest at 15 minutes post-rMS ( n = 4). (C) Western blotting data showing that the p-AMPK protein level in BV-2 cells increased significantly at 30 minutes after rMS ( n = 4). (D, E) Representative western blotting results show that STO-609 and compound C alleviated rMS-induced activation of AMPK in vitro ( n = 4). (F–H) Representative western blotting data showing that compound C abrogated rMS-induced upregulation of SOCS3 and inhibition of p38 and STAT3 in vitro ( n = 4). (I) Representative immunofluorescence images showing that rMS inhibited the translocation of NF-κB p65 (green, Alexa Fluor 488) from the cytosol to the nucleus after LPS treatment in vitro , where this effect was reversed by administration of compound C (20 μmol/mL) 0.5 hours before LPS pretreatment ( n = 3). Arrows indicate BV-2 cells. Scale bars: 10 μm. Data are expressed as mean ± SD. # P < 0.05, ## P < 0.01, vs . Control group; * P < 0.05, ** P < 0.01, **** P < 0.001, vs . LPS group; † P < 0.05, †† P < 0.01, vs. LPS + rMS group (one‐way analysis of variance followed by Tukey’s multiple comparisons tests). AMPK: Adenosine 5′-monophosphate-activated protein kinase; CaMKKβ: calmodulin-dependent protein kinase kinase beta; Compound C: an AMPK inhibitor; DAPI: 4′,6-diamidino-2-phenylindole; LPS: lipopolysaccharide; NF-κB: nuclear factor kappa-B; p-AMPK: phosphorylated AMPK(Thr172); p-CaMKKβ: phospho-CaMKKβ(Ser511); rMS: repetitive magnetic stimulation; SOCS3: suppressor of cytokine signaling 3; STAT3: transducer and activator of transcription 3; STO-609: a CaMKKβ inhibitor.

Article Snippet: Intrathecal injections of compound C, an adenosine 5′-monophosphate-activated protein kinase (AMPK) inhibitor (10 μg/10 μL; MedChemExpress, Newark, NJ, USA, Cat# HY-13418A), and SOCS3 siRNA (500 pmol/10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# SC-270156) were performed as described previously by Mestre et al. (1994).

Techniques: In Vitro, Western Blot, Activation Assay, Inhibition, Immunofluorescence, Translocation Assay, Control

Journal: Neural Regeneration Research

Article Title: Trans-spinal magnetic stimulation upregulates microglial SOCS3 to attenuate neuroinflammation in chronic constriction injury–induced neuropathic pain

doi: 10.4103/NRR.NRR-D-24-00912

Figure Lengend Snippet: The pain-relieving effect and upregulation of SOCS3 induced by focal rTSMS depended on the activation of AMPK. (A) Timeline diagram of CCI modeling, rTSMS treatment, mechanical withdrawal threshold assessment, and experimental analysis in rats. (B) Western blotting data illustrates that the p-AMPK protein level decreased in CCI model rats and increased in CCI + rTSMS + Vehicle rats ( n = 5). (C, D) Intrathecal injection of compound C largely prevented the pain-relieving effects of focal rTMS, when applied once or repeatedly, in rats with CCI neuropathic pain ( n = 8). (E–H) Representative western blotting results showing that compound C alleviated the rTSMS-induced activation of AMPK, increase in SOCS3, and downregulation of p38 and STAT3 ( n = 5). (I, J) Representative immunofluorescence images showing that compound C abrogated the rTSMS-induced upregulation of SOCS3 (green, Alexa Fluor 488) in the dorsal horn of the spinal cord ( n = 3). Dashed box indicates the region of interest in the dorsal horn of the spinal cord. Scale bars: 100 μm. Data are expressed as mean ± SD. # P < 0.05, ## P < 0.01, ### P < 0.001, vs. Sham + Vehicle group; * P < 0.05, ** P < 0.01, **** P < 0.0001, vs . CCI + Vehicle group; † P < 0.05, †† P < 0.01, ††† P < 0.001, †††† P < 0.0001, vs . CCI + rTSMS + Compound C group (C, D: two‐way analysis of variance; B, E–H, J: one‐way analysis of variance followed by Tukey’s multiple comparisons tests). AMPK: Adenosine 5′-monophosphate-activated protein kinase; CCI: chronic constrictive injury; DAPI: 4′,6-diamidino-2-phenylindole; Iba-1: ionized calcium binding adaptor molecule-1; p-AMPK: phosphorylated AMPK(Thr172); p-p38: phospho-p38 mitogen-activated protein kinase; p-STAT3: phospho-STAT3; rTSMS: repetitive trans-spinal magnetic stimulation; SOCS3: suppressor of cytokine signaling 3; STAT3: transducer and activator of transcription 3.

Article Snippet: Intrathecal injections of compound C, an adenosine 5′-monophosphate-activated protein kinase (AMPK) inhibitor (10 μg/10 μL; MedChemExpress, Newark, NJ, USA, Cat# HY-13418A), and SOCS3 siRNA (500 pmol/10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# SC-270156) were performed as described previously by Mestre et al. (1994).

Techniques: Activation Assay, Western Blot, Injection, Immunofluorescence, Binding Assay

Journal: Diabetes & Metabolism Journal

Article Title: Lactate-Induced Lipid Accumulation in Hepatocytes through GPR81 Activation

doi: 10.4093/dmj.2024.0531

Figure Lengend Snippet: Lactate-mediated G-protein-coupled receptor 81 (GPR81) activation regulates 5’ adenosine monophosphate-activated protein kinase (AMPK) in alpha mouse liver 12 (AML12) cells. (A) AML12 cells were treated with sodium L-lactate 20 and 40 mM for 3 days. (B) AML12 cells were treated with small interfering RNA (siRNA) 50 nM with or without lactate 20 mM for 4 days. Cell lysates were analyzed by Western blot to measure the protein levels of phosphorylation AMPK (p-AMPK) and AMPK. (C) AML12 cells were treated with lactate for 2 days and then co-treated with or without 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) 100 µM for next 2 days. Lipid accumulation was evaluated using Oil Red O staining. (D) AML12 cells were treated with lactate for 2 days and then co-treated with or without AICAR 100 µM for next 1 day. Representative images of immunoblots of mature form of sterol regulatory element-binding protein 1c (SREBP1c), CD36, and fatty acid binding protein 4 (FABP4). β-Actin or tubulin is a loading control. NS, not significant. Statistical significance compared with control is indicated by a P <0.05, b P <0.01, c P <0.0001. Statistical significance compared with si-scramble is indicated by d P <0.01, with siGPR81 indicated e P <0.01. Statistical significance compared with lactate 20 mM is indicated by f P <0.05, g P <0.01, h P <0.001.

Article Snippet: Antibodies for GPR81 (MBS821939, My BioSource, San Diego, CA, USA), MCT1 (sc-365501, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MCT4 (sc-376140, Santa Cruz Biotechnology), β-actin (sc-47778, Santa Cruz Biotechnology), phosphorylation 5’ adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172 #2535, Cell Signaling Technology, Danvers, MA, USA), AMPK (#2532, Cell Signaling Technology), peroxisome proliferator-activated receptor alpha (PPARα) (sc-398394 Santa Cruz Biotechnology), sterol regulatory element-binding protein 1 (SREBP1) (NB100-2215, Novus Biologicals, Centennial, CO, USA), stearoyl-CoA desaturase-1 (SCD1) (#2794, Cell Signaling Technology), acetyl-CoA carboxylase (ACC) (#3676, Cell Signaling Technology), and α-tubulin (#12351, Cell Signaling Technology) were used in this study.

Techniques: Activation Assay, Small Interfering RNA, Western Blot, Phospho-proteomics, Staining, Binding Assay, Control

Journal: Endocrinology and Metabolism

Article Title: Effects of Progranulin Deficiency on Inflammation and Fibrosis in the Kidneys and Liver of Diabetic Mice Fed a High-Fat Diet

doi: 10.3803/EnM.2025.2339

Figure Lengend Snippet: Sodium-glucose co-transporter-2 inhibitor- and progranulin knockout-induced autophagy via the mechanistic target of rapamycin complex 1 pathway in the kidneys of each group. Western blot analysis of the protein levels of (A) phosphorylated AMP-activated protein kinase (p-AMPK) and AMPK, (B) p-Akt and Akt, and (C) p-S6 and S6 in the kidneys. Comparisons among groups were performed with one-way analysis of variance followed by Tukey tests. Data are expressed as mean±standard deviation. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; WT, wild-type; DM, diabetes mellitus; Tofo, tofogliflozin; KO, knockout. a P <0.05, b P <0.01 vs. WT-DM; c P <0.05 vs. WT-Tofo.

Article Snippet: Membranes were blocked with a blocking reagent (0.1% Tween 20 and 5% bovine serum albumin in Tris-buffered saline) for 1 hour and subsequently incubated overnight at 4°C with the following primary antibodies (1:1,000): phospho-S6 (#2211; Cell Signaling Technology, Danvers, MA, USA), S6 (#2217; Cell Signaling Technology), phosphorylated AMP-activated protein kinase (p-AMPK) (#2531; Cell Signaling Technology), AMPK (#2532; Cell Signaling Technology), phospho-Akt (#4060; Cell Signaling Technology), Akt (#4691; Cell Signaling Technology), peroxisome proliferator-activated receptor α (PPARα) (#SC-398394; Santa Cruz Biotechnology, Dallas, TX, USA), and PPARγ (#2435; Cell Signaling Technology).

Techniques: Knock-Out, Western Blot, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: Effects of Progranulin Deficiency on Inflammation and Fibrosis in the Kidneys and Liver of Diabetic Mice Fed a High-Fat Diet

doi: 10.3803/EnM.2025.2339

Figure Lengend Snippet: Sodium-glucose co-transporter-2 inhibitor- and progranulin knockout-induced autophagy via the mechanistic target of rapamycin complex 1 pathway in the livers of each group. Western blot analysis of the protein levels of (A) phosphorylated AMP-activated protein kinase (p-AMPK) and AMPK, (B) p-Akt and Akt, and (C) p-S6 and S6 in the liver. Comparisons among groups were performed with one-way analysis of variance followed by Tukey tests. Data are expressed as mean±standard deviation. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; WT, wild-type; DM, diabetes mellitus; Tofo, tofogliflozin; KO, knockout. a P <0.05.

Article Snippet: Membranes were blocked with a blocking reagent (0.1% Tween 20 and 5% bovine serum albumin in Tris-buffered saline) for 1 hour and subsequently incubated overnight at 4°C with the following primary antibodies (1:1,000): phospho-S6 (#2211; Cell Signaling Technology, Danvers, MA, USA), S6 (#2217; Cell Signaling Technology), phosphorylated AMP-activated protein kinase (p-AMPK) (#2531; Cell Signaling Technology), AMPK (#2532; Cell Signaling Technology), phospho-Akt (#4060; Cell Signaling Technology), Akt (#4691; Cell Signaling Technology), peroxisome proliferator-activated receptor α (PPARα) (#SC-398394; Santa Cruz Biotechnology, Dallas, TX, USA), and PPARγ (#2435; Cell Signaling Technology).

Techniques: Knock-Out, Western Blot, Standard Deviation