Journal: Neural Regeneration Research

Article Title: Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

doi: 10.4103/1673-5374.360245

Figure Lengend Snippet: Metformin promotes vascular regeneration in aged mice post-SCI. (A) Representative image of spinal cord blood vessels immunostained for CD31 (green-Alexa Fluor® 488) in aged mice treated with metformin (100 mg/kg) or PBS. Metformin markedly enhanced vascular density in aged mice after metformin administration compared with the PBS group. Red lines indicate the epicenter of the injury site. (B) Quantification of the CD31-labeled blood vessels. (C) Representative images of blood vessels immunostained with CD31 (green-Alexa Fluor® 488) and Ki67 (red-Alexa Fluor® 594) in the sham mice and in aged metformin-treated and PBS-treated mice 7 days post-SCI. Metformin promoted more vascular regeneration in aged mice after metformin administration compared with the PBS group. Scale bars: 150 μm in A, 50 μm in C. (D) Quantification of CD31 + Ki67 + cells in the different groups. (E) Representative western blot images of p-AMPK, AMPK, p-eNOS, and eNOS expression in aged mice 7 days post-SCI. (F, G) Quantification of p-AMPK/AMPK and p-eNOS/eNOS expression in aged mice or the sham group treated with metformin or PBS. The data are presented as the mean ± SEM ( n = 3 per group). # P < 0.05, ## P < 0.01 (two-tailed Student’s t -test [B]), or one-way analysis of variance followed by Tukey’s multiple comparisons test [D, E–G]). PBS: Phosphate buffer saline; SCI: spinal cord injury.

Article Snippet: The membrane was then incubated overnight at room temperature (22–25°C) with antibodies against AMPK (rabbit, 1:500, Wanleibio, Shenyang, China, Cat# WL02254), phosphor-AMPK (p-AMPK; rabbit,1:500, Wanleibio, Cat# WL05103), eNOS (rabbit, 1:500; Wanleibio, Cat# WL01789), and phosphor-eNOS (p-eNOS; rabbit, 1:1000, CST, Beverly, MA, USA, Cat# 9571, RRID: AB_329837).

Techniques: Labeling, Western Blot, Expressing, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

doi: 10.4103/1673-5374.360245

Figure Lengend Snippet: Inhibition of the AMPK/eNOS pathway attenuates the beneficial effects of metformin on angiogenesis in SCMECs in vitro . (A) Representative western blot images of p-AMPK, AMPK, p-eNOS, and eNOS expression in SCMECs treated with metformin, metformin + compound C or PBS ( n = 3 per group). (B, C) Quantification of p-AMPK/AMPK and p-eNOS/eNOS expression in SCMECs subjected to different treatments. (D) Representative images of wound healing by SCMECs treated with PBS, metformin, or metformin + compound C. (E) Representative images of Transwell analysis of SCMECs treated with PBS, metformin, or metformin + compound C. (F) Quantification of the wound closure data shown in D. (G) Quantification of the migration data shown in E. (H) Representative images of tube formation by SCMECs treated with PBS, metformin, or metformin + compound C. Scale bars: 300 μm in D, 200 μm in E, 100 μm in H. (I, J) Quantification of the total branching length and tube mesh extension data shown in H. The data are presented as the mean ± SEM ( n = 3 per group in A–C, n = 6 per group in D–J). # P < 0.05, ## P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparisons test (B, C), or two-tailed Student’s t -test (F–J)). AMPK: Adenosine monophosphate-activated protein kinase; Compound C: an AMPK inhibitor; eNOS: endothelial nitric oxide synthase; p-AMPK: phosphor-AMPK; p-eNOS: phosphor-eNOS; PBS: phosphate buffer saline; SCMECs: spinal cord microvascular endothelial cells.

Article Snippet: The membrane was then incubated overnight at room temperature (22–25°C) with antibodies against AMPK (rabbit, 1:500, Wanleibio, Shenyang, China, Cat# WL02254), phosphor-AMPK (p-AMPK; rabbit,1:500, Wanleibio, Cat# WL05103), eNOS (rabbit, 1:500; Wanleibio, Cat# WL01789), and phosphor-eNOS (p-eNOS; rabbit, 1:1000, CST, Beverly, MA, USA, Cat# 9571, RRID: AB_329837).

Techniques: Inhibition, In Vitro, Western Blot, Expressing, Migration, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

doi: 10.4103/1673-5374.360245

Figure Lengend Snippet: Inhibition of the AMPK/eNOS pathway abolishes the beneficial effects of metformin on functional recovery in the aged mice following injury. (A, B) Functional evaluation of locomotor recovery in aged mice treated with metformin or metformin + compound C and the sham group as determined by BMS scoring over a 56-day period. Compound C reversed the beneficial effects of metformin on hindlimb functional recovery in aged mice after SCI. (C) Sensory functional analysis based on withdrawal threshold in aged mice treated with metformin or metformin + compound C and the sham group post-injury. (D) Representative electrophysiological traces in aged mice treated with metformin only or metformin + compound C post-injury at 56 days and in the sham group. The metformin + compound C group exhibited more severe MEP disruption compared with the metformin only group. (E, F) Quantification of the amplitude and latency period data from the different groups as shown in D. (G) Representative hematoxylin-eosin-stained transverse sections from the injury epicenter among the different groups. The metformin plus compound C group exhibited more severe pathological changes. Red lines indicate the lesion area. Scale bar: 200 μm. (H, I) Quantification of the healthy and lesioned tissue data shown in G. (J) Representative images of footprint analysis of aged mice before surgery and 56 days post-SCI treated with metformin or metformin + compound C. The metformin plus compound C group exhibited more severely impaired hindlimb motor recovery than the metformin only group based on footprint analysis. (K, L) Quantification of the stride length and paw rotation data shown in J. The data are presented as the mean ± SEM ( n = 6 per group). # P < 0.05; ¦ P < 0.05, ¦¦ P < 0.01, vs . aged mice post-SCI with metformin + compound C treatment (two-tailed Student’s t -test). AMPK: Adenosine monophosphate-activated protein kinase; BMS: Basso Mouse Scale; CC: Compound C, an AMPK inhibitor; eNOS: endothelial nitric oxide synthase; Met: metformin; MEP: motor evoked potential; SCI: spinal cord injury.

Article Snippet: The membrane was then incubated overnight at room temperature (22–25°C) with antibodies against AMPK (rabbit, 1:500, Wanleibio, Shenyang, China, Cat# WL02254), phosphor-AMPK (p-AMPK; rabbit,1:500, Wanleibio, Cat# WL05103), eNOS (rabbit, 1:500; Wanleibio, Cat# WL01789), and phosphor-eNOS (p-eNOS; rabbit, 1:1000, CST, Beverly, MA, USA, Cat# 9571, RRID: AB_329837).

Techniques: Inhibition, Functional Assay, Staining, Two Tailed Test

Journal: Neural Regeneration Research

Article Title: Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

doi: 10.4103/1673-5374.360245

Figure Lengend Snippet: Inhibition of the AMPK/eNOS pathway reverses the beneficial effects of metformin on angiogenesis in aged mice following injury. (A) Representative images of spinal cord blood vessels immunostained with CD31 (green-Alexa Fluor® 488) in aged mice treated with metformin (100 mg/kg) or metformin + compound C. The metformin + compound C group exhibited less vascular density than the metformin only group. Red lines indicate the epicenter of the injury site. (B) Quantification of the CD31-labeled blood vessels. (C) Representative images of blood vessels immunostained with CD31 (green-Alexa Fluor® 488) and Ki67 (red-Alexa Fluor® 594) in the sham group and in aged mice treated with metformin or metformin + compound C at 7 days post-SCI. The metformin + compound C group exhibited less vascular regeneration than the metformin only group. Scale bars: 150 μm in A, 50 μm in C. (D) Quantification of CD31 + Ki67 + cells. The data are presented as the mean ± SEM ( n = 6 per group). # P < 0.05 (two-tailed Student’s t -test). AMPK: Adenosine monophosphate-activated protein kinase; CC: Compound C, an AMPK inhibitor; DAPI: 4′,6-diamidino-2-phenylindole; eNOS: endothelial nitric oxide synthase; Met: metformin; SCI: spinal cord injury.

Article Snippet: The membrane was then incubated overnight at room temperature (22–25°C) with antibodies against AMPK (rabbit, 1:500, Wanleibio, Shenyang, China, Cat# WL02254), phosphor-AMPK (p-AMPK; rabbit,1:500, Wanleibio, Cat# WL05103), eNOS (rabbit, 1:500; Wanleibio, Cat# WL01789), and phosphor-eNOS (p-eNOS; rabbit, 1:1000, CST, Beverly, MA, USA, Cat# 9571, RRID: AB_329837).

Techniques: Inhibition, Labeling, Two Tailed Test

Journal: Diabetes & Metabolism Journal

Article Title: Beneficial Effects of a Curcumin Derivative and Transforming Growth Factor-β Receptor I Inhibitor Combination on Nonalcoholic Steatohepatitis

doi: 10.4093/dmj.2022.0110

Figure Lengend Snippet: EW-7197 relieves hepatocellular fibrosis but has side effects. Hepatocellular fibrosis was induced by treating human hepatic stellate cell (LX-2) cells with transforming growth factor β (TGF-β; 2 ng/mL). (A, B) The ability of EW-7197 to restore the original state was tested by expressing fibrosis markers such as p-SMAD2/3, α-smooth muscle actin (α-SMA), and ColI. (C, D) Alpha mouse liver 12 (AML12) cells were treated with EW-7197 combined with TGF-β. (E, F) AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (Erk), and Akt activities were measured to determine the effect of EW-7197 treatment on the TGF-β non-canonical pathway. a P <0.05 vs. control (Con), b P <0.05 vs. TGF-β, c P <0.001 vs. Con, d P <0.001 vs. TGF-β.

Article Snippet: Antibodies against p-SMAD2 (#3108), p-SMAD3 (#9520), phosphor-AMPK (p-AMPK) (#2531), AMPK (#2532), and Kelch-like ECH-associated protein 1 (Keap1) (#4678) were obtained from Cell Signaling Technologies.

Techniques: Expressing

Journal: Diabetes & Metabolism Journal

Article Title: Beneficial Effects of a Curcumin Derivative and Transforming Growth Factor-β Receptor I Inhibitor Combination on Nonalcoholic Steatohepatitis

doi: 10.4093/dmj.2022.0110

Figure Lengend Snippet: Curcumin 2005-8 (Cur5-8) and EW-7197 co-administration improves hepatic lipid accumulation. (A, B) Accumulation of lipid droplets and variations in liver sizes were measured using H&E staining and liver imaging, respectively. (C) Nonalcoholic fatty liver disease (NAFLD) activity was scored by quantifying the degree of steatosis, hepatocyte ballooning, and lobular inflammation based on the results of H&E staining. (D, E, F) The NAFLD activity score is assigned as described in the Methods section. Metabolism-related proteins were determined by measuring rho-associated coiled-coil kinase 1 (Rock1) and sterol regulatory elementbinding protein 1c (Srebp1c) levels, AMP-activated protein kinase (AMPK) activity, and reactive oxygen species (ROS) scavenging effect. Endoplasmic reticulum stress regulation was determined by measuring Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and activating transcription factor 6α (ATF6α) levels. a P <0.05 vs. control (Con), b P <0.05 vs. methionine-choline-deficient diet (MCD), c P <0.001 vs. Con, d P <0.001 vs. MCD.

Article Snippet: Antibodies against p-SMAD2 (#3108), p-SMAD3 (#9520), phosphor-AMPK (p-AMPK) (#2531), AMPK (#2532), and Kelch-like ECH-associated protein 1 (Keap1) (#4678) were obtained from Cell Signaling Technologies.

Techniques: Staining, Imaging, Activity Assay

Journal: Veterinary Research

Article Title: Curcumin-dependent phenotypic transformation of microglia mediates resistance to pseudorabies-induced encephalitis

doi: 10.1186/s13567-023-01149-x

Figure Lengend Snippet: CUR regulates energy metabolism through AMPK-dependent pathways in PRV-infected BV2 cells. A BV2 cells were infected with/without PRV for 24 h and then treated with/without 20 μM CUR for 24 h. Western blot analysis of p-AMPK Thr172 protein levels (normalized to the t-AMPK protein). β-Actin was used as the loading control ( n = 3). B Relative protein levels of p-AMPK Thr172 . C BV2 cells were treated with different concentrations of Compound C for 24 h, and cell viability was determined by the CCK-8 assay to determine the toxicity range ( n = 4). D BV2 cells were infected with/without PRV or were treated with PRV and an AMPK inhibitor (Compound C or siRNA) alone or in combination for 24 h, followed by treatment with/without 20 μM CUR for 24 h. Western blot analysis of p-AMPK (normalized to the t-AMPK protein), t-AMPK, Gpat4, and LDHa levels (Gpat4 and LDHa values were normalized to the β-actin protein); β-actin was used as the loading control ( n = 3). E Relative p-AMPKThr172 protein levels. F Relative LDHa protein levels. G Relative Gpat4 protein levels. H The extracellular acidification rate of BV2 cells after the different treatments (values normalized to the control) ( n = 4). I The OCR of BV2 cells after the different treatments (values normalized to the control) ( n = 4). J BV2 cell ATP levels in the different treatment groups ( n = 4). K The M1 phenotype-related inflammatory factor TNF-α in BV2 cells was detected using ELISA ( n = 4). L The levels of the M1 phenotype-related inflammatory factor IL-6 in BV2 cells ( n = 4). M The levels of the M2 phenotype-related anti-inflammatory factor IL-4 in BV2 cells ( n = 4). N The levels of the M2 phenotype-related anti-inflammatory factor IL-10 in BV2 cells ( n = 4). All experiments were performed in parallel. The results are presented as the mean ± SD. Statistical significance was determined using one-way ANOVA followed by an LSD post hoc test for multiple comparisons among the groups. * P < 0.05, ** P < 0.01, *** P < 0.001, and NS, not significant.

Article Snippet: Furthermore, the membrane was blocked with 5% nonfat dry milk or 5% BSA in tris-buffered saline containing Tween-20 (TBST) at 25 ℃ and then incubated overnight at 4 °C with the following antibodies: rabbit anti-AMPK (1:2000, bs-1115R, Bioss, China), phosphor(p)-AMPK Thr172 (1:1000, 2531, Cell Signaling, USA), lactate dehydrogenase A (LDHa, 1:1000, WL03271, Wanleibio, China), nuclear factor kappa-B p65 (NF-κB p65, 1:1000, WL01273b, Wanleibio, China), p-NF-κB p65 Ser536 (1:10 000, WL02169, Wanleibio, China), glycerol-3-phosphate acyltransferase 4 (GPAT4, 1:1000, bs-15587R, Bioss, China), Bax (1:10 000, 50,599–2-Ig, Proteintech, China), Caspase-3/p17/p19 (1:1000, 19,677–1-AP, Proteintech, China), Bcl-2 (1:4000, 26,593–1-AP, Proteintech, China), and β-actin (1:5000, 20,536–1-AP, Proteintech, China).

Techniques: Infection, Western Blot, CCK-8 Assay, Enzyme-linked Immunosorbent Assay