Journal: Scientific reports

Article Title: Combination of high-fat/high-fructose diet and low-dose streptozotocin to model long-term type-2 diabetes complications.

doi: 10.1038/s41598-017-18896-5

Figure Lengend Snippet: Figure 9. Effect of HF/HF/Stz regimen on neuronal and glial activation in dorsal root ganglia and spinal dorsal horn. Within DRG, CGRP-immunoreactive neurons (A), but not SP-labeled cells (B) are decreased in the HF/ HF/Stz group. Within the spinal cord, the HF/HF/Stz regimen increases the number of GFAP-labeled astrocytes (C) but not the number of IBA1-positive microglial cells (D). Moreover, the HF/HF/Stz regimen induces a significant increase in Fos expression within lamina II, used here as a marker of nociceptive neuronal activation (E). Error bars represent the SEM. *P < 0.05, ***P < 0.001, compared with the chow-diet group. Data (A,B,C and D) are analyzed using a two-tailed Mann-Whitney U-test whereas data from (E) are analyzed using a Kruskal-Wallis test followed by the Dunn’s multiple comparisons test.

Article Snippet: Cryostat sections of dorsal root ganglia (DRG) were incubated overnight at 4 °C with either the anti-Substance P or anti-calcitonin gene-related peptide (CGRP) antibodies (1/500, Guinea Pig anti-SP and anti-CGRP, Neuromics, USA).

Techniques: Activation Assay, Labeling, Expressing, Marker, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Calcitonin gene‐related peptide inhibits angiotensin II‐induced NADPH oxidase‐dependent ROS via the Src/STAT3 signalling pathway

doi: 10.1111/jcmm.15288

Figure Lengend Snippet: CGRP inhibited oxidative stress in Ang II‐induced VSMCs. Vascular smooth muscle cells (VSMCs), stimulated with CGRP (10 −6 ‐10 −8 mol/L) for 60 min, CGRP 8‐37 (3 × 10 −5 mol/L) for 30 min or apocynin (10 −6 mol/L) for 2 h, were treated with Ang II (10 −7 mol/L) for 30 min. A, Quantification of intracellular reactive oxygen species (ROS) levels. ROS levels were measured using DCFH‐DA as described in method. B, Quantitation of the intracellular NADP/NADPH ratio. C, Western blot analysis of membrane and cytoplasmic fractions of p47phox in VSMCs. D, Quantitative real‐time PCR analysis of mRNA levels of p47phox in VSMCs. Bar graphs show mean ± SEM values from three independent experiments. * P < .05, ** P < .01 and *** P < .001 vs Ang II. # P < .05 vs control

Article Snippet: VSMCs were first pre‐treated Ang II (10 −7 mol/L) for 30 minutes and then stimulated with or without and CGRP (10 −7 mol/L) for 60 minutes and CGRP 8–37 30 minutes. cAMP and PKA levels of the VSMCs were measured using parameter cAMP assay (R&D Systems) and protein kinase A (PKA) ELISA (Mlbio) kits, respectively, according to the manufacturers’ protocols.

Techniques: Quantitation Assay, Western Blot, Membrane, Real-time Polymerase Chain Reaction, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Calcitonin gene‐related peptide inhibits angiotensin II‐induced NADPH oxidase‐dependent ROS via the Src/STAT3 signalling pathway

doi: 10.1111/jcmm.15288

Figure Lengend Snippet: CGRP inhibited oxidative stress via receptors/ cAMP‐PKA‐dependent pathway. VSMCs, stimulated with CGRP (10 −6 ‐10 −8 mol/L) for 60 min or CGRP 8‐37 (3 × 10 −5 mol/L) for 30 min, were treated with Ang II (10 −7 mol/L) for 30 min. In some experiments, dibutyl‐cAMP (10 −3 mol/L) was applied for 60 min and H‐89 was applied 30 min before CGRP pre‐treatment. A, Quantitative real‐time PCR analysis of the mRNA levels of CRLR, RAMP1 and RCP in VSMCs. B, Western blot analyses of protein levels of CRLR, RAMP1 and RCP in VSMCs. C, Quantification of intracellular ROS levels in VSMCs after pre‐treatment with dibutyl‐cAMP or H‐89. D, Quantitative real‐time PCR analysis of mRNA levels of p47phox in VSMCs. E, Western blot analysis of membrane and cytoplasmic fractions of p47phox in VSMCs. Bar graphs show mean ± SEM values from three independent experiments. * P < .05, ** P < .01 and *** P < .001 vs Ang II. # P < .05 vs control. + P < .05 vs CGRP + Ang II

Article Snippet: VSMCs were first pre‐treated Ang II (10 −7 mol/L) for 30 minutes and then stimulated with or without and CGRP (10 −7 mol/L) for 60 minutes and CGRP 8–37 30 minutes. cAMP and PKA levels of the VSMCs were measured using parameter cAMP assay (R&D Systems) and protein kinase A (PKA) ELISA (Mlbio) kits, respectively, according to the manufacturers’ protocols.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Membrane, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Calcitonin gene‐related peptide inhibits angiotensin II‐induced NADPH oxidase‐dependent ROS via the Src/STAT3 signalling pathway

doi: 10.1111/jcmm.15288

Figure Lengend Snippet: CGRP attenuated Ang II‐induced Src/STAT3 activation in VSMCs. VSMCs, stimulated with CGRP (10 −7 mol/L) for 60 min or CGRP 8‐37 (3 × 10 −5 mol/L) for 30 min, were treated with Ang II (10 −7 mol/L) for 30 min. A and B, Phosphorylation of Src and STAT3 was evaluated via Western blot analysis. C and D, The distribution and levels of p‐Src and p‐STAT3 were detected via immunofluorescence. E, VSMCs were treated with PP2 (10 −5 mol/L, 20 min), and phosphorylation of STAT3 was evaluated via Western blot analysis. Bar graphs show mean ± SEM values from three independent experiments. * P < .05, ** P < .01 and *** P < .001 vs Ang II. # P < .05 vs control

Article Snippet: VSMCs were first pre‐treated Ang II (10 −7 mol/L) for 30 minutes and then stimulated with or without and CGRP (10 −7 mol/L) for 60 minutes and CGRP 8–37 30 minutes. cAMP and PKA levels of the VSMCs were measured using parameter cAMP assay (R&D Systems) and protein kinase A (PKA) ELISA (Mlbio) kits, respectively, according to the manufacturers’ protocols.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Immunofluorescence, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Calcitonin gene‐related peptide inhibits angiotensin II‐induced NADPH oxidase‐dependent ROS via the Src/STAT3 signalling pathway

doi: 10.1111/jcmm.15288

Figure Lengend Snippet: CGRP attenuated Ang II‐induced Src/STAT3 activation via receptor/cAMP‐PKA‐dependent pathway in VSMCs. VSMCs, stimulated with CGRP (10 −7 mol/L) for 60 min or CGRP 8‐37 (3 × 10 −5 mol/L) for 30 min, were treated with Ang II (10 −7 mol/L) for 30 min. In some experiments, dibutyl‐cAMP (10 −3 mol/L) and H‐89 (10 −5 mol/L) were applied 60 and 30 min before CGRP pre‐treatment, respectively. Phosphorylation of Src (A) and STAT3 (B) was evaluated via Western blot analysis. Bar graphs show mean ± SEM values from three independent experiments. * P < .05, ** P < .01 and *** P < .001 vs Ang II. # P < .05 vs control

Article Snippet: VSMCs were first pre‐treated Ang II (10 −7 mol/L) for 30 minutes and then stimulated with or without and CGRP (10 −7 mol/L) for 60 minutes and CGRP 8–37 30 minutes. cAMP and PKA levels of the VSMCs were measured using parameter cAMP assay (R&D Systems) and protein kinase A (PKA) ELISA (Mlbio) kits, respectively, according to the manufacturers’ protocols.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Calcitonin gene‐related peptide inhibits angiotensin II‐induced NADPH oxidase‐dependent ROS via the Src/STAT3 signalling pathway

doi: 10.1111/jcmm.15288

Figure Lengend Snippet: Src/STAT3 signalling pathway is involved in the antioxidant activity of CGRP. VSMCs, stimulated with CGRP (10 −7 mol/L) for 60 min or CGRP 8‐37 (3 × 10 −5 mol/L) for 30 min, were treated with Ang II (10 −7 mol/L) for 30 min. In some experiments, PP2 (10 −5 mol/L) or niclosamide (10 −5 mol/L) was applied 20 min before CGRP pre‐treatment. VSMCs were transfected with Src ORF cDNA clones (Src) or STAT3 ORF cDNA clones (STAT3) and incubated for 24 h. Increased Src (A) and STAT3 (B) protein levels in VSMCs were evaluated via Western blot analysis after transfection with Src or STAT3 ORF cDNA clones, respectively. (C), Quantification of intracellular ROS levels in VSMCs via DCFH‐DA. Src group, VSMCs were transfected with Src ORF cDNA clones; STAT3 group, VSMCs were transfected with STAT3 ORF cDNA clones; and control group, VSMCs were transfected with blank vector. Bar graphs show mean ± SEM values from three independent experiments. * P < .05, ** P < .01 and *** P < .001 vs Ang II. # P < .05 vs control. + P < .05 vs CGRP + Ang II

Article Snippet: VSMCs were first pre‐treated Ang II (10 −7 mol/L) for 30 minutes and then stimulated with or without and CGRP (10 −7 mol/L) for 60 minutes and CGRP 8–37 30 minutes. cAMP and PKA levels of the VSMCs were measured using parameter cAMP assay (R&D Systems) and protein kinase A (PKA) ELISA (Mlbio) kits, respectively, according to the manufacturers’ protocols.

Techniques: Antioxidant Activity Assay, Transfection, Clone Assay, Incubation, Western Blot, Control, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Calcitonin gene‐related peptide inhibits angiotensin II‐induced NADPH oxidase‐dependent ROS via the Src/STAT3 signalling pathway

doi: 10.1111/jcmm.15288

Figure Lengend Snippet: CGRP suppressed hypertrophy and hyperplasia of VSMCs in vitro and in vivo . VSMCs, stimulated with CGRP (10 −7 mol/L) for 60 min or CGRP 8‐37 (3 × 10 −5 mol/L) for 30 min, were treated with Ang II (10 −7 mol/L) for 30 min. In some experiments, NAC (10 −2 mol/L) or H 2 O 2 (10 −3 mol/L) was applied 30 min before CGRP or Ang II pre‐treatment. A and B, The proliferation of VSMCs was analysed using BrdU assays. C, Systolic blood pressure (SBP) was measured by tail‐cuff plethysmography in three groups: control group (saline‐treated), Ang II (750 µg/kg/d, in saline)‐treated group and Ang II + CGRP (50 nmol/d, in saline)‐treated group (n = 5). D, The medial thickness of the abdominal aortas was assayed in the Ang II‐treated and Ang II + CGRP‐treated groups (n = 5). E, The 8‐OHdG immunohistochemistry of the abdominal aortas in the Ang II‐treated and Ang II + CGRP‐treated groups (n = 5). Bar graphs show mean ± SEM values from three independent experiments. * P < .05, ** P < .05 vs Ang Ⅱ. # P < .05 vs control

Article Snippet: VSMCs were first pre‐treated Ang II (10 −7 mol/L) for 30 minutes and then stimulated with or without and CGRP (10 −7 mol/L) for 60 minutes and CGRP 8–37 30 minutes. cAMP and PKA levels of the VSMCs were measured using parameter cAMP assay (R&D Systems) and protein kinase A (PKA) ELISA (Mlbio) kits, respectively, according to the manufacturers’ protocols.

Techniques: In Vitro, In Vivo, Control, Saline, Immunohistochemistry