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

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) Representative Fura-2 ratiometric fields (left) and calcium traces (center) of DRG neurons responding to filtered supernatant from S. pyogenes M1 (5×109 cfu/mL), capsaicin (1 µm), and KCl (40 mM). Proportions (Right) of capsaicin non-responsive (Cap−) and capsaicin responsive (Cap+) neurons that responded to M1 supernatant (n=3–4 fields/condition). (B–D) Representative Fura-2 ratiometric fields (B) and calcium traces (C) of DRG neurons stimulated with filtered supernatant from S. pyogenes M1 (wt) or isogenic mutants lacking SLS (ΔsagA), both SLO and SLS (ΔsloΔsagA), or double mutant bacteria in which sagA expression was restored (ΔsloΔsagA+pDL:sagA). (D) Proportions of responding DRG neurons to bacterial supernatant from S. pyogenes M1 (wt) or isogenic mutant strains (n=3 fields/condition). (E) DRG neurons stimulated for 30 min with supernatant from S. pyogenes M1 (wt), isogenic mutants, or medium, analyzed for in vitro release of CGRP (n=5 samples/group). Statistical analysis: (A) Two-way ANOVA, Bonferroni post-tests. (D,E) One-way ANOVA, Tukey post-tests. *p<0.05 ***p<0.001 ****p<0.0001. ns=not significant. Scale bars, 50 µm. Mean±SEM. See Figure S2 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Mutagenesis, Bacteria, Expressing, In Vitro

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) Histopathology of flank biopsies from vehicle or RTX-treated mice 3 days after injection of S. pyogenes M1 (5×106 cfu). Scale bars, 50 µm. (B) Bacterial load recovery (log10 cfu) from flank lesions and spleens in RTX or vehicle-treated mice after S. pyogenes M1 injection (5×106 cfu, n=4/group). (C–E) Flow cytometry of leukocyte recruitment in necrotizing lesions 1 day after S. pyogenes M1 injection (5×106 cfu): (C) Representative FACS plots showing neutrophils (CD11b+Ly6G+ gates) in lesion samples. (D–E) Quantification of immune cell populations by flow cytometry in flank biopsies from infected Trpv1-Cre/Dta mice or control littermates (n=4/group), or from uninfected mice, infected vehicle-treated mice, or infected RTX-treated mice (n=4–5/group). (F–H) Measurement of CGRP release ex vivo from flank skin punch biopsies. (G) CGRP release from uninfected skin (0 h), 7 h, or 24 h after S. pyogenes M1 injection (5×106 cfu) (n=3/group). (H) CGRP release from uninfected skin or 7 h after S. pyogenes M1 (5×106 cfu) injection of Trpv1-Cre/Dta mice or control littermates, or Vehicle or RTX-treated mice (n=3/group). Statistical analysis: (B,D,E,H) Two-way ANOVA, Bonferroni post-tests. (G) One-way ANOVA, Tukey post-tests. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. ns=not significant. nd=none detected. Mean±SEM. See Figure S5 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Histopathology, Injection, Flow Cytometry, Infection, Control, Ex Vivo

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A–D) Subcutaneous administration of BoNT/A (25 pg/100 µL) or vehicle 6 days prior to S. pyogenes M1 injection in flank skin (5×106 cfu). (B) Representative images of lesions (day 8), (C) Dermonecrosis size measurements, and (D) Weight loss over time after injection of S. pyogenes (n=5–10/group). (E–H) Intrathecal administration of BoNT/A or vehicle 1 day prior to S. pyogenes M1 injection in flank skin (5×106 cfu). (F) Representative images of lesions (day 8), (G) Dermonecrosis size measurements, and (H) Weight loss over time after injection of S. pyogenes (n=6/group). (I) DRG neurons exposed to BoNT/A (25 pg/200 µL) or medium for 24 h were stimulated with S. pyogenes supernatant (5×109 cfu/mL) for 30 min, and CGRP was measured in neuronal supernatant (n=5/group). (J) CGRP release from skin punch biopsies of mice treated intrathecally or locally with BoNT/A, 7 h after S. pyogenes M1 (5×106 cfu) injection (n=3/group). Statistical analysis: (C,D,G,H) Two-way ANOVA, Bonferroni post-tests. (I,J) One-way ANOVA, Tukey post-tests. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. ns=not significant. Mean±SEM. See Figure S6 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Injection

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) DRG neurons were pretreated with BoNT/A for 24 h, or with CGRP antagonists (CGRP8–37 or BIBN4096) immediately before co-incubation with mouse neutrophils and S. pyogenes M1 for 1 h. Bacterial survival was measured as the multiplication factor of surviving colonies/starting inoculum (n=3–4 replicates/group). (B) Mouse neutrophils were incubated with S. pyogenes M1 in presence of CGRP or vehicle for 1 h, and bacterial survival measured (n=4/group). (C) Human whole blood was incubated with S. pyogenes M1 in presence of CGRP or vehicle for 3 h, and bacterial survival measured (n=3/group). (D) Representative images of lesions at day 8 (left) and dermonecrosis size (right) of mice treated 2 h after S. pyogenes M1 injection (5×106 cfu) with vehicle, BoNT/A, or BIBN4096 (n=6–7/group). (E–G) Mice were treated subcutaneously with BoNT/A or vehicle at day 2 and day 9 following flank injection of S. pyogenes M1 (5×106 cfu). Representative images show lesions before and after treatment (E). Dermonecrotic lesions (F) and abscess sizes (G) were measured over time (n=10/group). Blue dots show injection sites at day 2 and day 9. Arrows show BoNT/A treatments. Statistical analysis: (A–C) One-way ANOVA, Tukey post-tests. (D–G) Two-way ANOVA, Bonferroni post-tests. (A–C,F–G) *p<0.05 **p<0.01 ***p<0.01 ****p<0.0001. (D) BIBN4096 vs veh: *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001, BoNT/A vs veh: †p<0.05 ††p<0.01 †††p<0.001 ††††p<0.0001. ns=not significant. Mean±SEM. See Figure S7 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Incubation, Injection