Journal: Journal of Clinical and Translational Hepatology

Article Title: RAMP1 Protects Hepatocytes against Ischemia-reperfusion Injury by Inhibiting the ERK/YAP Pathway

doi: 10.14218/JCTH.2023.00339

Figure Lengend Snippet: (A) The CCK-8 assay was conducted on primary hepatocytes from WT mouse livers that were treated with inhibitors associated with apoptosis pathways, both under normoxia and after 6 h. (B) A CCK-8 assay was conducted on primary hepatocytes from RAMP1-KO mouse livers treated with inhibitors associated with apoptosis pathways, both in normoxia and after 6 h of H/R treatment. Western blot analysis was performed to detect the activation of MAPK signaling pathways and YAP phosphorylation in primary hepatocytes (C) and livers (H) from these mice, and statistical analysis was conducted for primary hepatocytes (D-G) and livers (I-L) (n=3–5 per group). All data are presented as the mean ± SD. For A, * p <0.05, ** p <0.01, *** p <0.001 compared with the WT group with the same treatment using Student’s two-tailed t-test. B, * p <0.05, ** p <0.01, *** p <0.001 compared with the groups under normoxia with the same treatment using Student’s two-tailed t-test; D and F, * p <0.05, compared with the sham/control groups; # p <0.05 compared with the WT I/R groups using Student’s two-tailed t-test. Truli and PY-60: YAP phosphorylation inhibitors, Temuterkib and SCH772984: ERK phosphorylation inhibitors, Stattic: STAT3 inhibitor, and MK2206: P-AKT inhibitor. CCK-8,cell counting Kit-8; CGRP,caltonin gene-related peptide; ERK1/2, extracellular signal–regulated kinase1/2; HIRI, hepatic ischemia-reperfusion injury; JNK1/2, c-Jun NH2-terminal kinase1/2; MAPK, mitogen-activated protein kinase; p-ERK1/2, phosphorated extracellular signal–regulated kinase1/2; p-JNK1/2, phosphorated c-Jun NH2-terminal kinase1/2; p-YAP, phosphorated yes-associated protein; STAT3, signal transducer and activator of transcription 3; VP, verteporfin; WT, wild-type; YAP, yes-associated protein.

Article Snippet: YAP phosphorylation inhibitor: Truli (E1061, Selleck, 0.2 nm) and PY-60 (HY-141644, MCE; 1.6 μm; 10 mg/kg for mice in vivo , i.p. ); ERK phosphorylation inhibitor: Temuterkib (HY-101494, MCE; 5 nm) and SCH772984 (HY-50846, MCE; 300 nm, 5 mg/kg for mice in vivo , i.p. ); ERK agonist: C16-PAF(HY-108635, MCE, 1 μm , ); STAT3 inhibitor : Sttatic (HY-13818, MCE; IC50:10 μm); p-AKT inhibitor: MK2206 (HY-10358, MCE; 65 nm); Caspase-3 inhibitor: Z-VAD (HY-16658B, MCE; 10 μm ; 10 mg/kg for mice in vivo , i.p. ); CGRP agonist: Calcitonin Gene Related Peptide (CGRP) II, rat TFA (HY-P1913A , MCE; 83 μm ); Verteporfin (HY-B0146, MCE; 5 μm ).

Techniques: CCK-8 Assay, Western Blot, Activation Assay, Protein-Protein interactions, Phospho-proteomics, Two Tailed Test, Control, Cell Counting

Journal: Journal of Clinical and Translational Hepatology

Article Title: RAMP1 Protects Hepatocytes against Ischemia-reperfusion Injury by Inhibiting the ERK/YAP Pathway

doi: 10.14218/JCTH.2023.00339

Figure Lengend Snippet: (A) Protein levels of RAMP1 in Control and H/R groups with and without CGRP agonist. (B) Relative cell activity was determined by CCK-8 assay of RAMP1 in the control and H/R groups using a CGRP agonist. (C) CCK-8 assay was performed using CGRP agonists, YAP inhibitors, and agonists in H/R. (D) CCK-8 assay was performed using a CGRP agonist combined with an ERK inhibitor or agonist in H/R. (E) CCK-8 experiments were performed using ERK inhibitors in combination with YAP inhibitors or agonists in H/R. (F) CCK-8 assays were performed using ERK agonists combined with a YAP inhibitor or agonist in H/R. (G) Statistical analysis of flow cytometry to detect the proportion of apoptosis in H/R with a CGRP agonist, YAP inhibitor and agonist, and ERK inhibitor and agonist. (H) Statistical data in H/R, ERK inhibitor combined with YAP inhibitor or agonist were used to detect the proportion of apoptosis by flow cytometry. (I) Statistical data in H/R using ERK agonists combined with YAP inhibitors or agonists to detect the proportion of apoptosis. CGRP agonist: Caltonin gene-related peptide (CGRP) II, rat TFA, ERK agonist (C16-PAF); ERK inhibitor (SCH772984); YAP agonist (PY-60); and YAP inhibitor (Verteporfin). The cells were treated with apoptosis pathway inhibitors and subjected to 6 h of reperfusion after hypoxia. All data are presented as the mean ± SD. * p <0.05, ** p <0.01, *** p <0.001 using Student’s two-tailed t-test. CCK-8, cell counting Kit-8; CGRP agonist, caltonin gene-related peptide agonist; HIRI, hepatic ischemia-reperfusion injury.

Article Snippet: YAP phosphorylation inhibitor: Truli (E1061, Selleck, 0.2 nm) and PY-60 (HY-141644, MCE; 1.6 μm; 10 mg/kg for mice in vivo , i.p. ); ERK phosphorylation inhibitor: Temuterkib (HY-101494, MCE; 5 nm) and SCH772984 (HY-50846, MCE; 300 nm, 5 mg/kg for mice in vivo , i.p. ); ERK agonist: C16-PAF(HY-108635, MCE, 1 μm , ); STAT3 inhibitor : Sttatic (HY-13818, MCE; IC50:10 μm); p-AKT inhibitor: MK2206 (HY-10358, MCE; 65 nm); Caspase-3 inhibitor: Z-VAD (HY-16658B, MCE; 10 μm ; 10 mg/kg for mice in vivo , i.p. ); CGRP agonist: Calcitonin Gene Related Peptide (CGRP) II, rat TFA (HY-P1913A , MCE; 83 μm ); Verteporfin (HY-B0146, MCE; 5 μm ).

Techniques: Control, Activity Assay, CCK-8 Assay, Flow Cytometry, Two Tailed Test, Cell Counting

Journal: International Journal of Molecular Sciences

Article Title: Plasma with Added Protease Inhibitors Improves Alpha- and Beta-CGRP Measurement Compared to Serum: Towards a Reliable Biomarker for Chronic Migraine

doi: 10.3390/ijms26209958

Figure Lengend Snippet: Differences in CGRP levels in plasma with and without PI. ( A ) Correlation of α-CGRP levels in PI-plasma and No PI-plasma. Dark blue line represents the linear regression, and the red dotted line represents the 95% confidence interval (CI). ( B ) Difference in α-CGRP levels in PI-plasma (represented in green) vs. No PI-plasma (represented in black); data are shown as median with CI 95%. Comparison was made using the Wilcoxon Signed-Rank test (PI-plasma median = 38.28 pg/mL, No PI-plasma median = 21.26 pg/mL, p ≤ 0.0001). ( C ) Correlation of β-CGRP levels in PI-plasma and No PI-plasma. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( D ) Difference in β-CGRP levels in PI-plasma (represented in light blue) vs. No PI-plasma (represented in pink); data are shown as median with CI 95%. Comparison was made using the Wilcoxon Signed-Rank test (PI-plasma median = 6.909 pg/mL, No PI-plasma median = 6.200 pg/mL, p = ns (non-significant)). **** p < 0.0001.

Article Snippet: α-CGRP and β-CGRP levels in plasma were measured using commercial ELISA tests, using Abbexa CGRP1 (CALCA) ELISA kits for α-CGRP (Abbexa Ltd., Cambridge, UK) [ ] and Cusabio Human Calcitonin Gene Related Peptide ELISA kits for β-CGRP (Cusabio Biotech Co., Wuhan, China) [ ], following the manufacturers’ instructions.

Techniques: Clinical Proteomics, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Plasma with Added Protease Inhibitors Improves Alpha- and Beta-CGRP Measurement Compared to Serum: Towards a Reliable Biomarker for Chronic Migraine

doi: 10.3390/ijms26209958

Figure Lengend Snippet: Correlation of α-CGRP and β-CGRP in plasma samples. ( A ) Correlation of α-CGRP levels vs. β-CGRP plasma levels in No PI-plasma. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( B ) Correlation of α-CGRP levels vs. β-CGRP levels in PI-plasma. Dark blue line represents the linear regression, and the red dotted line represents the CI.

Article Snippet: α-CGRP and β-CGRP levels in plasma were measured using commercial ELISA tests, using Abbexa CGRP1 (CALCA) ELISA kits for α-CGRP (Abbexa Ltd., Cambridge, UK) [ ] and Cusabio Human Calcitonin Gene Related Peptide ELISA kits for β-CGRP (Cusabio Biotech Co., Wuhan, China) [ ], following the manufacturers’ instructions.

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Plasma with Added Protease Inhibitors Improves Alpha- and Beta-CGRP Measurement Compared to Serum: Towards a Reliable Biomarker for Chronic Migraine

doi: 10.3390/ijms26209958

Figure Lengend Snippet: Comparison of α-CGRP levels in serum and plasma. ( A ) Correlation of α-CGRP serum vs. No PI-plasma levels. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( B ) Difference in α-CGRP levels in serum (represented in burgundy) vs. No PI-plasma (represented in black); data are shown as median with CI 95%. Comparison was made using the Wilcoxon Signed-Rank test (serum median = 24.32 pg/mL, No PI-plasma median = 24.94 pg/mL, p = ns). ( C ) Correlation of α-CGRP serum levels vs. PI-plasma levels. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( D ) Difference in α-CGRP levels in serum (represented in burgundy) vs. PI-plasma (represented in green); data are shown as median with CI 95%. Comparison was made using the Wilcoxon Signed-Rank test (serum median = 13.63 pg/mL, PI-plasma median = 38.29 pg/mL, p ≤ 0.01). ** p < 0.010.

Article Snippet: α-CGRP and β-CGRP levels in plasma were measured using commercial ELISA tests, using Abbexa CGRP1 (CALCA) ELISA kits for α-CGRP (Abbexa Ltd., Cambridge, UK) [ ] and Cusabio Human Calcitonin Gene Related Peptide ELISA kits for β-CGRP (Cusabio Biotech Co., Wuhan, China) [ ], following the manufacturers’ instructions.

Techniques: Comparison, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Plasma with Added Protease Inhibitors Improves Alpha- and Beta-CGRP Measurement Compared to Serum: Towards a Reliable Biomarker for Chronic Migraine

doi: 10.3390/ijms26209958

Figure Lengend Snippet: Comparison of β-CGRP levels in serum and plasma. ( A ) Correlation of β-CGRP serum vs. No PI-plasma levels; the dark blue line represents the linear regression, and the red dotted line represents the CI. ( B ) Difference in β-CGRP concentrations in serum (represented in burgundy) vs. plasma (represented in pink); data are shown as median with CI 95%. Comparisons were made using the Wilcoxon Signed-Rank test (serum median = 3.625 pg/mL, plasma median = 8.516 pg/mL, p ≤ 0.0001). ( C ) Correlation of β-CGRP serum vs. No PI-plasma levels when using PI; the dark blue line represents the linear regression, and the red dotted line represents the CI. ( D ) Difference in β-CGRP concentrations in serum (represented in burgundy) vs. No PI-plasma with PI (represented in blue); data are shown as median with CI 95%. Comparisons were made using the Wilcoxon Signed-Rank test (serum median = 1.716 pg/mL, plasma with PI median = 6.909 pg/mL, p ≤ 0.0001). **** p < 0.0001.

Article Snippet: α-CGRP and β-CGRP levels in plasma were measured using commercial ELISA tests, using Abbexa CGRP1 (CALCA) ELISA kits for α-CGRP (Abbexa Ltd., Cambridge, UK) [ ] and Cusabio Human Calcitonin Gene Related Peptide ELISA kits for β-CGRP (Cusabio Biotech Co., Wuhan, China) [ ], following the manufacturers’ instructions.

Techniques: Comparison, Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Plasma with Added Protease Inhibitors Improves Alpha- and Beta-CGRP Measurement Compared to Serum: Towards a Reliable Biomarker for Chronic Migraine

doi: 10.3390/ijms26209958

Figure Lengend Snippet: Dynamics of CGRP levels along migraine treatment. ( A ) Changes in α-CGRP concentrations in No PI-plasma before starting the treatment (M0), 6 months after starting treatment (M6) ; Wilcoxon Signed-Rank test ( p < 0.01) and ( B ) after 12 months of the beginning of the treatment (M12); Friedman test followed by Dunn’s test. ( C ) Changes in β-CGRP levels in No PI-plasma at M0 and M6 after starting treatment; Wilcoxon Signed-Rank test (ns) and ( D ) M12 Friedman test followed by Dunn’s test. * p < 0.050, ** p < 0.010.

Article Snippet: α-CGRP and β-CGRP levels in plasma were measured using commercial ELISA tests, using Abbexa CGRP1 (CALCA) ELISA kits for α-CGRP (Abbexa Ltd., Cambridge, UK) [ ] and Cusabio Human Calcitonin Gene Related Peptide ELISA kits for β-CGRP (Cusabio Biotech Co., Wuhan, China) [ ], following the manufacturers’ instructions.

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Plasma with Added Protease Inhibitors Improves Alpha- and Beta-CGRP Measurement Compared to Serum: Towards a Reliable Biomarker for Chronic Migraine

doi: 10.3390/ijms26209958

Figure Lengend Snippet: Analysis of CGRP levels in plasma depending on demographic variables. ( A ) Correlation of α-CGRP No PI-plasma levels and age. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( B ) Correlation of α-CGRP PI-plasma levels and age. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( C ) Correlation of β-CGRP No PI-plasma levels and age. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( D ) Correlation of β-CGRP PI-plasma levels and age. Dark blue line represents the linear regression, and the red dotted line represents the CI. ( E ) Comparison of α-CGRP No PI-plasma levels from patients sorted by sex (column A median = 23.31 pg/mL; column B median = 26.09 pg/mL; p = ns). ( F ) Comparison of α-CGRP PI-plasma levels from patients sorted by sex. ( G ) Comparison of β-CGRP No PI-plasma levels by sex (column A median = 8.350 pg/mL; column B median = 8.849 pg/mL; p = ns). ( H ) Comparison of β-CGRP PI-plasma levels sorted by sex. Data are shown as median with CI 95%. The comparisons in ( E – H ) were made using Mann–Whitney U test.

Article Snippet: α-CGRP and β-CGRP levels in plasma were measured using commercial ELISA tests, using Abbexa CGRP1 (CALCA) ELISA kits for α-CGRP (Abbexa Ltd., Cambridge, UK) [ ] and Cusabio Human Calcitonin Gene Related Peptide ELISA kits for β-CGRP (Cusabio Biotech Co., Wuhan, China) [ ], following the manufacturers’ instructions.

Techniques: Clinical Proteomics, Comparison, MANN-WHITNEY