cy5 labeled mouse c5a protein  (MedChemExpress)

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: A novel C5a-blocking cyclic peptide drug, Cp1, for the preventive treatment of sepsis. a Schematic illustration of the discovery and mechanism of Cp1 for preventive treatment of sepsis. This figure illustrates the outcomes of excessive C5a production following complement activation in sepsis, such as the formation of a cytokine storm, functional paralysis of neutrophils, and coagulation dysfunction, ultimately leading to multiple organ dysfunction or even failure. In this project, we devised and validated a novel long-acting C5a-blocking cyclic peptide drug (Cp1) via phage screening technology to block the upstream C5a-mediated amplification cascade of the inflammatory response. In early infection, we utilized the efficient neutralization of Cp1 against C5a to effectively curb the “waterfall effect” of inflammatory factors and mitigate the progression to dysregulated systemic inflammation, thereby providing effective prevention and therapeutic intervention for sepsis. A schematic diagram is shown in the figure. b Molecular structure of Cp1 obtained via phage screening technology. c Cartoon diagram of the binding of Cp1 to the mouse C5a protein. d 2D diagram of Cp1 binding to the mouse C5a protein. e Sequence alignment between mouse C5a and human C5a. The interactions involved are summarized with magenta (hydrogen bond) or blue (salt bridge) arrows. f Surface diagram of the binding of Cp1 to the mouse C5a protein. g 3D view of the binding of Cp1 to the mouse C5a protein. Cp1 is shown in purple. The mouse C5a protein is shown in yellow. Hydrogen bond interactions are shown as magenta dashed lines. The numbers indicate the hydrogen bond length. Salt–bridge interactions are shown as blue dashed lines. h Cartoon diagram of the mouse C5a/C5aR1 binding mode. i 3D view of the mouse C5a/C5aR1 binding mode: the mouse C5aR1 protein is shown in blue. The mouse C5a protein is shown in yellow. Hydrogen bond interactions are shown as red dashed lines

Article Snippet: A total of 10 ng of Cy5-labeled mouse C5a protein (MCE, Shanghai, China) was preincubated with linear peptides or cyclic peptides (10 or 100 ng) at room temperature for 0.5 h. Subsequently, 2×10 5 mouse neutrophils were incubated with Cy5-labeled mouse C5a or Cy5-labeled mouse C5a/peptide complexes at 4 °C for 0.5 h in the dark, followed by centrifugation, washing with PBS and flow cytometric analysis (Sony ID7000TM, Japan).

Techniques: Blocking Assay, Activation Assay, Functional Assay, Coagulation, Amplification, Infection, Neutralization, Binding Assay, Sequencing

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Validation of the binding affinity and blocking specificity of C5a-blocking Cp1. a SPR analysis of the mouse C5a protein with linear peptides K1, K2, K3 or K4. b Flow cytometry analysis of competitive inhibition assays for C5a-blocking linear peptides K1, K2, K3, and K4 at high (1 μg/mL) or low (10 ng/mL) concentrations, n = 3. c The inhibitory efficiency of C5a-blocking linear peptides K1, K2, K3 and K4 on mouse neutrophil migration determined by chemotaxis assay at a dose of 10 ng per well, n = 5. d The in vivo inhibitory efficiency of C5a-blocking linear peptides K1, K2, K3 and K4 on plasma proinflammatory cytokines and chemokines determined by ELISA at a dose of 100 μg/20 g via i.v. injection, n = 3. e SPR analysis of cyclic-peptide Cp1 with mouse C5a, human C5a, mouse C5a-desArg or mouse C5. f Flow cytometry analysis of competitive inhibition assays for Cp1 and Cp3 at high (1 μg/mL) or low (10 ng/mL) concentrations, n = 3. g Cyclic peptide Cp1, Cp3 or anti-C5 antibody (eculizumab)-induced erythrocyte hemolysis rate quantified by hemoglobin absorbance at 540 nm, n = 3. h Validation of Cp1-specific blockade of C5a-C5aR1 detected by flow cytometry. PMNs (without TNF-α stimulation) were preincubated with C5aR1-antibody, C5aR2-antibody, Cp1, Cp1 + C5aR1-antibody or Cp1 + C5aR2-antibody at room temperature for 0.5 h, followed by incubation with Cy5-C5a at 4 °C for 0.5 h in the dark, n = 3. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: A total of 10 ng of Cy5-labeled mouse C5a protein (MCE, Shanghai, China) was preincubated with linear peptides or cyclic peptides (10 or 100 ng) at room temperature for 0.5 h. Subsequently, 2×10 5 mouse neutrophils were incubated with Cy5-labeled mouse C5a or Cy5-labeled mouse C5a/peptide complexes at 4 °C for 0.5 h in the dark, followed by centrifugation, washing with PBS and flow cytometric analysis (Sony ID7000TM, Japan).

Techniques: Biomarker Discovery, Binding Assay, Blocking Assay, Flow Cytometry, Inhibition, Migration, Chemotaxis Assay, In Vivo, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Injection, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Plasma stability and pharmacokinetics study of C5a-blocking Cp1 and the functional modulation of PMNs by Cp1-mediated C5a blockade. a , b Stability of C5a-blocking linear peptide K1 and cyclic peptide Cp1 in 50% (v/v) normal plasma or CLP plasma for 24 h, n = 3. c , d Plasma residual Cp1 percentage‒time curves or plasma Cp1 concentration‒time curves during the first 24 h after intravenous administration (100 μg of Cp1), n = 5. e , f The plasma residual Cp1 percentage‒time curves or the plasma Cp1 concentration‒time curves at 14 days after intravenous administration (100 μg of Cp1), n = 5. g The ratio of plasma C5a to C5 in normal mice or CLP model mice was determined via ELISA after Cp1 (100 μg/20 g) administration, n = 3. h The quantitative results of cfDNA in plasma 24 h after CLP surgery after Cp1 treatment (100 μg/20 g), n = 4. i The quantitative results of neutrophil elastase (NE) in plasma 24 h after CLP surgery after Cp1 treatment (100 μg/20 g), n = 4. The quantitative results of the chemotaxis assay for PMNs ( j ) and mouse neutrophils ( l ) at 24 h, n = 6. The inhibitory effects of Cp1 on PMN ( k ) or mouse neutrophil ( m ) migration were determined via a chemotaxis assay with a Cp1 concentration gradient from 10 ng to 1 μg per well, n = 6. n The protective effects of Cp1 on C5a-induced phagocytic impairment in PMNs were quantified via flow cytometry, n = 3. PMNs (2 × 10 5 ) were prestimulated with 100 ng of hC5a or hC5a/Cp1 complexes for 6 h. PMNs were further incubated with 20 μL of Red E. coli BioParticles™ or Green S. aureus BioParticles™ (Invitrogen, USA) at 37 °C for 0.5 h in the dark. PMNs were incubated with DMAO-labeled E. coli or S. aureus at an MOI of 10 at 37 °C for 0.5 h in the dark, followed by gentamicin treatment ( n = 3). o Absolute numbers of neutrophils or peripheral monocytes in CLP model mice were detected via routine blood tests at predetermined time points (4 h, 12 h and 24 h) after intravenous administration of Cp1 (100 μg/20 g), n = 4. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: A total of 10 ng of Cy5-labeled mouse C5a protein (MCE, Shanghai, China) was preincubated with linear peptides or cyclic peptides (10 or 100 ng) at room temperature for 0.5 h. Subsequently, 2×10 5 mouse neutrophils were incubated with Cy5-labeled mouse C5a or Cy5-labeled mouse C5a/peptide complexes at 4 °C for 0.5 h in the dark, followed by centrifugation, washing with PBS and flow cytometric analysis (Sony ID7000TM, Japan).

Techniques: Clinical Proteomics, Drug discovery, Blocking Assay, Functional Assay, Enzyme-linked Immunosorbent Assay, Chemotaxis Assay, Migration, Concentration Assay, Flow Cytometry, Incubation, Labeling

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Inhibitory effects of C5a-blocking Cp1 on inflammatory factors and chemokines in both plasma and peritoneal lavage fluid (PLF) from CLP-induced septic mice. a Inflammatory factors and chemokines in plasma determined by the Luminex assay 24 h after CLP surgery with different doses of Cp1 administration (100 or 300 μg/20 g) via i.v. injection, n = 4. b Inflammatory factors and chemokines in the PLF determined by the Luminex assay 24 h after CLP surgery with different doses of Cp1 administration (100 or 300 μg/20 g) via i.v. injection, n = 4. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: A total of 10 ng of Cy5-labeled mouse C5a protein (MCE, Shanghai, China) was preincubated with linear peptides or cyclic peptides (10 or 100 ng) at room temperature for 0.5 h. Subsequently, 2×10 5 mouse neutrophils were incubated with Cy5-labeled mouse C5a or Cy5-labeled mouse C5a/peptide complexes at 4 °C for 0.5 h in the dark, followed by centrifugation, washing with PBS and flow cytometric analysis (Sony ID7000TM, Japan).

Techniques: Blocking Assay, Clinical Proteomics, Luminex, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Treatment with C5a-blocking Cp1 effectively prevented organ dysfunction and promoted bacterial clearance in CLP-induced septic mice. Liver ( a ) and kidney ( b ) functions were assessed in CLP model mice 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. c Plasma LDH or LAC levels in CLP model mice 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. d Coagulation function parameters in CLP model mice 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. e H&E staining results of lung tissue pathological sections collected 24 h post-CLP surgery, n = 3. The scale bar represents 100 μm. f The plasma VCAM-1, E-selectin or PAI-1 levels 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. g The wet/dry weight ratio of lung tissue in each group 24 h post-CLP surgery, n = 4. h The capillary leakage in each group was quantified by the ratio of HSA level in BALF to serum 24 h post-CLP surgery, n = 4. i The capillary leakage in each group was quantified by the ratio of Evans blue in PLF to serum 24 h post-CLP surgery, n = 3. j The figures of 24 h bacterial culture dishes with whole blood from each group collected 24 h post-CLP modeling, n = 4. k The quantitative results of CFUs/mL in blood in each group 24 h post-CLP modeling, n = 4. l The Kaplan–Meier survival curves of CLP model mice during the first 7 days after cyclic peptide (Cp1) or linear peptide (K1) administration (100 μg/20 g), n = 10. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: A total of 10 ng of Cy5-labeled mouse C5a protein (MCE, Shanghai, China) was preincubated with linear peptides or cyclic peptides (10 or 100 ng) at room temperature for 0.5 h. Subsequently, 2×10 5 mouse neutrophils were incubated with Cy5-labeled mouse C5a or Cy5-labeled mouse C5a/peptide complexes at 4 °C for 0.5 h in the dark, followed by centrifugation, washing with PBS and flow cytometric analysis (Sony ID7000TM, Japan).

Techniques: Blocking Assay, Clinical Proteomics, Coagulation, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Response of Cp1-treated mice to secondary infection and Cp1 treatment-induced changes in the mRNA levels of peripheral mononuclear cells. a Experimental timeline of CLP-treated mice receiving LPS stimulation. b Plasma IL-6 response to LPS stimulation in each group 24 h post-CLP surgery, n = 4. c Experimental timeline of LPS-treated mice receiving live bacterial infection after 14 d of exposure to Cp1 (100 μg/20 g), n = 3. d Clearance results of infection initiated after 14 d of exposure to Cp1 are presented in 24 h blood bacteria culture dishes, n = 3. e CFU count results of Fig. 6d, n = 3. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001. f Sample correlations among the three groups. g Statistical bar graph of differentially expressed genes (DEGs) between the Cp1-treated and untreated groups. h Cluster analysis of mRNA samples among the three groups. i Volcano plot of DEGs between the Cp1-treated and untreated groups. j The top 20 molecular function (MF) terms significantly enriched in DEGs between the Cp1-treated and untreated groups, as determined via GO analysis. k Bubble plot of REACTOME pathway enrichment between the Cp1-treated and untreated groups. l Bubble plot of the top 30 enriched metabolism-associated genes in the KEGG pathway between the Cp1-treated and untreated groups. m The top 12 upregulated and downregulated genes identified via KEGG pathway enrichment analysis between the Cp1-treated and untreated groups. n The subcellular localization of DEGs based on GO enrichment between the Cp1-treated and untreated groups. The control group, C5a-stimulated group and Cp1-treated group are represented as the M, M + S and M + S + C groups, respectively, n = 3. The screening criteria for DEGs were a |log2FoldChange | ≥ 1 and a P < 0.05

Article Snippet: A total of 10 ng of Cy5-labeled mouse C5a protein (MCE, Shanghai, China) was preincubated with linear peptides or cyclic peptides (10 or 100 ng) at room temperature for 0.5 h. Subsequently, 2×10 5 mouse neutrophils were incubated with Cy5-labeled mouse C5a or Cy5-labeled mouse C5a/peptide complexes at 4 °C for 0.5 h in the dark, followed by centrifugation, washing with PBS and flow cytometric analysis (Sony ID7000TM, Japan).

Techniques: Infection, Clinical Proteomics, Bacteria, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: A novel C5a-blocking cyclic peptide drug, Cp1, for the preventive treatment of sepsis. a Schematic illustration of the discovery and mechanism of Cp1 for preventive treatment of sepsis. This figure illustrates the outcomes of excessive C5a production following complement activation in sepsis, such as the formation of a cytokine storm, functional paralysis of neutrophils, and coagulation dysfunction, ultimately leading to multiple organ dysfunction or even failure. In this project, we devised and validated a novel long-acting C5a-blocking cyclic peptide drug (Cp1) via phage screening technology to block the upstream C5a-mediated amplification cascade of the inflammatory response. In early infection, we utilized the efficient neutralization of Cp1 against C5a to effectively curb the “waterfall effect” of inflammatory factors and mitigate the progression to dysregulated systemic inflammation, thereby providing effective prevention and therapeutic intervention for sepsis. A schematic diagram is shown in the figure. b Molecular structure of Cp1 obtained via phage screening technology. c Cartoon diagram of the binding of Cp1 to the mouse C5a protein. d 2D diagram of Cp1 binding to the mouse C5a protein. e Sequence alignment between mouse C5a and human C5a. The interactions involved are summarized with magenta (hydrogen bond) or blue (salt bridge) arrows. f Surface diagram of the binding of Cp1 to the mouse C5a protein. g 3D view of the binding of Cp1 to the mouse C5a protein. Cp1 is shown in purple. The mouse C5a protein is shown in yellow. Hydrogen bond interactions are shown as magenta dashed lines. The numbers indicate the hydrogen bond length. Salt–bridge interactions are shown as blue dashed lines. h Cartoon diagram of the mouse C5a/C5aR1 binding mode. i 3D view of the mouse C5a/C5aR1 binding mode: the mouse C5aR1 protein is shown in blue. The mouse C5a protein is shown in yellow. Hydrogen bond interactions are shown as red dashed lines

Article Snippet: PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark.

Techniques: Blocking Assay, Activation Assay, Functional Assay, Coagulation, Amplification, Infection, Neutralization, Binding Assay, Sequencing

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Validation of the binding affinity and blocking specificity of C5a-blocking Cp1. a SPR analysis of the mouse C5a protein with linear peptides K1, K2, K3 or K4. b Flow cytometry analysis of competitive inhibition assays for C5a-blocking linear peptides K1, K2, K3, and K4 at high (1 μg/mL) or low (10 ng/mL) concentrations, n = 3. c The inhibitory efficiency of C5a-blocking linear peptides K1, K2, K3 and K4 on mouse neutrophil migration determined by chemotaxis assay at a dose of 10 ng per well, n = 5. d The in vivo inhibitory efficiency of C5a-blocking linear peptides K1, K2, K3 and K4 on plasma proinflammatory cytokines and chemokines determined by ELISA at a dose of 100 μg/20 g via i.v. injection, n = 3. e SPR analysis of cyclic-peptide Cp1 with mouse C5a, human C5a, mouse C5a-desArg or mouse C5. f Flow cytometry analysis of competitive inhibition assays for Cp1 and Cp3 at high (1 μg/mL) or low (10 ng/mL) concentrations, n = 3. g Cyclic peptide Cp1, Cp3 or anti-C5 antibody (eculizumab)-induced erythrocyte hemolysis rate quantified by hemoglobin absorbance at 540 nm, n = 3. h Validation of Cp1-specific blockade of C5a-C5aR1 detected by flow cytometry. PMNs (without TNF-α stimulation) were preincubated with C5aR1-antibody, C5aR2-antibody, Cp1, Cp1 + C5aR1-antibody or Cp1 + C5aR2-antibody at room temperature for 0.5 h, followed by incubation with Cy5-C5a at 4 °C for 0.5 h in the dark, n = 3. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark.

Techniques: Biomarker Discovery, Binding Assay, Blocking Assay, Flow Cytometry, Inhibition, Migration, Chemotaxis Assay, In Vivo, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Injection, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Plasma stability and pharmacokinetics study of C5a-blocking Cp1 and the functional modulation of PMNs by Cp1-mediated C5a blockade. a , b Stability of C5a-blocking linear peptide K1 and cyclic peptide Cp1 in 50% (v/v) normal plasma or CLP plasma for 24 h, n = 3. c , d Plasma residual Cp1 percentage‒time curves or plasma Cp1 concentration‒time curves during the first 24 h after intravenous administration (100 μg of Cp1), n = 5. e , f The plasma residual Cp1 percentage‒time curves or the plasma Cp1 concentration‒time curves at 14 days after intravenous administration (100 μg of Cp1), n = 5. g The ratio of plasma C5a to C5 in normal mice or CLP model mice was determined via ELISA after Cp1 (100 μg/20 g) administration, n = 3. h The quantitative results of cfDNA in plasma 24 h after CLP surgery after Cp1 treatment (100 μg/20 g), n = 4. i The quantitative results of neutrophil elastase (NE) in plasma 24 h after CLP surgery after Cp1 treatment (100 μg/20 g), n = 4. The quantitative results of the chemotaxis assay for PMNs ( j ) and mouse neutrophils ( l ) at 24 h, n = 6. The inhibitory effects of Cp1 on PMN ( k ) or mouse neutrophil ( m ) migration were determined via a chemotaxis assay with a Cp1 concentration gradient from 10 ng to 1 μg per well, n = 6. n The protective effects of Cp1 on C5a-induced phagocytic impairment in PMNs were quantified via flow cytometry, n = 3. PMNs (2 × 10 5 ) were prestimulated with 100 ng of hC5a or hC5a/Cp1 complexes for 6 h. PMNs were further incubated with 20 μL of Red E. coli BioParticles™ or Green S. aureus BioParticles™ (Invitrogen, USA) at 37 °C for 0.5 h in the dark. PMNs were incubated with DMAO-labeled E. coli or S. aureus at an MOI of 10 at 37 °C for 0.5 h in the dark, followed by gentamicin treatment ( n = 3). o Absolute numbers of neutrophils or peripheral monocytes in CLP model mice were detected via routine blood tests at predetermined time points (4 h, 12 h and 24 h) after intravenous administration of Cp1 (100 μg/20 g), n = 4. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark.

Techniques: Clinical Proteomics, Drug discovery, Blocking Assay, Functional Assay, Enzyme-linked Immunosorbent Assay, Chemotaxis Assay, Migration, Concentration Assay, Flow Cytometry, Incubation, Labeling

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Inhibitory effects of C5a-blocking Cp1 on inflammatory factors and chemokines in both plasma and peritoneal lavage fluid (PLF) from CLP-induced septic mice. a Inflammatory factors and chemokines in plasma determined by the Luminex assay 24 h after CLP surgery with different doses of Cp1 administration (100 or 300 μg/20 g) via i.v. injection, n = 4. b Inflammatory factors and chemokines in the PLF determined by the Luminex assay 24 h after CLP surgery with different doses of Cp1 administration (100 or 300 μg/20 g) via i.v. injection, n = 4. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark.

Techniques: Blocking Assay, Clinical Proteomics, Luminex, Injection

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Treatment with C5a-blocking Cp1 effectively prevented organ dysfunction and promoted bacterial clearance in CLP-induced septic mice. Liver ( a ) and kidney ( b ) functions were assessed in CLP model mice 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. c Plasma LDH or LAC levels in CLP model mice 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. d Coagulation function parameters in CLP model mice 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. e H&E staining results of lung tissue pathological sections collected 24 h post-CLP surgery, n = 3. The scale bar represents 100 μm. f The plasma VCAM-1, E-selectin or PAI-1 levels 24 h post-CLP surgery after Cp1 (100 μg/20 g) administration, n = 4. g The wet/dry weight ratio of lung tissue in each group 24 h post-CLP surgery, n = 4. h The capillary leakage in each group was quantified by the ratio of HSA level in BALF to serum 24 h post-CLP surgery, n = 4. i The capillary leakage in each group was quantified by the ratio of Evans blue in PLF to serum 24 h post-CLP surgery, n = 3. j The figures of 24 h bacterial culture dishes with whole blood from each group collected 24 h post-CLP modeling, n = 4. k The quantitative results of CFUs/mL in blood in each group 24 h post-CLP modeling, n = 4. l The Kaplan–Meier survival curves of CLP model mice during the first 7 days after cyclic peptide (Cp1) or linear peptide (K1) administration (100 μg/20 g), n = 10. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark.

Techniques: Blocking Assay, Clinical Proteomics, Coagulation, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Response of Cp1-treated mice to secondary infection and Cp1 treatment-induced changes in the mRNA levels of peripheral mononuclear cells. a Experimental timeline of CLP-treated mice receiving LPS stimulation. b Plasma IL-6 response to LPS stimulation in each group 24 h post-CLP surgery, n = 4. c Experimental timeline of LPS-treated mice receiving live bacterial infection after 14 d of exposure to Cp1 (100 μg/20 g), n = 3. d Clearance results of infection initiated after 14 d of exposure to Cp1 are presented in 24 h blood bacteria culture dishes, n = 3. e CFU count results of Fig. 6d, n = 3. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001. f Sample correlations among the three groups. g Statistical bar graph of differentially expressed genes (DEGs) between the Cp1-treated and untreated groups. h Cluster analysis of mRNA samples among the three groups. i Volcano plot of DEGs between the Cp1-treated and untreated groups. j The top 20 molecular function (MF) terms significantly enriched in DEGs between the Cp1-treated and untreated groups, as determined via GO analysis. k Bubble plot of REACTOME pathway enrichment between the Cp1-treated and untreated groups. l Bubble plot of the top 30 enriched metabolism-associated genes in the KEGG pathway between the Cp1-treated and untreated groups. m The top 12 upregulated and downregulated genes identified via KEGG pathway enrichment analysis between the Cp1-treated and untreated groups. n The subcellular localization of DEGs based on GO enrichment between the Cp1-treated and untreated groups. The control group, C5a-stimulated group and Cp1-treated group are represented as the M, M + S and M + S + C groups, respectively, n = 3. The screening criteria for DEGs were a |log2FoldChange | ≥ 1 and a P < 0.05

Article Snippet: PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark.

Techniques: Infection, Clinical Proteomics, Bacteria, Control

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Effects of blocking the C5a/C5aR1 signaling on the early stage of lung fibrosis: ( A ) Scheme for the BLM-induced lung fibrosis. The mice were treated with PMX53 (1 mg/kg) or vehicle 1 h before BLM exposure on day 0, and then once a day. Mice were euthanized on day 7 after BLM modeling. ( B ) Weight changes of the mice; n = 8 for each group. ( C ) Lung coefficients of the mice; n = 8 for each group. ( D ) H&E staining of the lung tissues. Representative images are shown. Original magnification: 200×. ( E ) The number of nuclei per high-power field of lung tissues; n = 8 for each group. ( F ) The inflammatory index of the alveoli; n = 8 for each group. ( G ) The Ashcroft score; n = 8 for each group. ( H , I ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 in lung tissues was quantified by qPCR; n = 6 for each group. ( J ) The expression of αSMA protein in lung tissues. Representative immunohistochemical plots and statistical plots are shown. Original magnification: 200×; n = 8 for each group. ( K , L ) Immunohistochemical staining of collagen I in lung tissues. Representative images and relative IOD analyses are shown. Original magnification: 200×; n = 8 for each group. ( M ) Masson staining of the lung tissues. Representative images and collagen volume fraction analyses are shown. Original magnification: 200×; n = 8 for each group. ( N , O ) WB analysis of the ACSL4 and αSMA protein in lung tissues. Representative bands and statistical plots are shown; n = 6 for each group. ( P ) The expression of Acsl4 in lung tissues was quantified by qPCR; n = 6 for each group. ( Q ) Immunohistochemical staining of ACSL4 protein in the lung tissues. Representative images and relative IOD analyses are shown; n = 8 for each group. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA followed by adjustments for multiple comparisons in panel ( B ), or Student’s t -test in panels ( C , E – J , L , M , O – Q ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Blocking Assay, Staining, Expressing, Immunohistochemical staining

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Inhibition of C5a/C5aR1 signaling reduced ACSL4 levels and attenuated lung inflammation and fibrosis in the chronic stage of pulmonary fibrosis: ( A ) Scheme of BLM-induced lung fibrosis. Mice were euthanized on day 21 after BLM modeling. ( B ) Weight changes of the mice; n = 6 for each group. ( C ) Lung coefficients of the mice; n = 6 for each group. ( D ) H&E staining of the lung tissues. Representative images are shown. Original magnification: 200×. ( E ) Number of nuclei per high-power field of the lung tissues; n = 6 for each group. ( F ) The inflammatory index of the alveoli; n = 6 for each group. ( G ) The Ashcroft score; n = 6 for each group. ( H , I ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 in lung tissues was quantified by qPCR; n = 5 for each group. ( J ) Immunohistochemical staining of αSMA in lung tissues. Representative images and relative IOD analyses are shown. Original magnification: 200×; n = 6 for each group. ( K , L ) Immunohistochemical staining of collagen I in lung tissues. Representative images and relative IOD analyses are shown. Original magnification: 200×; n = 6 for each group. ( M ) Masson staining of lung tissues. Representative images and collagen volume fraction analyses are shown. Original magnification: 200×; n = 6 for each group. ( N , O ) WB analysis of the ACSL4 and αSMA proteins in lung tissues. Representative bands and statistical plots are shown; n = 6 for each group. ( P ) The expression of Acsl4 in lung tissues was quantified by qPCR; n = 6 for each group. ( Q ) Immunohistochemical staining of ACSL4 in lung tissues. Representative images and relative IOD analyses are shown; n = 6 for each group. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 by two-way ANOVA followed by adjustments for multiple comparisons in panel ( B ), or Student’s t -test in panels ( C , E – J , L , M , O – Q ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Inhibition, Staining, Expressing, Immunohistochemical staining

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Blocking ACSL4 reduced C5a/C5aR1 signaling-induced activation and migration of lung fibroblasts: ( A ) The expression of Acsl4 was quantified by qPCR; n = 6 for each group. ( B ) WB analysis of ACSL4 in lung fibroblasts. Representative bands and statistical plots are shown; n = 6 for each group. ( C , D ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 was quantified by qPCR; n = 6 for each group. ( E ) WB analysis of αSMA in lung fibroblasts. Representative bands and statistical plots are shown; n = 6 for each group. ( F ) The expression of αSMA was tested by immunofluorescence. Representative plots are shown. Original magnification: 400×; αSMA (green) and DAPI (blue). ( G ) The cell scratch assay was used to assess lung fibroblasts’ migration. Representative images are shown. ( H ) The transwell assay. Representative plot and statistical plots of the transwell assay results are shown; n = 3 for each group. ( I ) The proliferation of fibroblasts was tested by CCK8. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001 by Student’s t -test in panels ( A , B ), one-way ANOVA followed by adjustments for multiple comparisons in panels ( C – E , H ), or two-way ANOVA followed by adjustments for multiple comparisons in panel ( I ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Blocking Assay, Activation Assay, Migration, Expressing, Immunofluorescence, Wound Healing Assay, Transwell Assay

Journal: Biomolecules

Article Title: ACSL4 Drives C5a/C5aR1–Calcium-Induced Fibroblast-to-Myofibroblast Transition in a Bleomycin-Induced Mouse Model of Pulmonary Fibrosis

doi: 10.3390/biom15081106

Figure Lengend Snippet: Blockade of calcium signaling attenuated ACSL4 expression induced by the C5a/C5aR1 signaling: ( A ) The calcium concentration of lung fibroblasts was tested by calcium imaging. Representative plots and statistical plots are shown; n = 4 for each group. ( B , C ) The expression of Acta2 , Col1a1 , Col3 , and Fn1 was quantified by qPCR; n = 6 for each group. ( D ) WB analysis of ACSL4 and αSMA in lung fibroblasts. Representative bands and statistical plots are shown; n = 6 for each group. ( E ) The expression of αSMA was tested by immunofluorescence. Plots are shown. Original magnification: 400×; αSMA (green) and DAPI (blue). ( F , G ) The horizontal migration ability of lung fibroblasts was tested by the cell scratch assay. Representative plots and statistical plots are shown; n = 4 for each group. ( H ) The spatial migration ability of lung fibroblasts was tested by transwell assay. Representative plot and statistical plot are shown; n = 5 for each group. Data are the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, , **** p < 0.0001 by one-way ANOVA followed by adjustments for multiple comparisons in panels ( A – C , H ), Student’s t -test in panel ( D ), or two-way ANOVA followed by adjustments for multiple comparisons in panel ( G ); ns = not significant.

Article Snippet: In some experiments, TGF-β1 (Sinobiological, Beijing, China, Cat#: 80116-RNAH-5), PRGL493, C5a (MCE, New Jersey, USA, Cat#: HY-P7695), or FK506 (Selleck, Texas, USA, Cat#: S5003) was added to the cell culture medium at the indicated concentrations and time points.

Techniques: Expressing, Concentration Assay, Imaging, Immunofluorescence, Migration, Wound Healing Assay, Transwell Assay