PD-1-dependent therapeutic effect of Trichinella spiralis cystatin on myocardial infarction in a mice model

Figures

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  Fig. 1. Identification of knockout of the programmed death-1 (PD-1) in PD-1−/− mice by PCR with PD-1-specific primers. Lanes: bp, size of DNA ladder; 0, blank control; 1–9, mouse number. PD-1−/−, programmed PD-1 knockout mice; WT, wild-type mice

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  Fig. 2. Treatment with rTs-Cys significantly improved the survival rate of MI mice with PD-1 expression. The 28-day survival rate of each group of mice after surgery and treatment (n = 20) is shown. Asterisks indicate a statistically significant difference in survival rate between groups at *P < 0.05 and **P < 0.01; ns, not significant. MI, Myocardial infarction; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; WT, wild-type mice

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  Fig. 3. Treatment with rTs-Cys significantly improved the SV, LVEF and LVFS in MI mice with PD-1 expression. a Typical B-mode ultrasound image in mice from different groups. b Typical M-mode ultrasound image in mice from different groups. c Cardiac function parameters in different groups (n = 6). The results are presented as the mean ± standard error of the mean (SEM). Asterisks indicate a statistically significant difference in the respective parameter at *P < 0.05, **P < 0.01 and ***P < 0.001; ns, not significant. LVEF, Left ventricular ejection fraction; LVFS, left ventricular fractional shortening; MI, myocardial infarction; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; SV, stroke volume; WT, wild-type mice

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  Fig. 4. Treatment with rTs-Cys significantly reduced histopathological damage in the hearts of MI mice with PD-1 expression. a Representative shape of the heart from the different groups of mice. b Representative Masson staining sections of infarcted heart tissues from the different groups of mice (magnification 15× and 200×). c Percentage of left ventricular infarction area. d Left ventricular infarct wall thickness. The results are presented as the mean ± standard error of the mean (SEM) (n = 6). Asterisks indicate a statistically significant difference between groups at **P < 0.01 and ***P < 0.001; ns, not significant. MI, Myocardial infarction; PBS, phosphate-buffered saline; PD-1−/−,programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; WT, wild-type mice

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  Fig. 5. Hematoxylin/eosin (HE) staining of representative heart tissue sections within the infarcted area of the heart from the different groups of mice 7 days after the MI operation and treatment (magnification 200×, scale bar: 100 μm). MI, Myocardial infarction; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; WT, wild-type mice

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  Fig. 6. Transcriptional levels of vascular endothelial growth factor (VEGF) in the MI area of hearts from the different groups of mice relative to glyceraldehyde 3-phosphate dehydrogenase (GADPH) measured by real-time quantitative PCR. The results are presented as the mean ± standard error of the mean (SEM) (n = 6). Asterisks indicate a statistically significant difference at **P < 0.01; ns, not significant. MI, Myocardial infarction; mRNA, messenger RNA; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; VEGF, vascular endothelial growth factor; WT, wild-type mice

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  Fig. 7. rTs-Cys regulated inflammatory-related cytokine expression. a Serological levels of the pro-inflammatory cytokines TNF-α and IL-6 and regulatory cytokines IL-10 and TGF-β, as measured by quantitative enzyme-linked immunosorbent assay. b The messenger RNA transcriptional levels of TNF-α, IL-6, IL-10 and TGF-β relative to glyceraldehyde 3-phosphate dehydrogenase (GADPH) in infarcted heart tissue, as measured by real-time quantitative PCR. The results are presented as the mean ± standard error of the mean (SEM) (n = 6). Asterisks indicate a statistically significant difference at *P < 0.05 and ***P < 0.001; ns, not significant. IL, Interleukin; MI, myocardial infarction; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; VEGF, vascular endothelial growth factor; WT, wild-type mice

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  Fig. 8. rTs-Cys-induced PD-1-dependent M2 macrophage polarization in infarcted heart tissues. a Relative mRNA expression level of iNOS and Arg-1 in the infarcted cardiac tissues of WT and PD-1−/− mice. The results are presented as the mean ± standard error of the mean (SEM) (n = 6). Asterisks indicate a statistically significant difference at *P < 0.05, **P < 0.01 and ***P < 0.001; ns, not significant. b Representative immunofluorescence staining of cardiac tissue sections from the MI zone in the different groups at 7 days post-surgery and treatment. Scale bars: 1×, 1000 μm; 10×, 100 μm. Arg-1, Arginase-1; iNOS, inducible nitric oxide synthase; MI, myocardial infarction; mRNA, messenger RNA; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; VEGF, vascular endothelial growth factor; WT, wild-type mice

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  Fig. 9. Levels of inflammatory cytokines (TNF-α and IL-6) and regulatory cytokines (IL-10 and TGF-β) in the supernatant of BMDMs derived from WT and PD-1−/− mice co-incubated with LPS with or without rTs-Cys, as measured by quantitative enzyme-linked immunosorbent assay. The results are presented as the mean ± standard error of the mean (SEM). Asterisks indicate a statistically significant difference at *P < 0.05, **P < 0.01 and ***P < 0.001; ns, not significant. BMDMs, Bone marrow-derived macrophages, IL, interleukin; LPS, lipopolysaccharide; MI, myocardial infarction; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; WT, wild-type mice

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  Fig. 10. Flow cytometry was performed to detect CD86 (M1 phenotype) and CD206 (M2 phenotype) glycoproteins on BMDMs derived from both WT and PD-1−/− mice, co-incubated with LPS and rTs-Cys. The results are presented as the mean ± standard error of the mean (SEM) (n = 3). Asterisks indicate a statistically significant difference at *P < 0.05, **P < 0.01 and ***P < 0.001; ns, not significant. BMDMs, Bone marrow-derived macrophages; FITC, fluorescein isothiocyanate; FSC, SSC, forward and side scatter, respectively; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; PD-1−/−, programmed death-1 (PD-1) knockout mice; rTs-Cys, recombinant Trichinella spiralis cystatin; WT, wild-type mice

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  Fig. 11. rTs-Cys promotes M2-type polarization of macrophages through the PD-1 pathway to improve MI in mice. a Intraperitoneal (i.P.) injection of rTs-Cys in MI mice effectively alleviates the inflammatory response in the damaged heart and mitigates MI symptoms. b rTs-Cys regulates the release of inflammatory cytokines from macrophages, thereby improving the inflammatory microenvironment of the heart, reducing myocardial fibrosis, and enhancing VEGF expression levels. This regulation is PD-1 dependent, C rTs-Cys improves cardiac function and tissue damage in MI mice by promoting the polarization of macrophages from the M1 to M2 phenotype through PD-1 regulation. α-SMAL, Alpha-smooth muscle actin; IL, interleukin; PD-1, programmed death-1; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; rTs-Cys, recombinant Trichinella spiralis cystatin; SV, stroke volume; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor; WT, wild-type mice