Effect of programmed cell death-1/programmed death ligand-1 signaling pathway on myocardial ischemia-reperfusion injury
Objective To investigate the effect of programmed death receptor 1(PD-L1)and its ligand signaling pathway on myocardial ischemia-reperfusion injury(I/RI)and the underlying mechanism.Methods Totally,20 C57 BL mice aged 8-10 weeks were randomly divided into sham operation group(Sham group),I/RI group,I/RI+CPG-ODN group and I/RI+PD-L1 group,with 5 mice in each group.After modeling,the single cell sequencing was done.Serum interleukin(IL)-2,IL-6 and interferon-γ(IFN-γ)were determined.The protein expression levels of PD-L1,B cell lymphoma/leukemia-2(bcl-2)and bcl-2 associated X protein(bax)in tissues were detected by Western blotting.H9C2 cardiomyocytes were randomly divided into normal control group(control group),hypoxia/reoxygenation model group(H/R group),H/R+CPG-ODN group,H/R+PD-L1 group.Except for the control group,the H9C2 cells in the rest groups were pretreated with corresponding pretreatment for 30 min,and then treated with hypoxia for 3 h and reoxygenation for 3 h.The activity of H9C2 cells in each group was detected by Cell Counting Kit-8(CCK-8)method.The activity of LDH in H9C2 cells in each group was detected by LDH kit.The expression of PD-L1,bcl-2 and bax mRNA in H9C2 cells was detected by RT-PCR.One-way analysis of variance was used for comparison between groups.Results The proportion of T lymphocytes in the heart tissue of C57BL mice increased significantly after I/RI.The Pdcd1 gene,which is the target gene of programmed death factor-1(PD-1),has the highest coefficient of variation after I/RI;CCK-8 assay showed that the cell viability in H/R+CPG-ODN group was higher than that in H/R and H/R+PD-L1 groups(73.88±3.40 vs.55.24±2.17,45.89±1.99,F=988.50,P＜0.01).LDH levels in H/R group,H/R+CPG-ODN group and H/R+PD-L1 group were higher than in control group(171.972±2.551,140.276±8.204,231.931±12.932 vs.113.806±13.526,F=73.20,P＜0.05),those in H/R+CPG-ODN group were lower than in H/R group(t=7.879,P＜0.01),and those in H/R+PD-L1 group was higher than in H/R group(t=6.389,P＜0.05).IL-2,IL-6 and IFN-γ levels in I/RI group were higher than those in sham operation group(95.51±5.29,156.27±11.89,43.75±0.87 vs.38.13±2.67,59.38±5.55,26.66±0.23,t=37.11,12.80,41.15,P＜0.001).Western blotting showed that PD-L1 in CPG-ODN group was higher than that in H/R group(3.06±0.38 vs.1.79±0.11,t=5.66,P＜0.05);bcl-2 in CPG-ODN group was higher than that in H/R group(0.95±0.09 vs.0.66±0.02,t=5.18,P＜0.05);bax in PD-L1 group was higher than that in H/R group(1.54±0.02 vs.1.18±0.01,t=6.88,P＜0.05).The relative expression of PD-L1 in H/R+CPG-ODN group was higher than that in H/R group(4.39±0.45 vs.2.15±0.28,t=7.32,P＜0.05).The relative expression of bcl-2 in H/R+CPG-ODN group was higher than that in H/R+PD-L1 group(0.91±0.08 vs.0.45±0.08,t=7.40,P＜0.05).The relative expression of bax in H/R+PD-L1 group was higher than that in H/R+CPG-ODN group(1.826±0.138 vs.1.06±0.113,t=7.44,P＜0.05).Conclusion By activating the PD-1/PD-L1 signaling pathway to exert immunomodulatory effects,it can significantly reduce the inflamma-tory response caused by I/RI,thereby exerting myocardial protection.
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