Kallistatin deficiency exacerbates neuronal damage after cardiac arrest

Abstract

The purpose of study was to evaluate that kallistatin deficiency causes excessive production of reactive oxygen species and exacerbates neuronal injury after cardiac arrest. For in vitro study, kallistatin knockdown human neuronal cells were given ischemia-reperfusion injury, and the oxidative stress and apoptosis were evaluated. For clinical study, cardiac arrest survivors admitted to the ICU were divided into the good (CPC 1-2) and poor (CPC 3-5) 6-month neurological outcome groups. The serum level of kallistatin, Nox-1, H₂O₂ were measured. Nox-1 and H₂O₂ levels were increased in the kallistatin knockdown human neuronal cells with ischemia-reperfusion injury (p < 0.001) and caspase-3 was elevated and apoptosis was promoted (SERPINA4 siRNA: p < 0.01). Among a total of 62 cardiac arrest survivors (16 good, 46 poor), serum kallistatin were lower, and Nox-1 were higher in the poor neurological group at all time points after admission to the ICU (p = 0.013 at admission; p = 0.020 at 24 h; p = 0.011 at 72 h). At 72 h, H₂O₂ were higher in the poor neurological group (p = 0.038). Kallistatin deficiency exacerbates neuronal ischemia-reperfusion injury and low serum kallistatin levels were associated with poor neurological outcomes in cardiac arrest survivors.

Figure 1

Cell viability and kallistatin level of OGD/Reoxy-treated control and kallistatin knockdown HCN-2 cells. (A) The cell viability was measured with/without OGD/Reoxy processes in control and kallistatin knockdown human neuronal (HCN-2) cells. In both the control and kallistatin knockdown cells, exposure to OGD/Reoxy decreased cell viability. (Control siRNA: p < 0.01, SERPINA4 siRNA: p < 0.001). (B) The expression of kallistatin was decreased the most in the kallistatin knockdown HCN-2 cells with OGD/Reoxy. OGD: oxygen–glucose deprivation, reoxy: reoxygenation.

Figure 2

Nox-1, Hydrogen peroxide, Cleaved caspase 3 in control and kallistatin knockdown HCN-2 cells after exposure to OGD/Reoxy. Nox-1 expression and H₂O₂ levels were increased in the kallistatin knockdown human neuronal cells with OGD/Reoxy (p < 0.001) and cleaved caspase-3 expression was elevated and apoptosis was promoted (SERPINA4 siRNA: p < 0.01). OGD: oxygen–glucose deprivation, Reoxy: reoxygenation. Original blots/gels are presented in Supplementary Fig. 3.

Figure 3

Clinical investigation flow diagram. Out of 113 OHCA patients, a total of 62 patients were selected as the final study subjects. 16 patients had a good neurological outcome (CPC 1–2) and 46 patients had a poor neurological outcome (CPC 3–5) 6 months after cardiac arrest. OHCA: out of hospital cardiac arrest, CPC: cerebral performance category.

Figure 4

Serum kallistatin, Nox-1, hydrogen peroxide level among 6-month good CPC (CPC 1–2) and poor CPC (CPC 3–5). Serum levels of kallistatin, NADPH oxidase, and H₂O₂ were measured both good (blue line) and poor CPC (orange line) group at admission time, 24 h, and 72 h. Serum kallistatin levels were lower in poor CPC groups and Nox-1 levels were higher at all time points (0.60 μg/L vs. 2.41 μg/L, p = 0.013 at admission; 1.08 μg/L vs. 2.27 μg/L, p = 0.020 at 24 h; 1.17 μg/L vs. 2.67 μg/L, p = 0.011 at 72 h). At 72 h, H₂O₂ levels were higher in poor CPC groups (6.82 μmol/L vs. 7.27 μmol/L, p = 0.038). CPC: cerebral performance category.