Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia-Reperfusion Injury in Rats

Abstract

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia-reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli's four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.

Keywords: Animal model; Cerebral ischemia–reperfusion injury; LC–MS/MS; Mild hypothermia; Urine proteome.

Fig. 1

Protective effects of mild hypothermia on neurological damage after cerebral ischemia–reperfusion (IR) injury. a Hippocampal CA1 region, b Cerebral cortex. Sham Sham-operation group, IR cerebral I/R injury followed by normothermia (37 °C) group, IR + MH cerebral I/R injury followed by 4 h of MH (32 °C) group. Scale bars = 50 µm

Fig. 2

Differentially expressed urinary proteins after cerebral ischemia–reperfusion (IR) injury followed by normothermia or MH. a Hierarchical cluster analysis, b Venn diagram. Sham Sham-operation group, IR cerebral I/R injury followed by normothermia (37 °C) group, IR + MH cerebral I/R injury followed by 4 h of MH (32 °C) group

Fig. 3

GO analysis of the DEPs associated with mild hypothermia. a Biological process, b Cellular component, c Molecular function; (p value < 0.05)

Fig. 4

STRING protein–protein interaction network analysis of differentially expressed proteins associated with mild hypothermia. The number of nodes is 112, the average node degree is 3.54, and the average local clustering coefficient is 0.394 (p-value < 1.0e−16)

Fig. 5

Validation of two DEPs associated with MH by western blotting. a Representative blots visualizing the levels of FZD1 and B2M in the three groups. β-actin was used as the loading control. n = 4. b Quantitative analysis of the expression level of FZD1 and B2M in the hippocampus. The data passed normality test. FZD1, One way ANOVA, F (2, 9) = 8.683, p = 0.0079; Brown–Forsythe test, F (DFn, DFd) = 0.6464 (2,9), p = 0.5466; Tukey’s multiple comparisons test, Sham vs. IR: p = 0.0116, IR vs. IR + MH: p = 0.0177. B2M, One way ANOVA, F (2,9) = 7.884, p = 0.0105; Brown–Forsythe test, F (DFn, DFd) = 1.537 (2,9), p = 0.2666; Tukey’s multiple comparisons test, Sham vs. IR: p = 0.0264, IR vs. IR + MH: p = 0.0136. (* p < 0.05)