Cold-inducible RNA binding protein inhibits H₂O₂-induced apoptosis in rat cortical neurons

Abstract

Objective: The expression cold-inducible RNA-binding protein (CIRP) is significantly enhanced in neurons under hypothermia, but its roles remain unclear. This study aims to investigate whether the cerebral protection under hypothermia is mediated by the CIRP-mediated inhibition of neuronal apoptosis.

Methods: Primary rat cortical neurons were isolated, cultured, and transduced with lentiviral CIRP-RNAi. Apoptosis of the transduced neurons was induced with 100 μmol/L H₂O₂, the treated cells were divided into two groups, and cultured in 37 °C or 32 °C incubator respectively. Cell viability was detected by MTT colorimetric assay. Neuronal apoptosis was detected by flow cytometry after labeling the cells with Hoechst 33342 and Annexin V-FITC/PI. The protein expressions of CIRP, activated caspase-3, and thioredoxin (TRX) were detected by Western blot.

Results: Under 32 °C, CIRP protein is significantly induced in cortical neurons; the expression of activated caspase-3 decreases, while the TRX expression increases. The rate of neuronal apoptosis is 4.5±0.8%. Under 37 °C, CIRP expression is evidently reduced in cortical neurons; the expression of activated caspase-3 is significantly enhanced with reduced level of TRX expression. The rate of neuronal apoptosis reaches 53.5±1.7% (P < 0.05, compared to that in 32 °C group).

Conclusions: The induction of CIRP protein in rat cortical neurons under hypothermia inhibits H₂O₂-induced neuronal apoptosis and thereby exerts neuroprotective effect, which forms one of the cerebral protective pathways under hypothermia.