The Protective Effects Of Cold Induced RNA-binding Protein On Cardiomyocytes Apoptosis

Objective：Previews study showed that32°C has a protective effects on brain injury andmyocardial injury, It's used clinically for heart surgery and the treatment of severebrain damage. At present the mechanism about protective effect of hypothermia isstill unclear. Cold-induced RNA-binding protein (CIRP) is a cold shock proteinwhich was significantly induced at the temperature of32°C, its role is unclear either.A large number of studies have shown that abnormal apoptosis of myocardial cellsplay an important role in the development of many heart diseases. Our work wasdesigned to study the protective mechanism of hypothermia on myocardial injury byinvestigating the effect of CIRP on the apoptosis of H9c2cells.Method:H9c2cells were treated with H₂O₂which can make a cell model of apoptosis.Cell incubators with electronic temperature control were used respectively forintervention of room temperature (37°C) and hypothermia (32°C). Stealth RNAwas used for RNA interference, which can down-regulate expression of CIRP.Lactate dehydrogenase (LDH) activity was detected by collecting thesupernatant of cells; Cell viability was detected by MTT colorimetric assay.Apoptosis of h9c2cells was detected by flow cytometry after labeling the cells withAnnexin V-FITC/PI. mRNA expression of CIRP was detected by RT-PCR; Theprotein expression of CIRP was detected by Western blot. Experimental groups: thefirst part: â… normal control group (control), II. Room temperature H₂O₂group (RT+H₂O₂), III. Hypothermia H₂O₂group (HT+H₂O₂). The second part: â… Roomtemperature H₂O₂group (RT+H₂O₂), II Hypothermia H₂O₂group (HT+H₂O₂), IIIhypothermia+H₂O₂+RNAi group (HT+H₂O₂+RNAi)。 Results:mRNA and protein expression of CIRP can be detected in H9c2cells at Roomtemperature. The H9c2cells treated with H₂O₂were placed respectively at roomtemperature incubator and low temperature incubator, the results showed that cellsurvival rate in H₂O₂group at room temperature (RT+H₂O₂)was less than that ofH₂O₂group of hypothermia (HT+H₂O₂) and normal control group; LDH activity andapoptosis of RT+H₂O₂group were higher than HT+H₂O₂group and normalcontrol group, and the difference between the two treatment groups becamesignificant at6h(p<0.05); In the HT+H₂O₂group, mRNA and protein expressionlevels of CIRP were higher than RT+H₂O₂group, and the differences between thetwo groups became significant at6h(p<0.05);With the intervene of CIRP inHT+H₂O₂group, the CIRP protein expression was significantly decreased, the LDHactivity and apoptosis were increased compared with HT+H₂O₂group, the differencebetween HT+H₂O₂group and the CIRP-RNAi group was significant (p<0.05).Conclusions：Apoptosis can be induced by H₂O₂in H9c2cells. The induction of CIRP canruduce injury and apoptosis induced by H₂O₂in H9c2cells. The LDH activity andapoptosis rate of HT+H₂O₂group were reduced by RNA interference ofCIRP,which suggested that CIRP may have a protective effect on H9c2cells.