Myocardial Protection Of Cold Inducible RNA-binding Protein Against Hypoxia/Reoxygenation Injury

Objective:Reserch on the expression of the cold-induced RNA binding protein and its rolein myocardial cell under the hypoxia/reoxygenation condition.Method:（1）Use the H9c2myocardial cells cultured in vitro to establish thehypoxia/reoxygenation (H/R) model（95%N2and5%CO2）.（2）Use RT-PCR and Western blot to detect the expression of the mRNA andprotein of CIRP under H/R conditions respectively, and then pick the time points withhighest expression which will be used as the H/R periods in the follow-upexperiments.（3）Carry out the hypoxia and reoxygenation treatment to the cells at thedetermined time points above,14h for hypoxia and2h for reoxygenation. Thefollowing experiments can be divided into ten groups:①normal control group;②H/R group;③CIRP siRNA treated cell group;④CIRP siRNA treated cell+H/R group;⑤Negative interference fragment treated cell group;⑥Negative interference fragment treated cell+H/R group;⑦CIRP overexpressing plasmid treated cell group;⑧CIRP overexpressingplasmid treated cell+H/R group;⑨Control plasmid treated cell group;⑩Control plasmid treated cell+H/R group.（4）Use western blot to detect the expression of CIRP protein and apoptosisprotein caspas-3and use the flow cytometry to detect the apoptosis of each group.Results:(1) During the myocardial hypoxia treatment, the RT-PCR showed thesignificant increase of the CIRP mRNA expression. At2h, it declined slightly. At14h, it reached the peak value. The western blot showed the significant increasing trend ofthe CIRP protein expression during hypoxia and it reached the peak value at14h.Flow cytometry showed that the apoptosis increased over time during hypoxia. At6hand8h, it reached the peak value and, at24h, it declined.(2) After14h of hypoxia, CIRP showed peak expression at2h duringreoxygenation.(3) Cells in the exogenous transfected CIRP overexpressing plasmid+H/Rgroup had lower capase-3expression and apoptosis rate than those in the H/R groupwith statistically significant result, P <0.05;Cells in the transfected CIRP siRNA+H/R group had higher capase-3expression and apoptosis rate than those in the H/Rgroup with statistically significant result, P <0.05. Cells in the CIRP overexpressinggroup had lower capase-3expression and apoptosis rate than the Control group withstatistically significant result, P <0.05. Cells in interfered CIRP expression group hadhigher caspase-3expression and apoptosis rate than the Control group, withstatistically significant result, P <0.05.Conclusions:(1) The CIRP gene expresses in H9c2cells in a low level.(2) Under H/R conditions, the H9c2cardiac cells have gradually increasingcold-induced RNA binding protein expression over time during hypoxia. It reachesthe peak value at14h of hypoxia and2h of reoxygenation.(3) Overexpression of CIRP can inhibit the apoptosis of the H9c2myocardialcells during the14h of hypoxia and2h of reoxygenation;(4) After14h of hypoxia and2h of reoxygenation of the H9C2myocardial cells,interfered CIRP expression can promote apoptosis.(5) CIRP overexpression can mitigate the apoptosis of the H9c2myocardial cellsunder normal conditions.(6) Interfered CIRP expression can aggravate the apoptosis of the H9c2myocardial cells under normal conditions.In conclusion,CIRP can protect H9c2myocardial cells against H/R injury.