Effect of CIPC and intervention of Ca(2+)-regulated factors on CaN, cbl-b and p-AKT expression in neurons

Abstract

CaN induces the apoptosis in neurons, but the influence of CIPC and the intervention of pretreament with Ca(2+)-regulated factors, such as nimodipine, MK801 and cyclosporine A, on CaN expression is not clear. We also do not know whether cbl-b takes part in the induction of ischemia or induces an expression change of cbl-b in CIPC. So we will discuss the effect of CIPC, pretreatment with nimodipine, MK801 and cyclosporine A on the expression of the CaN, cbl-b and p-AKT in the hippocampus neurons. In our study, we established rat models including sham, ischemia, CIPC, nimodipine, MK801 and cyclosporine A. The neurological deficit scores were processed. The right hippocampus was removed and stained with TTC, and the volume of cerebral infarction was calculated. The apoptotic neurons were detected by TUNEL staining. The expressions of CaN, cbl-b and p-AKT at the protein level were examined by Western blotting, and the transcription of cbl-b by RT-PCR, respectively. The results showed that the neurological deficit scores, the volume of the cerebral infarction, the numbers of the apoptotic neurons, the protein expression of CaN, cbl-b and the transcription of cbl-b were the highest in the ischemia and MK801 groups, there were no difference between the two groups(P>0.05); these factors in CIPC group were all lower than those in the ischemia group(P<0.05); and much lower in the nimodipine and cyclosporine A group than those in the CIPC group (among them, the volume of the cerebral infarction in the nimodipine and cyclosporine A groups P<0.01, the expression of CaN in nimodipine group P<0.01, others were P<0.05), but no significant difference existed between the nimodipine and cyclosporine A groups(P>0.05). The expression of p-AKT was the lowest in the ischemia and MK801 groups, and there was no difference between the two groups (P>0.05), This factor was higher in CIPC group than that in the ischemia group (P<0.05); it was the highest in the nimodipine and cyclosporine A groups among these groups (the nimodipine group P<0.01, the cyclosporine A group P<0.05), no significant difference existed between the nimodipine and cyclosporine A groups (P>0.05. Continuous ischemia increases the expression of CaN, and the transcription and the protein expression of cbl-b. Cbl-b reduces the phosphorylated expression of AKT, ultimately activating apoptosis. CIPC inhibits above process and reduces the expression of CaN and cbl-b, and increases the expression of p-AKT, thereby inhibiting apoptosis in neurons. Nimodipine and cyclosporine A can reduce the expression of CaN and cbl-b, and increase the expression of p-AKT, via a moderate increase in the concentration of intracellular calcium and inhibition of the activity of CaN; MK801 counteracts the effect of CIPC.