Department of Neurology, Second Affiliated Hospital, Dalian Medical University, Dalian 116027, China.
Brain Res. 2011 May 10;1389:1-8. doi: 10.1016/j.brainres.2011.02.085. Epub 2011 Mar 4.
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.
CaN 可诱导神经元凋亡，但 CIPC 的影响以及钙调节因子（如尼莫地平、MK801 和环孢素 A）预处理对 CaN 表达的影响尚不清楚。我们也不知道 cbl-b 是否参与了 CIPC 诱导的缺血或诱导 cbl-b 在 CIPC 中的表达变化。因此，我们将讨论 CIPC、尼莫地平、MK801 和环孢素 A 预处理对海马神经元中 CaN、cbl-b 和 p-AKT 表达的影响。在我们的研究中，我们建立了包括假手术、缺血、CIPC、尼莫地平、MK801 和环孢素 A 的大鼠模型。对神经功能缺损评分进行了处理。取出右侧海马并用 TTC 染色，并计算脑梗死体积。通过 TUNEL 染色检测凋亡神经元。通过 Western blot 分别检测 CaN、cbl-b 和 p-AKT 的蛋白表达水平，通过 RT-PCR 检测 cbl-b 的转录。结果显示，在缺血和 MK801 组中，神经功能缺损评分、脑梗死体积、凋亡神经元数量、CaN、cbl-b 蛋白表达和 cbl-b 转录最高，两组间无差异（P>0.05）；CIPC 组的上述因素均低于缺血组（P<0.05）；尼莫地平组和环孢素 A 组的这些因素均明显低于 CIPC 组（其中尼莫地平组和环孢素 A 组的脑梗死体积 P<0.01，尼莫地平组的 CaN 表达 P<0.01，其余 P<0.05），但尼莫地平组和环孢素 A 组之间无差异（P>0.05）。p-AKT 在缺血和 MK801 组中表达最低，两组间无差异（P>0.05）；CIPC 组高于缺血组（P<0.05）；尼莫地平组和环孢素 A 组的表达最高（尼莫地平组 P<0.01，环孢素 A 组 P<0.05），尼莫地平组和环孢素 A 组之间无差异（P>0.05）。持续缺血增加 CaN 的表达，以及 cbl-b 的转录和蛋白表达。Cbl-b 降低 AKT 的磷酸化表达，最终激活凋亡。CIPC 抑制上述过程并降低 CaN 和 cbl-b 的表达，增加 p-AKT 的表达，从而抑制神经元凋亡。尼莫地平组和环孢素 A 组通过适度增加细胞内钙浓度和抑制 CaN 活性，降低 CaN 和 cbl-b 的表达，增加 p-AKT 的表达，从而抑制凋亡；MK801 拮抗 CIPC 的作用。
