参附注射液对缺氧缺血性脑损伤新生大鼠皮质区钙网蛋白表达及神经元凋亡的影响

Effects of Shenfu injection on calreticulin expression and neuronal apoptosis in the cerebral cortex of neonatal rats with hypoxic-ischemic brain damage

摘要:

目的 探讨参附注射液对缺氧缺血性脑损伤(HIBD)新生大鼠皮质区钙网蛋白(CRT)的表达、细胞内游离Ca²⁺ 浓度以及神经元凋亡的影响及可能的脑保护机制。方法 7 日龄新生大鼠随机分为对照组、缺氧缺血组和参附干预组,各组进一步分为缺氧缺血后3、6、12、24、72 h 5 个亚组,每组10 只。参照Rice 法制备HIBD 模型;对照组不予缺血缺氧处理;参附干预组于造模后立即腹腔注射参附注射液(10 mL/kg),每日1 次,连用3 d。RT-PCR 及Western blot 法检测CRT mRNA 及蛋白的表达变化;荧光显微镜法检测脑细胞中游离Ca²⁺ 浓度;流式细胞术检测神经元凋亡率。结果 缺氧缺血组和参附干预组各时间点CRT mRNA 及蛋白表达水平均较对照组升高(P<0.05),且参附干预组较缺氧缺血组升高更加明显(P<0.05);不同时间点,缺氧缺血组细胞内游离Ca²⁺ 浓度和神经元凋亡率均较对照组明显升高(P<0.05),而参附干预组细胞内游离Ca²⁺ 浓度和神经元凋亡率均较缺氧缺血组显著降低(P<0.05),但仍高于对照组(P<0.05)。结论 参附注射液可能通过上调CRT 的表达,降低细胞内钙超载来减轻新生大鼠缺氧缺血性脑损伤。

Abstract:

Objective To examine the expression of calreticulin (CRT) and the changes of intracellular free calcium and neuronal apoptosis in the cerebral cortex of neonatal rats with hypoxic-ischemic brain damage (HIBD), and to investigate the intervention effects of Shenfu injection. Methods Seven-day-old rats were randomly assigned to three groups: control, hypoxic-ischemia (HI) and Shenfu-treated. Each group (n=50) was subdivided into 5 groups sacrificed at 3, 6, 12, 24 and 72 hours. Rat models of HIBD were prepared according to the Rice's method. Rats in the control group only underwent the separation of right common carotidartery. Shenfu injection was administered by intraperitoneal injections right after HI insults and then once daily at a dosage of 10 mL/kg for 3 days in the Shenfu-treated group. The expression of CRT in the cerebral cortex was detected by RT-PCR and Western blot. The free calcium concentrations were determined under a fluorescent microscope. The apoptosis rate was measured by the flow cytometry. Results Compared with the control group, the expression levels of CRT in the HI and the Shenfu-treated groups were obviously up-regulated (P<0.05), and the expression levels of CRT in the Shenfu-treated group were notably higher than those in the HI group (P<0.05) at all time points. The concentrations of intracellular free calcium and the apoptosis rate of neurons in the cerebral cortex in the Shenfu-treated group were significantly reduced compared with those in the HI group (P<0.05), but increased significantly compared with those in the control group at all time points (P<0.05). Conclusions Shenfu injection may have neuroprotective effects against HIBD by up-regulation of CRT expression and relief of calcium overload.