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目的探讨Bax-抑制肽(BIP)对缺氧缺血性脑损伤(HIBD)新生鼠神经细胞凋亡的抑制作用及可能有效治疗的时间窗。方法选取182只7日龄新生Wistar大鼠,随机分为Sham组(假手术组,n=14)、HIBD组(对照组,n=84)、BIP组(实验组,n=84)3组。HIBD组和BIP组通过结扎左侧颈总动脉以及置于含氧8%的氮氧混合气中2 h建立HIBD模型,Sham组仅游离左侧颈总动脉,不结扎、不低氧处理。HIBD组和BIP组分别经左侧侧脑室注射生理盐水5μl和BIP5μg(5μl)。根据HIBD模型建立后注射BIP和生理盐水的时间不同,将HIBD组和BIP组分为0 h、6 h、12 h、24 h、48 h、72 h 6个亚组(n=14)。Sham组不注射任何药物。所有实验动物均于HIBD模型建立后7天处死,进行大脑海马组织病理检测、蛋白免疫印迹法(Western blot)检测、逆转录PCR(RT-PCR)测定,观察脑组织改变、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白及Caspase-3 mRNA的含量。结果 BIP组0 h、6 h、12 h、24 h、48 h亚组病理损伤程度均较HIBD组相应亚组轻,两组72 h亚组无明显差异。BIP组0 h、6 h、12 h、24 h、48 h亚组Caspase-3蛋白及Caspase-3 mRNA含量均明显明显低于HIBD组,差异有统计学意义[Caspase-3蛋白:0 h(0.164±0.308)比(0.473±0.088),6 h(0.262±0.304)比(0.473±0.054),12 h(0.339±0.020)比(0.472±0.119),24 h(0.379±0.181)比(0.481±0.068),48 h(0.417±0.026)比(0.478±0.081);Caspase-3 mRNA:0 h(0.220±0.008)比(0.245±0.019),6 h(0.285±0.018)比(0.365±0.027),12 h(0.300±0.017)比(0.398±0.045),24 h(0.565±0.026)比(0.850±0.080),48 h(0.546±0.028)比(0.651±0.023),P<0.001],两组72 h亚组差异无统计学意义(P>0.05)。结论BIP可明显减少新生鼠HIBD后Caspase-3蛋白及Caspase-3 mRNA含量,抑制缺氧缺血后脑组织神经细胞凋亡;缺氧缺血后48 h内是BIP治疗HIBD的有效治疗时间窗,且越早应用治疗效果越好。
Objective To investigate the inhibitory effect of Bax-inhibitor peptide (BIP) on neuronal apoptosis in neonatal rats with hypoxic-ischemic brain damage (HIBD) and the possible time window for its possible treatment. Methods A total of 182 7-day-old Wistar rats were randomly divided into three groups: Sham group (sham operation group, n = 14), HIBD group (control group, n = 84), BIP group . HIBD group and BIP group were established HIBD model by ligating the left common carotid artery and placed in 8% oxygenated oxygen and nitrogen mixture for 2 h. Sham group only free left common carotid artery, not ligated, not hypoxia treatment. The HIBD group and the BIP group were injected with 5μl of saline and BIP5μg (5μl) through the left lateral ventricle respectively. HIBD and BIP groups were divided into six subgroups (n = 14) of 0 h, 6 h, 12 h, 24 h, 48 h and 72 h after HIBD model was established. Sham group did not inject any drugs. All experimental animals were sacrificed 7 days after establishment of HIBD model. The pathological changes of hippocampus were observed, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the changes of brain tissue, cysteine Caspase-3 protein and Caspase-3 mRNA content. Results The pathological changes of BIP group at 0 h, 6 h, 12 h, 24 h and 48 h were lighter than that of HIBD group, and there was no significant difference between the two groups in 72 h subgroup. Caspase-3 protein and caspase-3 mRNA in BIP group were significantly lower than those in HIBD group at 0 h, 6 h, 12 h, 24 h and 48 h (P <0.05), the difference was statistically significant [Caspase-3 protein: 0 h 0.164 ± 0.308, 0.473 ± 0.088, 0.426 ± 0.304, 0.421 ± 0.119, 0.372 ± 0.304, 0.379 ± 0.181, 0.481 ± 0.304, (0.245 ± 0.019), 6 h (0.285 ± 0.018) vs (0.365 ± 0.027) at 0 h (0.220 ± 0.008), 12 h (0.300 ± 0.017) vs 0.398 ± 0.045, 24 h (0.565 ± 0.026) vs (0.850 ± 0.080), 48 h (0.546 ± 0.028) vs (0.651 ± 0.023), P <0.001] h subgroup there was no significant difference (P> 0.05). Conclusions BIP can significantly reduce the content of Caspase-3 protein and Caspase-3 mRNA in HIBD neonatal rats and inhibit the apoptosis of neural cells in hypoxic-ischemic brain tissue. Within 48 hours after hypoxic-ischemic ischemia, BIP can effectively treat HIBD with BIP, And the sooner the application of treatment better.