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目的目前认为神经细胞凋亡在新生儿缺氧缺血性脑病(hypoxicischemicencephalopathy,HIE)的病理过程中起重要作用,细胞色素C(CytC)是重要的促凋亡蛋白因子之一。近年研究发现,肌苷对成年大鼠脑缺血损伤有保护作用,肌苷可降低CytCmRNA的表达从而抑制神经细胞凋亡。本实验通过观察肌苷对新生大鼠缺氧缺血性脑损伤(HIBD)后神经细胞凋亡和细胞色素C基因表达的影响,以初步探讨肌苷对HIBD新生大鼠脑保护作用和可能的机制。方法健康7日龄SD大鼠140只被随机分为3组:正常对照组(n=40),肌苷治疗组(n=50)和HIBD组(n=50),其中肌苷治疗组和HIBD组分别再随机分为缺氧缺血(HI)后6h,12h,1d,3d,7d5个亚组(各亚组n=10)。通过分离、结扎左颈总动脉和8%低氧暴露制备HIBD动物模型。正常对照组不进行缺氧缺血处理,按肌苷治疗组和HIBD组各相同时间点随机分为5个亚组(各亚组n=8)。肌苷治疗组于模型制备后即刻开始腹腔注射肌苷注射液100mg/kg,每天2次,连续7d。以TUNEL法检测神经元凋亡情况,原位杂交技术测定CytCmRNA表达情况。结果正常对照组皮质区和海马区可见少许凋亡细胞和CytC阳性细胞,HI后6hHIBD组皮质区和CA1区凋亡细胞和CytC阳性细胞即见增多,于HI后1d达高峰,之后逐渐下降,HI后7d凋亡细胞和CytC阳性细胞数仍明显高于正常对照组,各时间点与正常对照组相比较,差异有显著性意义(P0.05)。经肌苷治疗后凋亡细胞数和神经细胞CytCmRNA表达均减少,各时间点与HIBD组相应时间点比较,差异有显著性意义(P0.05)。直线相关分析显示HIBD后,凋亡细胞数与CytCmRNA表达呈显著正相关(r=0.88,P0.01)。结论HI损伤后给予肌苷干预能减少HI导致的神经细胞凋亡,下调CytCmRNA表达。肌苷治疗后,新生HIBD大鼠的凋亡细胞数减少与CytCmRNA表达下调呈显著正相关,提示肌苷可能通过抑制CytCmRNA表达从而起到减少细胞凋亡、保护神经元的作用。
OBJECTIVE: Neuronal apoptosis plays an important role in the pathogenesis of neonatal hypoxicischemic encephalopathy (HIE). CytC is one of the important pro-apoptotic protein factors. Recent studies have found that inosine has a protective effect on cerebral ischemia injury in adult rats, inosine can reduce CytC mRNA expression and thus inhibit apoptosis of nerve cells. In this study, we observed the effects of inosine on neuronal apoptosis and cytochrome C gene expression after hypoxic-ischemic brain damage (HIBD) in neonatal rats to explore the possible protective effects of inosine on neonatal rats with HIBD mechanism. Methods 140 healthy 7-day-old SD rats were randomly divided into 3 groups: normal control group (n = 40), inosine treatment group (n = 50) and HIBD group (n = 50) HIBD group were randomly divided into 5 subgroups (n = 10 in each subgroup) 6h, 12h, 1d, 3d and 7d after hypoxia-ischemia (HI). HIBD animal models were prepared by isolation, ligation of the left common carotid artery and exposure to 8% hypoxia. Normal control group without hypoxic-ischemic treatment, according to inosine treatment group and HIBD group at the same time points were randomly divided into 5 subgroups (each subgroup n = 8). Inosine treatment group began intraperitoneal injection of inosine 100mg / kg immediately after the model was prepared, twice daily for 7 days. Neuronal apoptosis was detected by TUNEL method and the expression of CytC mRNA was detected by in situ hybridization. Results Apoptotic cells and CytC positive cells were observed in the cortex and hippocampus in normal control group. The apoptotic cells and CytC positive cells in cortex and CA1 area of HIBD group increased at 6 h after HI, reached a peak at 1 d after HI, and then decreased gradually. The number of apoptotic cells and CytC positive cells on day 7 after HI was still significantly higher than that of the normal control group. There was significant difference between the control group and the control group at each time point (P <0.05). After inosine treatment, the number of apoptotic cells and the expression of CytC mRNA in nerve cells decreased. The differences were statistically significant (P <0.05) at each time point compared with the corresponding time point in HIBD group. Linear correlation analysis showed that after HIBD, the number of apoptotic cells was positively correlated with the expression of CytC mRNA (r = 0.88, P <0.01). Conclusion Inosine administration after HI injury can reduce the apoptosis induced by HI and decrease the expression of CytC mRNA. After inosine treatment, the decrease of apoptotic cells in neonatal HIBD rats was significantly and positively correlated with the down-regulation of CytC mRNA, suggesting that inosine may play a role in reducing apoptosis and protecting neurons by inhibiting the expression of CytC mRNA.