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目的:通过检测缺氧性脑损伤新生鼠脑组织中p53蛋白的表达,从而探究珍宝丸对缺氧性脑损伤的保护作用。方法:取新生7天Wistar乳鼠200只,雌雄不限,随机分正常对照组(40只)、模型缺氧1 h组(40只)、模型缺氧4 h组(40只)、珍宝丸缺氧1 h治疗组(40只)、珍宝丸缺氧4 h治疗组(40只)。将新生Wistar大鼠置于8%O2+92%N2(V/V)的缺氧箱内建立新生鼠缺氧模型。在造模前每日1次连续7天灌胃给予生理盐水和珍宝丸,缺氧期间也同样剂量给药,分别于缺氧后12、24、48、72 h处死取脑组织待测。采用HE染色观察脑组织的病理变化,采用免疫组织化学方法检测各组大鼠脑组织中p53蛋白的表达。结果:HE染色结果显示:与正常对照组比较,模型缺氧1、4 h组均在缺氧后12 h时间点出现脑组织排列松散、细胞明显水肿、毛细血管周围间隙增宽,24 h达高峰,差异有统计学意义(P<0.01),然后至48、72 h出现缓解但无明显改善且这两个时间点无明显差异(P>0.05),模型缺氧1 h组在各时间点状态好于模型缺氧4 h组,差异有统计学意义(P<0.05);珍宝丸缺氧1、4 h治疗组均在缺氧后12 h时间点出现少量脑神经细胞轻微水肿且24 h达高峰,差异有统计学意义(P<0.01),随时间缓解、恢复至正常,1 h治疗组各时间点状态略好于4 h治疗组但差异无统计学意义(P>0.05);与模型组比较,珍宝丸各治疗组在各时间点的状态均明显改善差异有统计学意义(P<0.01)。免疫组化检测结果显示:与正常对照组比较,模型缺氧1 h和4 h组p53蛋白的阳性表达均在缺氧后12 h时间点显著增高且24 h达高峰,差异有统计学意义(P<0.01),48、72 h时间点p53蛋白的阳性表达低于12 h时间点但仍明显高于正常对照组,差异有统计学意义(P<0.05);珍宝丸缺氧1 h和4 h治疗组p53蛋白的阳性表达也均在缺氧后12 h时间点增高且24h达高峰,差异有统计学意义(P<0.01),48、72 h时间点p53蛋白的阳性表达低于12 h时间点但差异无统计学意义(P>0.05);模型缺氧1 h和4 h组在各个时间点上p53蛋白的表达均明显高于珍宝丸缺氧1 h和4 h治疗组,差异有统计学意义(P<0.01)。结论:珍宝丸可显著降低缺氧性脑损伤大鼠脑组织中p53蛋白的表达水平,这可能是其对缺氧性脑损伤的保护机制之一。
Objective: To explore the protective effect of Zhenbao Pill on hypoxic brain injury by detecting the expression of p53 protein in neonatal rats with hypoxic brain injury. Methods: Two hundred and seventy newborn Wistar rats were randomly divided into normal control group (n = 40), hypoxia model group (n = 40), model group (n = 40) Hypoxia 1 h treatment group (40), Treasure pill hypoxia 4 h treatment group (40). Newborn Wistar rats were placed in 8% O2 + 92% N2 (V / V) hypoxia chamber to establish newborn rat hypoxia model. The model rats were given saline and Zhenbao pills orally once daily for 7 consecutive days prior to model establishment. The rats were also sacrificed at 12, 24, 48 and 72 h after hypoxia, respectively. The pathological changes of brain tissue were observed by HE staining. The expression of p53 protein in brain tissue of each group was detected by immunohistochemical method. Results: The results of HE staining showed that compared with the normal control group, the rats in the hypoxia and hypoxia groups were loosely arranged in the brain at 12 h after hypoxia, the cells were edematous and the perivascular spaces widened. At 24 h (P <0.01) .At the time of 48 h and 72 h, there was no significant difference between the two groups (P> 0.05) (P <0.05). Compared with model group, the rats in hypoxia for 4 h had a statistically significant difference (P <0.05) (P <0.01), with the time to ease, returned to normal, 1 h treatment group at each time point slightly better than the 4 h treatment group but the difference was not statistically significant (P> 0.05); and Compared with the model group, the state of each treatment group of Zhenbao Pill was significantly improved at all time points (P <0.01). The results of immunohistochemistry showed that compared with the normal control group, the positive expression of p53 protein in hypoxia 1 h and 4 h groups was significantly increased at 12 h after hypoxia and peaked at 24 h (P <0.05), the difference was statistically significant ( P <0.01). The positive expression of p53 protein at 48 and 72 h was lower than that at 12 h, but still significantly higher than that of the normal control group (P <0.05) h treatment group, the expression of p53 protein also increased at 12 h after hypoxia and reached the peak at 24 h, the difference was statistically significant (P <0.01). At 48 and 72 h, the positive expression of p53 protein was lower than 12 h (P> 0.05). The expression of p53 protein at 1 hour and 4 hours of hypoxia in each group was significantly higher than that of Zhenbao pill in 1 and 4 hours of hypoxia Statistical significance (P <0.01). Conclusion: Zhenbao pill can significantly reduce the expression of p53 protein in the brain tissue of rats with hypoxic brain injury, which may be one of the protective mechanisms for hypoxic brain injury.