Page 45 - 南京医科大学学报自然科学版

第43卷第6期 扎西吉，张 赟，马凤美，等. 基于PKC/ERK通路探讨败酱总黄酮对缺氧缺血性脑病新生大鼠神经元
2023年6月 凋亡的影响［J］. 南京医科大学学报（自然科学版），2023，43（6）：786-794 ·787 ·

by gavage，once daily for 7 consecutive days. The neurological deficit score was used to detect the nerve function of rats. Hematoxylin⁃
eosin（HE）staining was used to observe the pathological changes in the hippocampus of brain tissue. TdT⁃mediated dUTP nick end
labeling（TUNEL）/neuronal nuclei antigen（NeuN）fluorescent double staining was used to observe the apoptosis of hippocampal
neurons. Western blot was used to detect the expression of protein kinase C（PKC）/extracellular signal ⁃ regulated kinase（ERK）
pathway and apoptosis⁃related proteins in brain tissue. Oxygen⁃glucose deprivation/reperfusion（OGD/R）was used to construct a cell
model. CCK⁃8 and lactate dehydrogenase（LDH）assays were performed to analyze the neuronal effect of total flavonoids of patriniae
radix on primary hippocampal neurons. Cell apoptosis was analyzed by flow cytometry，and Western blot was used to detect the
expression of apoptosis⁃related proteins. Results：In the in vivo study，compared with the sham operation group，the rats in the model
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group showed different degrees of neurological deficits，and the neurological deficit score，the numbers of NeuN TUNEL cells，the
expression of Bax and Caspase⁃3 in brain tissue increased significantly，while the expression of Bcl⁃2，the ratios of p⁃PKC/PKC and
p⁃ERK1/2/ERK1/2 reduced significantly（P < 0.05）. The neurons in the CA1 area of the hippocampus were swollen and decreased in
number. Compared with the model group，the neurological function of the total flavonoids of patriniae radix low⁃dose and high⁃dose
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group improved，and the neurological deficit score，the numbers of NeuN TUNEL cells，the expression of Bax and Caspase⁃3 in brain
tissue reduced significantly，while the expression of Bcl⁃2，the ratios of p⁃PKC/PKC and p⁃ERK 1/2/ERK1/2 increased significantly（P <
0.05）. The neurons in the CA1 area of the hippocampus were increased in density. And on the basis of the intervention of total
flavonoids of patriniae radix，combined with chelerythrine can significantly reduce the inhibitory effect of total flavonoids of patriniae
radix on hippocampal neuronal apoptosis after HIE. In an in vitro study，total flavonoids of patriniae radix increased cell viability after
OGD/R injury，reversed neuronal damage and reduced hippocampal neuronal apoptosis，while increasing hippocampal neuronal p⁃PKC/
PKC and p⁃ERK1/2 /ERK1/2 ratio（P < 0.05），verified the activation of PKC/ERK pathway by total flavonoids of patriniae radix in OGD/R⁃
induced primary hippocampal neurons. Conclusion：Total flavonoids of patriniae radix can inhibit hippocampal neuron apoptosis in
neonatal rats with HIE，and its mechanism may be related to the activation of PKC/ERK pathway.
［Key words］ total flavonoids of patriniae radix；hypoxic⁃ischemic encephalopathy；neuronal apoptosis；protein kinase C；extracellu⁃
lar signal⁃regulated kinase
［J Nanjing Med Univ，2023，43（06）：786⁃794］

围产期窒息是新生儿科的一个重大问题，会影 经元凋亡的影响，以期为HIE的治疗提供参考。
响胎儿的发育和成长，严重时可导致胎儿缺氧、窒
1 材料和方法
［1］
息甚至死亡 。缺氧缺血性脑病（hypoxic⁃ischemic
encephalopathy，HIE）是临床婴幼儿围产期窒息引起 1.1 材料
的一种相对常见的恶性并发症，是导致新生儿死亡、 10只SD孕鼠购自湖南斯莱克景达实验动物有
［2］
神经系统损伤及脑发育滞后的主要原因 。缺氧缺血 限公司，许可证号为SCXK（湘）2019⁃0004，每只可产
可导致ATP耗竭、神经元氧化应激和细胞死亡 ［3-4］ 。蛋 仔 10~12 只。单独饲养在无菌的鼠笼中，保持温度
白激酶C（protein kinase C，PKC）/细胞外信号调节激 22~24 ℃、湿度 45%~50%，12 h 明暗周期，可自由进
酶（extracellular signal⁃regulated kinase，ERK）通路在 食和饮水。取出生 10 d 的 SD 大鼠幼崽 90 只（体重
大脑神经系统调节中起着重要作用，与神经发育障 14~20 g）进行实验。本研究经青海省妇女儿童医院
［5］
碍和围产期学习、记忆障碍有关 。据报道，激活 医学研究中心实验动物使用与管理委员会批准
PKC/ERK 信号通路可改善小鼠脑出血后的氧化应 （IACUC：20200413002）。
［6］
激和神经元凋亡 。败酱草作为具有清热解毒、活 败酱草总黄酮（含量51.76%，批号ZL20150513，
血化瘀作用的中药在中国已有数千年的历史，有抗 南京泽朗医药科技有限公司）；白屈菜红碱（PKC 抑
癌、抗炎、抗氧化、抗菌等药理活性 。败酱总黄酮 制剂，纯度98.56%，HY⁃12048，Med Chem Express 公
［7］
是败酱草的主要活性成分，在修复受损神经元，促 司，美国）；罗氏原位末端转移酶标记（TdT⁃mediated
进神经功能恢复方面作用显著，可减少脑缺血再灌 dUTP nick end labeling，TUNEL）染色试剂盒（红色荧
注大鼠神经元凋亡 ［8-10］ 。然而，败酱总黄酮能否改 光）（11684817910，Roche Diagnostics 公司，美国）；
善 HIE 引起的神经元凋亡还未见相关报道。因此， RIPA 裂解液、二辛可宁酸（bicinchoninic acid，BCA）
本研究旨在探讨败酱总黄酮对HIE新生大鼠海马神 试剂盒、CCK⁃8试剂盒、膜联蛋白V⁃异硫氰酸荧光素/