Yang Dan, Wang Gang, Yang Li-Jun, Duan Ren-Ze, Chen Xian-Bing
Department of Pathology, Minda Hospital of Hubei University for Nationalities, Enshi, Hubei 445000, China/Medical School, Hubei Minzu University, Enshi, Hubei 445000, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2023 Jul 15;25(7):751-758. doi: 10.7499/j.issn.1008-8830.2302026.
To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism.
Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62).
Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (<0.05), and a significant increase in the protein expression level of P62 (<0.05).
Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.
研究川芎嗪注射液对新生缺氧缺血性脑病（HIE）大鼠线粒体自噬的影响及其分子机制。
将7日龄新生Sprague-Dawley大鼠随机分为假手术组8只、模型组12只、川芎嗪组12只。模型组和川芎嗪组大鼠采用结扎左颈总动脉并进行缺氧处理建立新生大鼠HIE模型，假手术组大鼠暴露血管但不做其他处理。川芎嗪组大鼠在缺氧缺血后每日腹腔注射川芎嗪（20mg/kg），假手术组和模型组大鼠每日腹腔注射等体积生理盐水。处理7天后采集样本。采用苏木精-伊红染色和尼氏染色观察脑组织神经元的病理变化；采用免疫组织化学染色观察海马和皮质中PINK1和Parkin的阳性表达；采用TUNEL染色检测神经元凋亡；采用蛋白质免疫印迹法检测线粒体自噬通路蛋白PINK1和Parkin以及自噬相关蛋白Beclin-1、微管相关蛋白1轻链3（LC3）和泛素结合蛋白（P62）的表达水平。
与假手术组相比，模型组神经元数量显著减少，细胞间隙增大，排列疏松，出现脂质空泡化，尼氏体减少。模型组海马中PINK1和Parkin的阳性表达增加、神经元凋亡率升高、PINK1、Parkin、Beclin1和LC3的蛋白表达水平升高（<0.05），P62的蛋白表达水平降低（<0.05）。与模型组相比，川芎嗪组神经元排列有序数量显著增加，尼氏体增加，PINK1和Parkin的阳性表达、神经元凋亡率以及PINK1、Parkin、Beclin1和LC3的蛋白表达水平显著降低（<0.05），P62的蛋白表达水平显著升高（<0.05）。
川芎嗪可减轻新生大鼠缺氧缺血性脑损伤，在一定程度上抑制神经元凋亡，可能是通过抑制PINKl/Parkin介导的自噬实现的。
