川芎嗪通过PINK1/Parkin通路调控自噬对新生大鼠缺氧缺血性脑病的影响

Effect of ligustrazine on hypoxic-ischemic encephalopathy in neonatal rats by regulating autophagy through the PINK1/Parkin pathway

目的研究川芎嗪注射液对缺氧缺血性脑病新生大鼠自噬的影响及其分子机制。方法将7日龄Sprague-Dawley新生大鼠随机分为假手术组(n=8)、模型组(n=12)、川芎嗪组(n=12)。模型组和川芎嗪组行左侧颈总动脉结扎后再缺氧处理,制备新生大鼠缺氧缺血性脑病模型。假手术组只暴露血管不做其他处理。川芎嗪组在缺氧缺血后每日腹腔注射川芎嗪注射液20 mg/kg,假手术组与模型组每日腹腔注射等体积的0.9%氯化钠溶液。在治疗7 d后取材,采用苏木精-伊红染色及尼氏染色观察脑组织神经元病理变化情况。采用免疫组织化学染色观察各组新生大鼠大脑海马及皮质区域PTEN诱导假定激酶1(PTEN-induced putative kinase 1,PINK1)、Parkin表达。采用TUNEL染色检测神经元凋亡情况。通过免疫印迹法检测PINK1、Parkin、自噬特异性基因Beclin1、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)和泛素结合蛋白(Protein 62,P62)的表达。结果与假手术组相比,模型组新生大鼠神经元数量减少,细胞间隙增大,排列疏松,胞质空泡化,尼氏小体减少;PINK1、Parkin阳性表达增加,神经元凋亡率升高(均P<0.05);PINK1、Parkin、Beclin1、LC3蛋白表达增加,P62蛋白表达减少(均P<0.05)。与模型组相比,川芎嗪组新生大鼠神经元数量增多,细胞排列整齐,尼氏小体增加;PINK1、Parkin阳性表达减少,神经元凋亡率降低(均P<0.05);PINK1、Parkin、Beclin1、LC3蛋白表达减少,P62蛋白表达增加(均P<0.05)。结论川芎嗪在一定程度上缓解了新生大鼠缺氧缺血性脑损伤,抑制神经元凋亡,其机制可能与抑制PINK1/Parkin介导的自噬相关。

Objective To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy(HIE)and its molecular mechanism.Methods 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).Results 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(P<0.05)and the decreased protein expression level of P62 in the hippocampus were also observed in the model group(P<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(P<0.05),and a significant increase in the protein expression level of P62(P<0.05).Conclusions 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.