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凋亡的影响
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青海省妇女儿童医院

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Exploring the effect of total flavonoids of patriniae radix on neuronal apoptosis in neonatal rats with hypoxic-ischemic encephalopathy based on PKC/ERK pathwayZhaxiji, Zhang Yun, Ma Fengmei, Wang Zhengling (Department of Neonatology, Qinghai women"s and children"s Hospital, Xining, Qinghai, 810000)
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    摘要:

    目的:探究败酱总黄酮对缺氧缺血性脑病(HIE)新生大鼠海马神经元凋亡的影响及其作用机制。方法:体内研究:72只新生大鼠分为假手术组、模型组、白屈菜红碱组(5 mg/kg)、败酱总黄酮低(50 mg/kg)、高(100 mg/kg)剂量组、败酱总黄酮+白屈菜红碱组(100 mg/kg败酱总黄酮+5 mg/kg白屈菜红碱),每组12只。采用结扎右颈总动脉和低氧处理2.5 h的方法构建HIE模型。白屈菜红碱组大鼠腹腔注射白屈菜红碱,败酱总黄酮各剂量组灌胃相应剂量的败酱总黄酮,败酱总黄酮+白屈菜红碱组大鼠在灌胃败酱总黄酮的同时腹腔注射白屈菜红碱,1次/d,连续给药7 d。通过神经功能缺损评分、负趋地性实验和翻正反射检测大鼠神经功能;HE染色观察脑组织海马区病理学变化;Fluoro-Jade C染色观察退化神经元;TUNEL/NeuN荧光双染观察海马神经元凋亡;Western blot法检测脑组织蛋白激酶C(PKC)/细胞外信号调节激酶(ERK)通路及凋亡相关蛋白[PKC、p-PKC、ERK1/2、p-ERK1/2、Bcl-2、Bax、Caspase-3]的表达。体外研究:采用氧-葡萄糖剥夺/再灌注(OGD/R)构建细胞模型,进行了CCK-8和乳酸脱氢酶(LDH)测定,以分析败酱总黄酮对原代海马神经元的神经保护作用,并采用流式细胞仪分析细胞凋亡,Western blot用于检测PKC、p-PKC、ERK1/2、p-ERK1/2蛋白表达。结果:在体内研究中,与假手术组相比,模型组大鼠出现不同程度的神经功能缺损现象,神经功能缺损评分、翻正反射时间和趋地性反射时间、Fluoro-Jade C+阳性和NeuN+TUNEL+阳性细胞数、脑组织Bax及Caspase-3表达显著增加,Bcl-2表达、p-PKC/PKC、p-ERK1/2/ERK1/2的比值显著降低(P<0.05),海马CA1区神经元肿胀、数量减少;与模型组相比,败酱总黄酮低、高剂量组神经功能改善,神经功能缺损评分、翻正反射时间和趋地性反射时间、Fluoro-Jade C+阳性和NeuN+TUNEL+阳性细胞数、脑组织Bax及Caspase-3表达显著降低,Bcl-2表达、p-PKC/PKC、p-ERK1/2/ERK1/2的比值显著增加(P<0.05),海马CA1区神经元密度增加;且在败酱总黄酮干预的基础上联用白屈菜红碱可显著减弱败酱总黄酮对HIE后海马神经元凋亡的抑制作用。在体外研究中,败酱总黄酮显著增加OGD/R损伤后的细胞活力,逆转神经元损伤并减少海马神经元细胞凋亡,同时增加了海马神经元p-PKC/PKC、p-ERK1/2/ERK1/2比值(P<0.05),在OGD/R诱导的原代海马神经元中验证了败酱总黄酮对PKC/ERK通路的激活。结论:败酱总黄酮可抑制HIE新生大鼠海马神经元凋亡,其作用机制可能与激活PKC/ERK通路有关。

    Abstract:

    Objective: To explore the effect of total flavonoids of patriniae radix on hippocampal neuron apoptosis in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its mechanism. Methods: In vivo studies: Seventy-two neonatal rats were divided into sham operation group, model group, chelerythrine group (5 mg/kg), and total flavonoids of patriniae radix low- (50 mg/kg) and high- (100 mg/kg) dose groups, total flavonoids of patriniae radix + chelerythrine group (100 mg/kg total flavonoids of patriniae radix + 5 mg/kg chelerythrine), with 12 rats in each group. The HIE model was constructed by ligating the right common carotid artery and hypoxic treatment for 2.5 h. Rats in the chelerythrine group were intraperitoneally injected with chelerythrine, and the total flavonoids of patriniae radix groups were given the corresponding doses of total flavonoids of patriniae radix by gavage, rats in the total flavonoids of patriniae radix + chelerythrine group were injected intraperitoneally with chelerythrine while giving total flavonoids of patriniae radix by gavage, once daily for 7 consecutive days. The neurological deficit score, negative geotaxis test and righting reaction were used to detect the nerve function of rats; HE staining was used to observe the pathological changes in the hippocampus of brain tissue; Fluoro-Jade C staining was used to observe degenerated neurons; TUNEL/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 [PKC, p-PKC, ERK1/2, p-ERK1/2, Bcl-2, Bax, Caspase-3] in brain tissue. In vitro studies: oxygen-glucose deprivation/reperfusion (OGD/R) was used to construct a cell model, and 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 protein expressions of PKC, p-PKC, ERK1/2, and p-ERK1/2. Results: In the in vivo study, compared with the sham operation group, the rats in the model group showed different degrees of neurological deficits, the neurological deficit score, righting reaction time and geotaxis reaction time, the numbers of Fluoro-Jade C+ positive cells and NeuN+TUNEL+ positive cells, the expression of Bax and Caspase-3 in brain tissue increased significantly, 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 group improved, the neurological deficit score, righting reaction time and geotaxis reaction time, the numbers of Fluoro-Jade C+ positive cells and NeuN+TUNEL+ positive cells, the expression of Bax and Caspase-3 in brain tissue reduced significantly, 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 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, 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.

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  • 收稿日期:2022-03-21
  • 最后修改日期:2022-06-06
  • 录用日期:2023-05-23
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