电针通过调控神经珠蛋白及PI3K/AKT信号通路抑制胆红素脑病诱导的SD乳鼠脑颞叶皮质细胞凋亡  

Electroacupuncture Inhibits Neuronal Apoptosis Induced by Bilirubin Encephalopathy in SD Suckling Rats by Regulating NGB and PI3K/Akt Signaling Pathways

作　　者：胡嘉恒 樊萍[2] 张丽荣 王依滢 隆令 胡智涵 袁玮民 赵婧雯 朱淑娟 黄娟 邱国平 甘胜伟 HU Jia-Heng;FAN Ping;ZHANG Li-Rong;WANG Yi-Ying;LONG Ling;HU Zhi-Han;YUAN Wei-Min;ZHAO Jing-Wen;ZHU Shu-Juan;HUANG Juan;QIU Guo-Ping;GAN Sheng-Wei(Institute of Neurosciences,Chongqing Medical University,Chongqing 400016,China;Department of Gynecology and Obstetrics,The Fifth People’s Hospital of Chongqing,Chongqing 400062,China)

机构地区：[1]重庆医科大学基础医学院神经科学研究中心,重庆400016 [2]重庆市第五人民医院妇产科,重庆400062

出　　处：《中国生物化学与分子生物学报》2021年第6期772-781,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.31300911,81601051);重庆市自然科学基金(No.CSTC2016jcyjA0073);重庆市卫生局(No.2015MSXM109);重庆市教委(No.CY200401)基金资助。

摘　　要：本研究通过电针刺激介导胆红素脑病(bilirubin encephalopathy,BE)模型鼠大脑颞叶皮质神经珠蛋白(neuroglobin,NGB)及PI3K/AKT通路、凋亡通路相关蛋白质的表达变化,以明确电针(electroacupuncture,EA)对于胆红素脑病的治疗作用,并探讨神经珠蛋白在该过程中的作用机制。将39只7日龄SD乳鼠分为假手术(Sham)组、BE模型组、电针治疗(BE+EA)组。经小脑延髓池注射胆红素溶液(10μg胆红素/g体重)制备BE模型,假手术组注射等量生理盐水作为对照,BE+EA组选取百会穴与曲池穴以频率2/15 Hz的疏密波,于造模前12 h、造模完成时及造模后12 h进行3次电针干预,每次15 min。使用HE、尼氏(Nissl)染色及透射电镜检测各组鼠脑颞叶皮质的病理变化及神经元超微结构的改变,结果显示电针处理可减轻BE乳鼠脑颞叶皮质神经元的损伤及增加尼氏体的数量,透射电镜则证实电针处理可改善神经元线粒体的水肿程度。应用免疫荧光染色法检测各组鼠脑颞叶皮质NGB的表达部位及其表达的细胞种类,结果显示NGB主要表达于颞叶皮质神经元中。进一步通过免疫印迹法检测各组鼠脑颞叶皮质中NGB蛋白及PI3K/AKT通路、线粒体凋亡信号通路相关蛋白质的表达,结果显示电针处理可增加NGB、PI3K p110α和pAKT Ser473的表达(分别为P<0.05、0.05和0.01),上调凋亡相关蛋白Bcl-2/Bax比值(P<0.001),进而抑制切割胱天蛋白酶3的激活(P<0.05)。通过TUNEL染色法检测各组凋亡细胞的数量,结果证实电针处理使凋亡细胞的数量减少(BE模型组186.00±13.86 vs BE+EA组78.67±11.85,P<0.01)。该研究初步证实,电针刺激可促进胆红素脑病鼠脑颞叶皮质中神经珠蛋白的表达,并进一步激活PI3K/AKT通路,而发挥其神经细胞保护功能,抑制凋亡反应的发生,电针可能成为胆红素脑病治疗潜在的方法。Objective In this study,electroacupuncture(EA)was used to analyze the expression changes of related proteins in neuroglobin(NGB),PI3K/AKT and apoptotic pathways in the temporal cortex of bilirubin encephalopathy(BE)rats,so as to investigate the therapeutic effect of EA on BE and the relevant mechanism of NGB in this process.Totally 39 seven-day-old SD rats were divided into Sham,BE model and BE+EA groups.The neonatal BE model was established by injecting bilirubin solution(10μg UCB/g Weight)into the cerebellomedullary cistern,Sham group was injected with the same amount of normal saline.BE rats were treated with EA at Baihui(GV20)and Quchi(LI11)acupoints with the frequency of 2/15 Hz for 15 min.Treatment was performed 12 h before modeling,followed by treatment every 12 h,in a total of three times.HE,Nissl staining and electron microscopy(TEM)were used to observe the pathological and ultrastructural changes of nerve cells in each group.Results showed that EA treatment reduced the damage of cortical neurons of BE rats and increase the number of Nissl bodies.TEM confirmed that EA treatment could alleviate the degree of mitochondria edema.Immunofluorescence staining was used to detect the expression sites and cell types of NGB.Results showed that NGB was mainly expressed in cortical neurons.Western blotting showed that EA treatment increased the expression of NGB,PI3K(p110 alpha),pAKT(Ser473)(P<0.05,P<0.05 and P<0.01,respectively)and the ratio of apoptosis-related protein Bcl-2/Bax(P<0.001),decreased the expression of Cleaved Caspase-3(P<0.05)in the temporal cortex of rats.TUNEL staining showed that EA reduced the number of apoptotic cells(BE group 186.00±13.86 vs BE+EA group 78.67±11.85,P<0.01).This study confirms that EA can promote the expression of NGB in the temporal cortex of BE rats,then activate the PI3K/AKT pathway to exert its neuroprotective function and inhibit the occurrence of apoptosis.EA may become a potential treatment method for BE.

关 键 词：胆红素脑病 神经珠蛋白 电针 细胞凋亡 

分 类 号：R744.1[医药卫生—神经病学与精神病学] Q291[医药卫生—临床医学]