胶质细胞源性神经营养因子/PI3K/AKT通路参与电针促进面神经压榨损伤模型兔的面神经再生

文题释义： 胶质细胞源性神经营养因子(glial cell-derived neurotrophic factor，GDNF)：是于1993年发现的由二硫键结合的同源二聚体蛋白质，含有134个氨基酸，分子质量为33-35 kD，是神经营养因子中的一种，属于转化生长因子超家族的远系成员，在全身各组织中均有表达，尤其在中枢神经系统内分布较广，具有显著的提高多巴胺能神经元存活与分化、抑制运动神经元凋亡、营养外周神经元、调整细胞间信息等作用。 PI3K：是由Sugimoto和Macara等(1984)发现的一种胞内磷脂酰肌醇激酶，由一个催化亚基(110 kD，p110 )和一个调节亚基(85 kD，p85 )构成，可特异地使肌醇环上的3位羟基磷酸化，其产物是在3位上有磷酸根的多磷酸肌醇脂，包括3，4-二磷酸磷脂酰肌醇(PI-3，4-P2)和3，4，5-三磷酸磷脂酰肌醇(PI-3，4，5-P3)等，它们都是位于质膜上的第二信使。PI3K是许多生命活动中关键的信号分子，PI3K介导的信号转导通路调节细胞的分裂、分化、凋亡等活动。 背景：多穴位电针治疗周围性面瘫的机制不明。胶质细胞源性神经营养因子(glial cell-derived neurotrophic factor，GDNF)是促进体外运动神经元存活的最有效因子，PI3K/AKT信号通路在保护受损神经元方面发挥着重要作用。目前暂没有GDNF/PI3K/AKT通路参与电针促进兔周围面神经再生的研究。 目的：观察电针对周围面神经压榨损伤后再生的影响，并从GDNF/PI3K/AKT介导的信号通路角度探讨电针对面神经元的保护机制。 方法：成年健康新西兰大白兔由西南医科大学动物实验中心提供，随机分成正常组和模型组。模型组制备右侧面神经颊支压榨性损伤的病理模型，造模成功后随机分为模型对照组和电针组，模型对照组自然康复，电针组取右侧翳风、颊车、四白、地仓、阳白、颧髎穴位进行电针治疗，1次/d，每次30 min。观察动物面瘫症状的改善情况并进行评分；正常组直接取材，模型组分别于术后 1，4，7，14，28 d取含面神经元的脑桥组织，进行苏木精-伊红染色观察神经元形态，尼氏染色观察尼氏小体，免疫组化测定GDNF的阳性表达，Western blotting检测3组各时间点面神经元组织中GDNF、PI3K、AKT、P-AKT的蛋白表达。实验方案经西南医科大学动物伦理学委员会批准，批准号为20170120001。 结果与结论：①与模型对照组相比，电针组动物的面瘫症状(患侧口角歪斜下垂，触须倒伏且运动减弱，眼睑上抬不能)恢复较快且完全；②电针组面神经元形态学改变、尼氏体变化均较模型对照组病理变化轻；③术后各时间点，面神经元GDNF免疫反应较强，阳性细胞数多于模型对照组(除术后1 d，其余各时间点P < 0.001)，且面神经元组织中GDNF、PI3K和P-AKT蛋白表达明显增高(P < 0.05/0.01/0.001)。提示：电针可有效治疗由压榨面神经颊支引起的周围性面瘫，促进面神经元形态恢复，其面神经元保护机制可能与上调GDNF在面神经元中的表达，激活PI3K/AKT信号通路有关。 ORCID: 0000-0002-9531-7400(李雷激) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Involvement of GDNF/PI3K/AKT pathway in promoting facial nerve regeneration using electroacupuncture in a rabbit model of facial nerve crush injury

BACKGROUND:The mechanism of electroacupuncture on multi-acupoints in the treatment of peripheral facial paralysis is still unknown.Glial cell-derived neurotrophic factor(GDNF)is currently the most effective factor in promoting the survival of motoneurons in vitro,and the PI3K/AKT pathway plays an important role in protecting damaged motoneurons.There is yet no research on GDNF/PI3K/AKT pathway involved in electroacupuncture promoting facial nerve regeneration in rabbits.OBJECTIVE:To observe the effect of electroacupuncture on regeneration after peripheral facial nerve crush injury,and to explore the protective mechanism of electroacupuncture on facial motoneurons through the GDNF/PI3K/AKT signaling pathway.METHODS:Sixty-six adult healthy New Zealand white rabbits provided by the Animal Experimental Center of Southwest Medical University were randomly divided into a normal group and a model group.The facial nerves on the right side in the model group were subjected to a crush injury.Then the animal models were randomly divided into a model control group and an electroacupuncture group.Animals in the model control group recovered naturally,while those in the electroacupuncture group underwent electroacupuncture at Yifeng,Jiache,Sibai,Dicang,Yangbai,and Quanliao acupoints daily for 30 minutes.The improvement of facial paralysis symptoms in experimental animals were observed and scored.Tissue samples were directly taken form the normal group,and pons tissues with facial neurons were taken in the model group at 1,4,7,14,and 28 days postoperatively.The morphologies of facial motoneurons and Nissl bodies were observed by hematoxylin-eosin staining and Nissl staining,respectively.Immunohistochemical techniques and western blot assay were used to detect the protein expression of GDNF,PI3K,AKT,and p-AKT in the facial motoneurons.The study protocol was approved by the Animal Ethics Committee of Southwest Medical University with approval No.20170120001.RESULTS AND CONCLUSION:The symptoms of facial paralysis were that the animal’s mouth was drooped at the affected side,with lodging tentacles and the movement being weakened,and the eyelids that could not be lifted,which recovered faster and more completely in the electroacupuncture group than the model control group.The morphological changes of facial neurons and changes of Nissl bodies in the electroacupuncture group were lighter than those in the model control group.At each time point postoperatively,the stronger GDNF immune response could be seen in the electroacupuncture group,and the number of GDNF-positive cells was higher than that of the model control group except 1 day postoperatively(P<0.001).The expressions of GDNF,PI3K,p-AKT proteins in the facial motoneurons were significantly increased in the electroacupuncture group compared with the model control group(P<0.05;P<0.01;P<0.001).To conclude,electroacupuncture can effectively treat the peripheral facial paralysis caused by the crushed injury of facial nerve and promote the recovery of facial neurons.The up-regulation of GDNF expression in the facial motoneurons and the activation of PI3K/AKT signaling pathway may be the underlying protective mechanism of electroacupuncture.