文章快速检索     高级检索
  新医学  2018, Vol. 49 Issue (7): 507-510  DOI: 10.3969/j.issn.0253-9802.2018.07.010
0

引用本文 [复制中英文]

余高锋, 李会仁, 金尚怡. PD-1/PD-L1通路在七氟醚致大鼠术后认知功能障碍中的作用[J]. 新医学, 2018, 49(7): 507-510.
Yu Gaofeng, Li Huiren, Jin Shangyi. Effect of programmed death 1/programmed death ligand 1 signaling pathway on sevoflurane-induced postoperative cognitive dysfunction in rats[J]. Journal of New Medicine, 2018, 49(7): 507-510.

基金项目

广东省医学科学技术研究基金项目(A2017107)

文章历史

收稿日期：2018-03-20
Contents              Abstract              Full text              Figures/Tables              PDF
PD-1/PD-L1通路在七氟醚致大鼠术后认知功能障碍中的作用
余高锋 , 李会仁 , 金尚怡     
510623 广州，广州市妇女儿童医疗中心麻醉手术中心 (余高锋，李会仁)；510530 广州，中山大学附属第三医院岭南医院心血管内科 (金尚怡)
收稿日期：2018-03-20
基金项目：广东省医学科学技术研究基金项目(A2017107)
摘要: 目的 探讨程序性死亡受体1/程序性死亡受体配体1 (PD-1/PD-L1)通路在七氟醚致大鼠术后认知功能障碍(POCD)中的作用。方法 将18只雄性SD大鼠随机分为3组各6只，生理盐水组(NS组)于海马区注射生理盐水2 μl，七氟醚致POCD组(POCD组)则注射β-淀粉样蛋白1-40 (Aβ1-40) 2 μl，PD-1/PD-L1通路阻断组(B组)注射Aβ1-40 2 μl的同时给予PD-L1单克隆抗体。POCD组和B组大鼠于术后30 d给予七氟醚麻醉4 h，NS组给予同等时长的空气对照。术后51 d行Morris水迷宫行为学测试后处死大鼠，测其海马内PD-1及PD-L1的表达，IL-1β及IL-10的含量以及海马神经元细胞凋亡率，比较3组结果。结果 与NS组比较，POCD组与B组大鼠上台前路程增加、逃避潜伏期延长(P均＜0.05)；与POCD组比较，B组大鼠上台前路程减少、逃避潜伏期缩短(P均＜0.05)；3组大鼠游泳速度比较差异无统计学意义(P＞0.05)。与NS组比较，POCD组大鼠海马内PD-1和PD-L1表达增强、IL-1β含量增加、IL-10含量减少、神经元细胞凋亡率升高(P均＜0.05)；与POCD组比较，B组大鼠海马内PD-L1表达减弱、IL-1β含量减少、IL-10含量增加、神经元细胞凋亡率降低(P均＜0.05)；POCD组与B组大鼠海马内PD-1比较差异无统计学意义(P＞0.05)。结论 PD-1/PD-L1通路在七氟醚致大鼠POCD中起关键作用，阻断PD-1/PD-L1通路可抑制POCD大鼠海马内免疫炎症反应，降低神经元细胞凋亡率。
关键词: 程序性死亡受体1    程序性死亡受体配体1    术后认知功能障碍    七氟醚    炎症反应    
Effect of programmed death 1/programmed death ligand 1 signaling pathway on sevoflurane-induced postoperative cognitive dysfunction in rats
Yu Gaofeng, Li Huiren, Jin Shangyi     
Department of Anesthesiology, Guangzhou Women and Children Medical Center, Guangzhou 510623, China
Abstract: Objective To investigate the role of programmed death 1/programmed death ligand 1(PD-1/PD-L1) signaling pathway in the postoperative cognitive dysfunction (POCD) induced by sevoflurane in rats. Methods Eighteen male Sprague-Dawley rats were randomly divided into three groups (n=6 for each group). In the normal saline group (NS group), 2 μl of normal saline was injected with in the hippocampus. In the sevoflurane-induced POCD group (POCD group), 2 μl of β-amyloid1-40(Aβ 1-40) amyloid protein was injected. In the PD-1/PD-L1 pathway blockage group (B group), 2 μl of Aβ1-40 was injected supplemented with PD-L1 monoclonal antibody. The rats in the POCD and B groups were anesthesized by sevoflurane for 4 h at postoperative 30 d, and air treatment of the same duration was given in the NS group. At postoperative 51 d, the rats were subject to Morris water maze and then sacrificed. The expression of PD-1 and PD-L1, the levels of IL-1β and IL-10 and the apoptosis rate of hippocampal neuronal cells were quantitatively measured and statistically compared among three groups. Results Compared with the NS group, the rats in the POCD and B groups exhibited significantly longer distance before entering the platform and evidently longer escape latency (all P < 0.05). Compared with the POCD group, the rats in the B group had significantly shorter distance before entering the flat form and considerbably shorter escape latency (both P < 0.05). No statistical significance was noted in terms of swimming speed among three groups (P > 0.05). Compared with the NS group, the expression of PD-1 and PD-L1 in hippocampus were significantly up-regulated, the IL-1β content was evidently increased, the IL-10 content considerably decreased, and the apoptosis rate of neuronal cells was dramatically increased in the POCD group (all P < 0.05). Compared with the POCD group, the expression of PD-L1 in hippocampus was significantly down-regulated, the content of IL-1β was evidently decreased, the content of IL-10 was considerably increased and the apoptosis rate of neuronal cells was significantly decreased in group B (all P < 0.05). The expression of PD-1 in the hippocampus did not significantly differ between the POCD and B groups (P > 0.05). Conclusions The PD-1/PD-L1 signaling pathway plays a key role in sevoflurane-induced POCD in rat models. Blocking the PD-1/PD-L1 signaling pathway can inhibit the immuno-inflammatory responses in the hippocampus and reduce the apoptosis rate of neuronal cells in POCD rats.
Key words: Programmed death receptor 1    Programmed death receptor ligand 1    Postoperative cognitive dysfunction    Sevoflurane    Inflammatory response    

术后认知功能障碍(POCD)是麻醉和术后出现的一种中枢神经系统并发症，其临床症状主要包括认知能力降低、理解水平下降、记忆减退等[1-2]。研究证实七氟醚与POCD的发生有密切联系，七氟醚致POCD的机制与β-淀粉样蛋白(Aβ)刺激胶质细胞产生炎症因子和细胞因子有关[3-4]。程序性死亡受体1 (PD-1)参与细胞凋亡并可从多个层面阻止T、B淋巴细胞的激活[5-6]。PD-1与其内源性配体程序性死亡受体配体1 (PD-L1)结合后发挥作用，但PD-1/PD-L1通路在七氟醚致POCD中的作用尚未明确。本研究旨在探讨PD-1/PD-L1通路在七氟醚致大鼠POCD中的作用。

材料与方法 一、动物及分组

取18只4月龄清洁级雄性SD大鼠，体质量为260~290 g，购自广东省动物实验基地。在动物房内饲养7 d，室温25 ℃，湿度40%，不控制饮水，常规饮食。将其随机分为3组各6只，包括生理盐水组(NS组)、七氟醚致POCD组(POCD组)和PD-1/PD-L1通路阻断组(B组)。

二、方法 1. 七氟醚致POCD模型制备

于大鼠海马区注射Aβ1-40制备模型，腹腔注射10%水合氯醛350 mg/kg进行麻醉，应用大鼠脑立体定向仪(深圳瑞沃德生命科技有限公司)定位双侧海马CA1区，钻开颅骨暴露硬脑膜，用微量进样器(上海激光医学仪器厂)从脑表面定位点垂直进针，依次将Aβ1-40 (5 μg/μl，使用前37 ℃孵育7 d) 2 μl注入POCD组及B组大鼠双侧海马，注射时长5 min，留针5 min，确保Aβ1-40完全浸润局部组织，NS组则等速注射等体积生理盐水。注射完后所有实验大鼠局部消毒后缝合皮肤，腹腔注射庆大霉素2万单位后常规饲养。B组大鼠海马内注射PD-L1单克隆抗体E1J2J 1 μl (货号15165S，美国CST公司)，POCD组和NS组大鼠海马内注射等体积生理盐水。术后30 d按文献[7]制备七氟醚致POCD模型，给予POCD组和B组大鼠2.0最低肺泡有效浓度(MAC)的七氟醚+氧气(浓度50%)处理4 h；NS组空气处理4 h。各组气体流量均设为2 L/min。

2. 行为学测定及标本制备

大鼠术前5 d开始Morris水迷宫(中国医学科学院药物研究所)行为学实验训练，4次/日，持续5 d。以池壁上4个等距离点把水池平分成4个象限(Ⅰ、Ⅱ、Ⅲ、Ⅳ)，在Ⅳ象限中心位置安放平台，直径15 cm，高度33 cm，没入水下1 cm。大鼠从上述4个象限面向池壁入水直至其找到平台，运用any-Maze动物行为学自动摄像分析系统记录大鼠游泳速度、逃避潜伏期、上台前路程以及搜索策略。若90 s内未发现平台，则引导其登上平台休息30 s，并将逃避潜伏期记为90 s。于术后51 d行Morris水迷宫行为学测试，测试后予各组大鼠腹腔注射水合氯醛350 mg/kg进行麻醉，开颅灌流取其海马组织进行测定。

3. 大鼠海马细胞凋亡荧光原位检测

采用原位缺口末端标记法(TUNEL)，按照试剂盒说明操作。使用Image-pro Plus 6.0软件进行分析，镜下细胞核棕色染色细胞为阳性细胞，每只大鼠取3张切片, 各切片在损伤区任意釆集5个高倍(400倍)视野。高倍视野下计算凋亡指数=调亡细胞数/总细胞数×100 %。

4. 蛋白免疫印迹法测定海马内PD-1及PD-L1的表达

用细胞裂解液提取蛋白后采用Bradfor法蛋白定量测定试剂盒(上海美季生物技术公司)进行蛋白定量。通过Scion Image软件检测PD-1和PD-L1目的条带，对比内参照β-actin条带灰度值，其比值为PD-1和PD-L1的相对表达量。

5. ELISA法测定IL-1β和IL-10的含量

用匀浆器研磨组织后于冰浴下超声波粉碎制匀浆，4 ℃离心取上清液，参照ELISA测定试剂盒说明，测定IL-1β、IL-10含量。用MK3酶标仪测吸光度OD值，对应标准曲线得出IL-1β和IL-10的含量。

三、统计学处理

使用SPSS 21.0处理数据，符合正态分布的计量资料用x±s表示，组间差异比较采用方差分析，有统计学意义时，进一步用SNK法作两两比较。P＜0.05为差异有统计学意义。

结果 一、大鼠Morris水迷宫行为学测试结果

术后51 d，与NS组比较，POCD组及B组大鼠上台前路程增加、逃避潜伏期延长(P均＜0.05)；与POCD组比较，B组大鼠上台前路程减少、逃避潜伏期缩短(P均＜0.05)；3组大鼠游泳速度比较差异无统计学意义(P＞0.05)，见表 1。

表 1 NS组、POCD组及B组大鼠术后51 d Morris水迷宫行为学测试结果比较(x±s)
二、NS组、POCD组及B组大鼠海马内PD-1、PD-L1、炎症因子及神经元细胞凋亡率比较

与NS组比较，POCD组大鼠海马内PD-1和PD-L1表达增强、炎症因子IL-1β含量增加、抑炎因子IL-10含量减少、神经元细胞凋亡率升高(P均＜0.05)；与POCD组比较，B组大鼠海马内PD-L1表达减弱、炎症因子IL-1β含量减少、抑炎因子IL-10含量增加、神经元细胞凋亡率降低(P均＜0.05)；POCD组与B组大鼠海马内PD-1比较差异无统计学意义(P均＞0.05)，见表 2。

表 2 NS组、POCD组及B组大鼠海马内PD-1、PD-L1、炎症因子及神经元细胞凋亡率比较(x±s)
讨论

双侧海马注射Aβ1-40可致大鼠海马神经元线粒体受损并导致其记忆减退和认知障碍[8]。本研究采用既往研究方法制备七氟醚致大鼠POCD模型，大鼠认知行为学的改变提示海马区注射Aβ1-40后复合七氟醚处理，可导致大鼠发生POCD。海马是学习记忆的重要核团，海马内Aβ沉积、神经元细胞外Aβ沉积是早老性痴呆的主要病理特征。Aβ能直接激活胶质细胞分泌和释放TNF-α、IL-1、IL-6、集落刺激因子、环氧化酶2和前列腺素等炎症趋化因子，最终导致神经元变性坏死[9-10]。七氟醚处理可激活大鼠海马N-甲基-D-天冬氨酸(NMDA)受体，增加钙离子内流并促进大量氧自由基生成，从而诱发海马神经元细胞凋亡和脂质过氧化损伤，七氟醚可通过抑制突触后膜乙酰胆碱传递的方式抑制神经元突触可塑性[11-13]。此外，七氟醚能刺激Aβ的聚集与沉积，对老年大鼠空间学习及记忆能力产生伤害[14-15]。本研究结果表明，NS组大鼠海马内炎症反应以及PD-1与PD-L1的含量均处于较低水平，而POCD组大鼠海马内炎症反应加重并最终导致神经元细胞凋亡，同时其PD-1和PD-L1表达增强，这种伴随性升高提示PD-1/PD-L1通路参与了POCD过程。

PD-1蛋白由N端细胞外结合域、跨膜结构域和C端胞浆结构域3部分组成，胞浆结构域中含有一个免疫受体酪氨酸交换模体和一个免疫受体酪氨酸抑制模体[16-17]。当PD-L1与PD-1受体结合后，可促进PD-1胞浆结构域尾端酪氨酸磷酸化，继而招募非跨膜型蛋白酪氨酸磷酸酶(SHP-2)，最终SHP2可使T淋巴细胞抗原受体相关蛋白CD-3ζ和ζ链相关蛋白-70去磷酸化，从而调控了下游病理生理过程，一方面抑制炎症因子的分泌，另一方面抑制PI3K/Akt、mTOR、S6、Erk2等信号通路的激活[18-20]。在本研究中，POCD大鼠海马内炎症因子IL-1β含量增加，同时炎症因子IL-10含量减少，导致神经元细胞凋亡，通过给予PD-L1单克隆抗体，抑制PD-L1与PD-1的结合从而阻断PD-1/PD-L1通路后，上述病理生理过程得以反转，提示PD-1/PD-L1通路是调控POCD炎症反应和细胞凋亡的上游关键通路。

综上所述，PD-1/PD-L1通路在七氟醚致大鼠POCD中起关键作用，阻断PD-1/PD-L1通路可抑制POCD大鼠海马内免疫炎症反应，从而降低神经元细胞凋亡率。

参考文献
[1]
吴祥, 高涛, 徐义国, 朱伟, 周东升, 姚琴, 张文武. 术后认知功能障碍的研究进展[J]. 新医学, 2015, 46(10): 650-652.
[2]
Zhang N, Liang M, Zhang DD, Xiao YR, Li YZ, Gao YG, Cai HD, Lin XZ, Lin CZ, Zeng K, Wu XD. Effect of goal-directed fluid therapy on early cognitive function in elderly patients with spinal stenosis[J]. Int J Surg, 2018, 54(Pt A): 201-205.
[3]
Hu N, Wang C, Zheng Y, Ao J, Zhang C, Xie K, Li Y, Wang H, Yu Y, Wang G. The role of the W:nt/β-catenin-Annexin A1 pathway in the process of sevoflurane-induced cognitive dysfunction[J]. J Neurochem, 2016, 137(2): 240-252. DOI:10.1111/jnc.2016.137.issue-2
[4]
Zhang Q, Li Y, Bao Y, Yin C, Xin X, Guo Y, Gao F, Huo S, Wang X, Wang Q. Pretreatment with nimodipine reduces incidence of POCD by decreasing calcineurin mediated hippocampal neuroapoptosis in aged rats[J]. BMC Anesthesiol, 2018, 18(1): 42. DOI:10.1186/s12871-018-0501-0
[5]
Zou W, Wolchok JD, Chen L. PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations[J]. Sci Transl Med, 2016, 8(328): 328rv4. DOI:10.1126/scitranslmed.aad7118
[6]
钱小庆, 张民杰. PD-1/PD-L1在脓毒症免疫中的作用[J]. 新医学, 2017, 48(10): 692-696.
[7]
田悦, 郭善斌, 黄威, 赵平. 七氟醚对β淀粉样蛋白诱导的大鼠认知功能障碍及海马组织氧化应激反应的影响[J]. 中华麻醉学杂志, 2014, 34(4): 462-465.
[8]
Zhao B, Pan Y, Wang Z, Xu H, Song X. Hyperbaric oxygen pretreatment improves cognition and reduces hippocampal damage via p38 mitogen-activated protein kinase in a rat mode[J]. Yonsei Med J, 2017, 58(1): 131-138. DOI:10.3349/ymj.2017.58.1.131
[9]
Jean YY, Baleriola J, Fà M, Hengst U, Troy CM. Stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus[J]. J Vis Exp, 2015, 17(100): e52805.
[10]
Parfejevs V, Debbache J, Shakhova O, Schaefer SM, Glausch M, Wegner M, Suter U, Riekstina U, Werner S, Sommer L. Injury-activated glial cells promote wound healing of the adult skin in mice[J]. Nat Commun, 2018, 9(1): 236. DOI:10.1038/s41467-017-01488-2
[11]
Zheng SQ, An LX, Cheng X, Wang YJ. Sevoflurane causes neuronal apoptosis and adaptability changes of neonatal rats[J]. Acta Anaesthesiol Scand, 2013, 57(9): 1167-1174. DOI:10.1111/aas.2013.57.issue-9
[12]
Zhang SB, Liu TJ, Pu GH, Li BY, Gao XZ, Han XL. MicroRNA-374 exerts protective effects by inhibiting SP1 through activating the PI3K/Akt pathway in rat models of myocardial ischemia-reperfusion after sevoflurane preconditioning[J]. Cell Physiol Biochem, 2018, 46(4): 1455-1470. DOI:10.1159/000489186
[13]
Zhang LM, Zhao XC, Sun WB, Li R, Jiang XJ. Sevoflurane post-conditioning protects primary rat cortical neurons against oxygen-glucose deprivation/resuscitation via down-regulation in mitochondrial apoptosis axis of Bid, Bim, Puma-Bax and Bak mediated by Erk1/2[J]. J Neurol Sci, 2015, 357(1-2): 80-87. DOI:10.1016/j.jns.2015.06.070
[14]
Li B, Sun J, Lv G, Yu Y, Wang G, Xie K, Jiao Y, Yu Y. Sevoflurane postconditioning attenuates cerebral ischemia-reperfusion injury via protein kinase B/nuclear factor-erythroid 2-related factor 2 pathway activation[J]. Int J Dev Neurosci, 2014, 38: 79-86. DOI:10.1016/j.ijdevneu.2014.08.005
[15]
Zhang Y, Li D, Luo J, Chen S, Dou X, Han M, Zhang H. Pharmacological postconditioning with sevoflurane activates PI3K/AKT signaling and attenuates cardiopulmonary bypass-induced lung injury in dog[J]. Life Sci, 2017, 15(173): 68-72.
[16]
Goodman A, Patel SP, Kurzrock R. PD-1-PD-L1 immune-checkpoint blockade in B-cell lymphomas[J]. Nat Rev Clin Oncol, 2017, 14(4): 203-220. DOI:10.1038/nrclinonc.2016.168
[17]
Wang Y, Wu L, Tian C, Zhang Y. PD-1-PD-L1 immune-checkpoint blockade in malignant lymphomas[J]. Ann Hematol, 2018, 97(2): 229-237. DOI:10.1007/s00277-017-3176-6
[18]
Ohaegbulam KC, Assal A, Lazar-Molnar E, Yao Y, Zang X. Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway[J]. Trends Mol Med, 2015, 21(1): 24-33. DOI:10.1016/j.molmed.2014.10.009
[19]
Gandini S, Massi D, Mandalà M. PD-L1 expression in cancer patients receiving anti PD-1/PD-L1 antibodies: a systematic review and meta-analysis[J]. Crit Rev Oncol Hematol, 2016, 100: 88-98. DOI:10.1016/j.critrevonc.2016.02.001
[20]
Chinai JM, Janakiram M, Chen F, Chen W, Kaplan M, Zang X. New immunotherapies targeting the PD-1 pathway[J]. Trends Pharmacol Sci, 2015, 36(9): 587-595. DOI:10.1016/j.tips.2015.06.005