胆碱能抗炎通路对大鼠脑缺血再灌注损伤的影响

doi:10.3969/j.issn.1006-7795.2017.03.003

摘要/Abstract

摘要： 目的本研究应用不同的胆碱能受体干扰剂M1受体激动剂McN-A-343（MA343）、M2受体拮抗剂美索曲明（methoctramine，MET）以及α7亚单位的N型受体激动剂胆碱（choline，CHO）分别作用于胆碱能抗炎通路（cholinergic anti-inflammatory pathway，CAP），通过检测不同部位肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白介素-1β（interleukin-1β，IL-1β）浓度，进一步探讨CAP对大鼠脑缺血再灌注损伤的影响。方法健康成年雄性SD大鼠25只，采用数字表法随机分为5组（n=5）：假手术组（sham operation group，Sham）、脑缺血再灌注组（ischemia reperfusion group，I/R）、MET组、MA343组和CHO组。四血管结扎法（four-vessel occlusion，4-VO）建立全脑缺血再灌注损伤模型，电凝大鼠双侧椎动脉（Sham组除外），于缺血前15 min分别经侧脑室向MET组、MA343组及CHO组注射MET、MA343及CHO，Sham组及I/R组注射0.9%（质量分数）氯化钠注射液10μL。除Sham组外，各组夹闭双侧颈总动脉20 min后再灌注。再灌注6 h后，断头处死动物，采集标本。放射免疫法（radio-immunoassay，RIA）检测左侧海马、心、肝、肺、肾和血浆中TNF-α、IL-1β浓度。TdT介导的dUTP缺口末端标记技术染色（terminal deoxynucleotidyl-trallsferase meiated dUTP nick end labeling，TUNEL）检测右侧海马CA1区细胞凋亡数。结果再灌注后，MET组和MA343组海马、心、肝、肾组织匀浆及血浆TNF-α、IL-1β浓度显著低于I/R组（P<0.05）。CHO组海马TNF-α浓度较I/R组有所降低（P<0.05），而心、肝、肾及血浆TNF-α、IL-1β浓度比较，差异无统计学意义（P>0.05）。MET、MA343和CHO均未能降低肺组织TNF-α、IL-1β浓度。与I/R组相比，MET、MA343及CHO组海马CA1区凋亡细胞数目减少（P<0.05）。结论 TNF-α、IL-1β的释放是脑缺血再灌注损伤的重要机制之一，CAP对脑缺血再灌注引发的局部及全身炎性反应均具有保护作用。其机制与MET、MA343和CHO抑制缺血再灌注后TNF-α、IL-1β的合成与释放有关。

关键词: 胆碱能抗炎通路, 迷走神经, 脑缺血再灌注, 肿瘤坏死因子-α, 白介素-1β, 美索曲明, McN-A-343, 胆碱

Abstract: Objective To investigate the protective effect of cholinergic anti-inflammatory pathway (CAP) via the M1 receptor agonist,the M2 receptor antagonist and the nAChR7 agonist. during cerebral ischemia-reperfusion injury in rats. Methods Twenty-five male healthy Sprague-Dawley rats were randomly divided into five equal groups:sham operation (Sham) group, ischemia reperfusion (I/R) group, methoctramine (MET) group, McN-A-343(MA343) group and choline(CHO) group. Rats were subjected to four-vessel occlusion (4-VO) global cerebral ischemia.by electrocauterization of the bilateral vertebral arteries, except Sham group. 15 min before ischemia-reperfusion, we administered intracerebroventricularly (i.c.v.) Methoctramine(500 ng/kg,10 μL), McN-A-343(500 ng/kg,10 μL) and Choline(500 ng/kg,10 μL) to MET group, MA343 group and CHO group, other groups receiving saline (0.9%). Then forebrain ischemia was induced by tightening of the clasps that around the common carotid arteries for 20 minutes, except Sham group. Blood and tissue samples were collected after reperfusion for 6 h in all groups. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels of hippocampus, heart, liver, lung,kidney and plasma were measured by radio-immunoassay(RIA).Apoptosis was detected by terminal deoxynucleotidyl-trallsferase meiated dUTP nick end labeling(TUNEL). Results Methoctramine and McN-A-343 could markedly inhibit the increase of TNF-α and IL-1βcontent in hippocampus, heart, liver, kidney and plasma after ischemia.Choline could also attenuated TNF-α and IL-1βexpression in hippocampus but could not inhibit them in heart, liver, kidney and serum. TNF-α and IL-1β levels in lung could not be suppressed by methoctramine, McN-A-343 or choline. Compared with I/R, the apoptotic cells in MET group, MA343 group and CHO group were significantly decreased. Conclusion It is indicated that CAP plays a potential role in alleviating local and systemic inflammatory response during cerebral ischemia-reperfusion injury. The mechanisms of anti-inflammatory were likely to suppress the expression of TNF-α and IL-1β.

Key words: cholinergic anti-inflammatory pathway, vagus nerve, cerebal ischemia-reperfusion, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), methoctramine, McN-A-343, choline

中图分类号:

R743.31

王潇, 苏跃, 李天佐, 赵斌江. 胆碱能抗炎通路对大鼠脑缺血再灌注损伤的影响[J]. 首都医科大学学报, 2017, 38(3): 342-347.

Wang Xiao, Su Yue, Li Tianzuo, Zhao Binjiang. Effect of cholinergic anti-inflammatory pathway on cerebral ischemia-reperfusion injury in the rat[J]. Journal of Capital Medical University, 2017, 38(3): 342-347.

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