大麻素受体2激动剂AM1241抑制大鼠脊髓损伤后纤维瘢痕形成  

Cannabinoid receptor 2 agonist AM1241 inhibits fibrous scarring after spinal cord injury in rats

作　　者：凌成明 储卫华 袁继超 陈欣 刘静静 张洪燕 林江凯 LING Chengming;CHU Weihua;YUAN Jichao;CHEN Xin;LIU Jingjing;ZHANG Hongyan;LIN Jiangkai(Department of Neurosurgery,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Neurology,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Neurosurgery,Hospital of Sichuan Armed Police Corps,Leshan,Sichuan Province,614000,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院:神经外科,重庆400038 [2]陆军军医大学(第三军医大学)第一附属医院:神经内科,重庆400038 [3]武警四川省总队医院神经外科,四川乐山614000

出　　处：《陆军军医大学学报》2022年第10期991-1000,共10页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81571214);重庆市技术创新与应用示范项目(cstc2018jscx-msybX0092)。

摘　　要：目的探讨AM1241对大鼠脊髓损伤后纤维瘢痕形成的作用及其机制。方法选取雌性6~8周龄Sprague-Dawley大鼠144只,分为4组:①假手术组(n=24)只移除椎板以暴露硬脊膜,不切开硬脊膜,术后给予生理盐水3 mL/kg;②载体组(n=40)建立大鼠脊髓背侧半切损伤模型,术后给予生理盐水3 mL/kg;③AM1241组(n=40)在载体组基础上给予大麻素受体2(cannabinoid receptor 2,CBR2)激动剂(AM1241,3 mg/kg)治疗;④AM1241+AM630组(n=40)在术后AM1241治疗前30 min给予CBR2拮抗剂(AM630,3 mg/kg)。各组药物均腹腔注射,1次/d,连续14 d。采用免疫荧光染色、Western blot、ELISA等方法评估AM1241对大鼠纤维瘢痕形成的抑制作用,同时采用行为学、电生理检测AM1241对大鼠神经功能的影响。结果与载体组比较,AM1241组大鼠脊髓损伤后,主要纤维瘢痕成分的表达降低,脊髓损伤后,纤维瘢痕面积减少,给予CBR2拮抗剂AM630后逆转了上述现象,差异均有统计学意义(P<0.05)。tubulinβⅢ免疫荧光染色、行为学评估、电生理实验结果显示:AM1241组促进了脊髓神经元细胞的存活,改善了脊髓损伤后的神经功能,包括Basso-Beattie-Bresnahan(BBB)评分[载体组(15.38±0.38)vs AM1241组(18.38±0.50)vs AM1241+AM630组(15.50±0.33),P<0.05]、斜板实验角度[载体组(54.25±1.25)°vs AM1241组(64.25±1.05)°vs AM1241+AM630组(54.75±0.49)°,P<0.05]等;ELISA实验结果显示:AM1241组抑制M1型巨噬细胞标记物iNOS、TNF-α、IL-1β的表达(P<0.05),增强M2型巨噬细胞标记物CD206、Arg-1的表达(P<0.05),降低促纤维化因子TGF-β1的表达(P<0.01);而应用CBR2拮抗剂AM630后逆转了上述作用。结论刺激大鼠CBR2能减少脊髓损伤后纤维瘢痕、改善神经功能,这一作用可能是通过影响巨噬细胞极化来实现的。ObjectiveTo determine the role of cannabinoid receptor 2(CBR2)agonist,AM1241,in the formation of fibrous scars in rats after spinal cord injury and elucidate the underlying mechanism.MethodsA total of 144 female Sprague-Dawley rats were selected and divided into 4 groups:sham operation group(n=24,only the lamina was removed to expose the dura mater,and 3 mL/kg normal saline was given postoperatively),vehicle group(n=40,the rats was inflicted to establish a model of dorsal hemisection and then given 3 mL/kg of normal saline),AM1241 group(n=40,the rats were given 3 mg/kg AM1241 on the basis of the vehicle group),and AM1241+AM630 group(n=40,after modeling,the rats were given 3 mg/kg AM630,CBR2 antagonist,in 30 min before AM1241 treatment).The corresponding drugs were injected intraperitoneally to each group,once a day,for 14 consecutive days.Immunofluorescence staining,Western blotting and ELISA were performed to evaluate the inhibitory effects of AM1241 on fibrous scar formation,and behavioral electrophysiology were carried out to detect its effect on neurological function.ResultsAfter spinal cord injury,AM1241 significantly reduced the expression of the main molecules in fibrous scars,as well as the area of fibrous scar,while the treatment of AM630 reversed above phenomena,with statistical significances(P<0.05).The results of tubulinβⅢimmunofluorescence staining,behavioral assessment as well as electrophysiological experiments demonstrated that AM1241 could promote the survival of spinal cord neurons,improve neurological function after injury,such as Basso-Beattie-Bresnahan(BBB)score and angle of oblique board test[vehicle group vs AM1241 group vs AM1241+AM630 group:15.38±0.38 vs 18.38±0.50 vs 15.50±0.33,P<0.05;(54.25±1.25)°vs(64.25±1.05)°vs(54.75±0.49)°,P<0.05].ELISA indicated that AM1241 inhibited the expression of iNOS,TNF-αand IL-1β(markers of M1-type macrophages,P<0.01),enhanced the expression of CD206 and Arg-1(markers of M2-type macrophages,P<0.05),and decreased the expression of TGF-β1(fibro

关 键 词：大麻素受体2/CBR2 大麻素受体2激动剂 脊髓损伤 纤维瘢痕 巨噬细胞极化 

分 类 号：R364.31[医药卫生—病理学] R651.2[医药卫生—基础医学]