柚皮素对创伤性脊髓损伤后运动功能的修复作用及其机制  

Effect and mechanism of naringenin on motor function repair after spinal cord injury

作　　者：魏子然 王超[2] 张璐[3] 陈云庆[4] 林倩[5] 张浩运 孙申杰 单烁 于腾波 Wei Ziran;Wang Chao;Zhang Lu;Chen Yunqing;Lin Qian;Zhang Haoyun;Sun Shenjie;Shan Shuo;Yu Tengbo(Department of Spinal Surgery,Qingdao Municipal Hospital,Qingdao 266000,China;Department of Spinal Surgery,The Affiliated Hospital of Qingdao University,Qingdao 266000,China;Medical Research Center,The Affiliated Hospital of Qingdao University,Qingdao 266000,China;Department of Pathology,The Affiliated Hospital of Qingdao University,Qingdao 266000,China;Department of Sports Medicine,The Affiliated Hospital of Qingdao University,Qingdao 266000,China;Department of Emergency Medicine,Qingdao Municipal Hospital,Qingdao 266000,China;Department of Orthopedics and Sports Medicine,Qingdao Municipal Hospital,Qingdao 266000,China)

机构地区：[1]青岛市市立医院脊柱外科,青岛266000 [2]青岛大学附属医院脊柱外科,青岛266000 [3]青岛大学附属医院医学研究中心,青岛266000 [4]青岛大学附属医院病理科,青岛266000 [5]青岛大学附属医院运动医学科,青岛266000 [6]青岛市市立医院急诊科,青岛266000 [7]青岛市市立医院骨关节与运动医学科,青岛266000

出　　处：《中华航海医学与高气压医学杂志》2023年第5期601-607,共7页Chinese Journal of Nautical Medicine and Hyperbaric Medicine

基　　金：国家自然科学基金(81902209);中国博士后科学基金(2020M672002)。

摘　　要：目的探讨柚皮素(NGN)对创伤性脊髓损伤后运动功能修复的作用及其机制。方法选取36只6~8周龄的雄性C57BL/6小鼠,按照随机数字表法分为假手术组、生理盐水组和NGN组,每组12只。假手术组小鼠仅行椎板切除术,不损伤脊髓;生理盐水组和NGN组小鼠利用钳夹法进行脊髓损伤手术。3组小鼠均从术前3 d开始每天灌胃给药(假手术组和生理盐水组小鼠给予生理盐水灌胃,NGN组小鼠给予50 mg/kg NGN灌胃给药),手术当天不给药,术后第1天恢复灌胃给药,1次/d,持续7 d,之后隔天灌胃给药,持续28 d。采用BMS评分法评估小鼠运动功能的恢复情况。冰冻切片行HE和尼式染色评估脊髓组织学改变。Western blotting和免疫荧光染色实验检测组织自噬蛋白的表达水平。体外培养小鼠小胶质细胞BV2,分为NGN组(NGN处理)与对照组(空白溶剂处理),采用CCK-8法检测细胞活性,利用腺病毒荧光示踪体系评估自噬流情况。与神经元细胞N2a建立共培养体系后,采用CCK-8法检测细胞活性,采用细胞划痕实验检测细胞迁移愈合能力。结果给予同等护理条件及康复活动下,损伤后第14、21、28天,NGN组小鼠BMS评分明显优于生理盐水组小鼠(P<0.05)。NGN可改善脊髓损伤后神经元数量和形态,并减少损伤组织中LC3B和p62的蓄积(P<0.05或P<0.001)。体外实验表明NGN呈剂量依赖性(6.25、12.5、25、50 mmol/L)增加BV2细胞活性(P<0.01),同时促进BV2细胞自噬发生和自噬溶酶体形成。细胞共培养条件下,NGN组BV2细胞可明显提高N2a细胞活性(P<0.05)并促进细胞迁移(P<0.01)。结论NGN可促进脊髓损伤后运动功能修复,其可能的机制是NGN通过提高小胶质细胞的自噬作用,促进神经元修复。Objective To investigate the effect and mechanism of naringenin(NGN)on neural function repair after traumatic spinal cord injury(SCI).Methods A total of 36 male C57BL/6 mice aged 6-8 weeks were selected and randomly divided into sham surgery group,saline group,and NGN group.The mice in the sham surgery group underwent laminectomy without causing any SCI,while the spinal cords of the mice in the other two groups were injured by the forceps compression method.Starting from three days prior to the laminectomy,all mice of the three groups received medication by intragastric administration on a daily basis(the sham surgery and saline groups received intragastric administration of saline,while the NGN group received intragastric administration of naringin at a dosage of 50 mg/kg).No medication was given on the day of the laminectomy.Intragastric administration resumed on the first day after the surgery,once a day,for a total of seven days.Subsequently,intragastric administration was done every other day for 28 days.The recovery of motor function in mice was evaluated using the Basso mouse scale(BMS).Histological changes in the spinal cord tissue were evaluated using frozen sections subjected to HE staining and Nissl staining.Western blot and immunofluorescence staining were used to evaluate the expression levels of autophagy-related proteins.Mouse microglia cell BV2 was cultured in vitro and divided into NGN group(treated with NGN)and control group(treated with blank solvent).The cell activity was evaluated by CCK-8 assay,and the autophagic flux was evaluated by an adenovirus fluorescent tracking system.A co-culture system with neuronal cells N2a was established,the cell activity was evaluated by CCK-8 assay,and the cell migration and healing abilities were evaluated by cell scratch assay.Results Under equal conditions of care and rehabilitation activities,the BMS scores of mice in the NGN group were significantly better than those in the saline group on 14,21,and 28 days after injury(P<0.05).NGN improved the neuronal

关 键 词：柚皮素 脊髓损伤 小胶质细胞 自噬 

分 类 号：R285.5[医药卫生—中药学]