三羟乙基芦丁对大鼠颈脊髓损伤的抗氧化应激作用  

Antioxidative stress of trihydroxyethyl rutin on cervical spinal cord injury in rats

作　　者：刘雅普 苏圆圆[1,2] 刘祺 杨舟[1] 李榕 黄祖成[1] 黄志平[1] 吴晓亮[1] 朱青安[1] Liu Yapu;Su Yuanyuan;Liu Qi;Yang Zhou;Li Rong;Huang Zucheng;Huang Zhiping;Wu Xiaoliang;Zhu Qingan(Department of Spinal Surgery,Nanfang Hospital,Southern Medical University,Guangzhou 510515,Guangdong Province,China;Second Affiliated Hospital of Luohe Medical College,Luohe 462300,Henan Province,China)

机构地区：[1]南方医科大学南方医院脊柱外科,广东省广州市510515 [2]漯河医学高等专科学校第二附属医院,河南省漯河市462300

出　　处：《中国组织工程研究》2022年第36期5868-5874,共7页Chinese Journal of Tissue Engineering Research

基　　金：河南省高校重点科研项目(21B320004),项目负责人:刘雅普;河南省医学科技攻关计划联合共建项目(LHGJ20200887),项目负责人:刘雅普;漯河市青年拔尖人才项目(2018QNBJRC01004),项目负责人:刘雅普;漯河医学高等专科学校创新能力提升项目(2019-LYZKYZD004&2019-LYZKYYB016),项目负责人:刘雅普。

摘　　要：背景:既往动物实验研究发现三羟乙基芦丁可改善颈脊髓损伤大鼠的神经功能,但缺乏其作用机制的相关研究。目的:通过体内体外实验研究三羟乙基芦丁对颈脊髓损伤的抗氧化应激作用,并对其可能机制进行初步探讨。方法:①细胞实验:大鼠肾上腺PC12细胞购于美国模式菌种收集中心(ATCC)细胞库。MTT法检测梯度浓度(0,25,75,100,150,200,250μmol/L)叔丁基过氧化氢(tert-butyl hydroperoxide,TBHP)、梯度浓度三羟乙基芦丁(0,50,100,200,400μmol/L)对PC12细胞活性的影响,确定TBHP和三羟乙基芦丁的实验浓度。②动物实验:将雄性SD大鼠分为假手术组、损伤组、三羟乙基芦丁50 mg/kg组、三羟乙基芦丁100 mg/kg组、STAT3抑制剂组,每组12只。除假手术组外行大鼠颈脊髓损伤,三羟乙基芦丁50 mg/kg组和100 mg/kg组损伤造模后分别予以腹腔注射三羟乙基芦丁50 mg/kg(1次/d)和100 mg/kg(1次/d);抑制剂组在损伤造模后给予STAT3抑制剂2000μg/kg(1次/d);假手术组动物行椎板切除术,但未行挫伤打击操作。假手术组和损伤组大鼠予以腹腔注射生理盐水,连续注射3 d。采用DCFH荧光探针检测三羟乙基芦丁对TBHP刺激PC12细胞中活性氧合成的影响;采用Western blot方法检测三羟乙基芦丁和STAT3抑制剂对PC12细胞及颈脊髓损伤组织中氧化应激相关蛋白Catalase和锰超氧化物歧化酶表达水平的影响;免疫荧光染色检测三羟乙基芦丁对颈脊髓损伤组织中STAT3和锰超氧化物歧化酶蛋白表达的影响。结果与结论:①三羟乙基芦丁可显著降低由TBHP诱导的PC12细胞内活性氧形成(P<0.05);②三羟乙基芦丁和STAT3抑制剂可显著上调PC12细胞和颈脊髓损伤组织内的Catalase蛋白和锰超氧化物歧化酶蛋白的表达量(P<0.05),同时显著抑制STAT3和NCK1蛋白表达量(P<0.05);③三羟乙基芦丁具有抗氧化应激作用,可降低脊髓损伤后氧化应激的破坏作用,其机制可能是通过调节SBACKGROUND:Previous animal studies have found that trihydroxyethyl rutin can improve the neurological function of rats with cervical spinal cord injury,but there is a lack of relevant studies on the mechanism of action.OBJECTIVE:To investigate the antioxidant stress effect of trihydroxyethyl rutin on spinal cord injury in vitro and in vivo,and to explore the possible mechanism.METHODS:(1)Cell experiment:The rat adrenal gland PC12 cells were purchased from cell bank of American Type Culture Collection.MTT assay was used to detect the effects of gradient concentrations of tert-butyl hydroperoxide(0,25,75,100,150,200,and 250μmol/L)and trihydroxyethyl rutin(0,50,100,200,and 400μmol/L)on the activity of PC12 cells.Experimental concentrations of tert-butyl hydroperoxide and trihydroxyethyl rutin were determined.(2)Animal experiment:Male Sprague-Dawley rats were assigned to sham operation group,injury group,trihydroxyethyl rutin 50 mg/kg group,trihydroxyethyl rutin 100 mg/kg group,and STAT3 inhibitor group(n=12).In addition to the sham operation group,cervical spinal cord injury was made in rats.Rats in the trihydroxyethyl rutin 50 mg/kg group and trihydroxyethyl rutin 100 mg/kg group were intraperitoneally given trihydroxyethyl rutin 50 mg/kg(once a day)and 100 mg/kg(once a day)after model establishment,respectively.The rats in the inhibitor group were given STAT3 inhibitor 2000μg/kg(once a day)immediately after model establishment.Animals in the sham operation group were subjected to laminectomy,but no contusion operation was performed.Rats in the sham operation group and the injury group were injected intraperitoneally with normal saline for 3 consecutive days.DCFH probe was used to detect the effects of trihydroxyethyl rutin on reactive oxygen species production in tert-butyl hydroperoxide-induced PC12 cells.Western blot assay was used to detect the effects of trihydroxyethyl rutin and STAT3 inhibitor on the expression levels of oxidative stress related proteins Catalase and MnSOD in PC12 cells and cervical spina

关 键 词：三羟乙基芦丁 颈脊髓损伤 大鼠 氧化应激 STAT3 

分 类 号：R459.9[医药卫生—治疗学] R496[医药卫生—临床医学]