基于PTEN/mTOR/STAT3通路探讨夹脊电针对脊髓损伤大鼠自噬的影响  

Mechanism of Electroacupuncture at Jiaji Acupoints on Autophagy in Rats with Spinal Cord Injury Based on PTEN/mTOR/STAT3 Signaling Pathway

作　　者：尹洪娜[1,2] 仲思潼 李竹馨 张硕 周新宇 崔杨 孙忠人[1,2] YIN Hongna;ZHONG Sitong;LI Zhuxin;ZHANG Shuo;ZHOU Xinyu;CUI Yang;SUN Zhongren(Heilongjiang University of Chinese Medicine,Harbin 150040,Heilongjiang,China;Second Affiliated Hospital of Heilongjiang University of Chinese Medicine,Harbin 150001,Heilongjiang,China)

机构地区：[1]黑龙江中医药大学,黑龙江哈尔滨150040 [2]黑龙江中医药大学附属第二医院,黑龙江哈尔滨150001

出　　处：《中华中医药学刊》2024年第11期1-7,I0001-I0004,共11页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81873378,81704181);国家重点研发计划中医药现代化研究专项(2022YFC3500405)。

摘　　要：目的观察夹脊电针对脊髓损伤(spinal and injury,SCI)大鼠第10号染色体上缺失的磷酸酶与同源张力蛋白(phosphatase and tensinhomolog,PTEN)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)/信号转导和转录激活子3(signal transducer and activator of transcription 3,STAT3)通路相关因子(p-PTEN、p-mTOR、p-STAT3)、自噬相关因子(LC3-Ⅱ、p62、Atg5)以及星形胶质细胞中星形胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、自噬标志物微管相关蛋白1轻链3(light chain 3,LC3)共定位信号的表达情况,探讨夹脊电针调控SCI细胞自噬促进神经功能恢复的可能机制,为临床运用夹脊电针治疗SCI提供新的思路和分子生物学理论依据。方法将72只雄性SD大鼠随机分为假手术(Sham)组、模型(Model)组、夹脊电针(EA)组、抑制剂+夹脊电针(bpV)组,每组按照造模后3、7和14 d治疗时间点分为3个亚组,每个亚组6只大鼠。采用Allen’s法建立SCI大鼠模型。EA组予双侧胸(T)9、T11夹脊穴,电针30 min,强度1 mA,密波(100 Hz),1次/d,分别连续治疗3、7、14 d。bpV组在电针治疗前10 min注射PTEN通路抑制剂bpV(HOpic),200μL/kg,1次/d,分别连续治疗3、7、14 d。采用Basso-Beattie-Bresnahan(BBB)评分观察并比较各组大鼠后肢运动功能;HE染色观察各组大鼠脊髓组织的病理形态学变化;Nissl染色观察各组大鼠脊髓组织前角运动神经元的形态和数量;IHC检测各组大鼠脊髓组织p-PTEN、p-mTOR、p-STAT3、LC3-Ⅱ、p62、Atg5的免疫组化染色累积光密度值(imaging object definition,IOD)值;WB检测各组大鼠p-PTEN、p-mTOR、p-STAT3蛋白相对表达量;IF检测脊髓组织星形胶质细胞GFAP、LC3共定位情况。结果大鼠行为学检测结果示:与Model组比,在3、7、14 d时,EA组、bpV组BBB评分均增高(P<0.05);HE结果示:Model组脊髓组织出现大量内含片状出血以及坏死组织的空腔,神经细胞松散排列,3 d后EA组脊髓组织结构逐渐�Objective To observe the expressions of phosphatase and tensin homolog deleted on chromosome ten(PTEN)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3)pathway-related factors(p-PTEN,p-mTOR,p-STAT3),autophagy-related factors(LC3-Ⅱ,p62,Atg5)and astrocyte glial fibrillary acidic protein(GFAP)and LC3 co-localization signals in rats with spinal cord injury(SCI),explore the possible mechanism of regulating the autophagy of SCI cells and promoting the recovery of nerve function by electroacupuncture(EA)at Jiaji acupoints and provide a new idea and theoretical basis of molecular biology for the clinical application of EA at Jiaji acupoints to treat SCI.Methods A total of 72 male SD rats were randomly divided into sham operation(Sham)group,model(Model)group,EA at Jiaji acupoints group and inhibitor(bpV)combined with EA at Jiaji acupoints group.Each group was divided into three subgroups according to the treatment time points of 3 d,7 d and 14 d after modeling,with 6 rats in each subgroup.SCI rat model was established by Allen's method.The EA at Jiaji acupoints group was treated with EA at bilateral T9 and T11 Jiaji acupoints for 30 min.The intensity was1 mA with dense wave(100 Hz),once a day,continuously treating for 3 d,7 d and 14 d respectively.The bpV group was injected with PTEN pathway inhibitor bpV(HOpic)10 min before EA treatment,200μL/kg,once a day,continuously treating for 3 d,7 d and 14d respectively.BBB score was used to observe and compare the hind limb motor function of rats in each group.HE staining was used to observe the pathological changes of spinal cord tissue in each group.Nissl staining was used to observe the morphology and number of motor neurons in the anterior horn of spinal cord tissue of rats in each group.The imaging object definition(IOD)values of p-PTEN,p-mTOR,p-STAT3,LC3-Ⅱ,p62 and Atg5 in spinal cord tissue of rats in each group were detected by IHC.Western Blot was used to detect the relative expressions of p-PTEN,p-mTOR and p-STAT3 protein

关 键 词：夹脊电针 脊髓损伤 PTEN/mTOR/STAT3 自噬 星形胶质细胞 

分 类 号：R245.97[医药卫生—针灸推拿学]