摘要
目的探讨循经按摩通过缺氧诱导因子-1α(Hypoxia inducible factor-1α,HIF-1α)/腺病毒E1BI9kDa相互作用蛋白3(BCL2/adenovirus E1B 19kDa interacting protein 3,BNIP3)通路对缺血性脑卒中大鼠线粒体自噬的影响。方法采用线栓法阻塞大鼠右侧大脑中动脉(Middle cerebral artery,MCA)制作缺血模型,将建模成功的缺血性脑卒中大鼠均分为空白对照组、按摩组、通路激动剂组、通路抑制剂组以及按摩+抑制剂组,空白对照组大鼠以非循经穴位(包括百会、风府、人中)进行常规按摩,按摩组采用循经按摩(包括髀关、太冲、丰隆、足三里),通路激动剂组采用HIF-1α/BNIP3激活剂进行干预,通路抑制剂组采用HIF-1α/BNIP3抑制剂进行干预,按摩+抑制剂组采用循经按摩和HIF-1α/BNIP3抑制剂进行联合干预,另选10只健康大鼠进行仅分离血管不插线栓的假手术干预。通过Zea Longa评分系统评估神经功能缺损;TTC脑片染色测定大鼠脑梗死体积和脑半球肿胀程度;HE染色观察大鼠海马组织的病理变化;免疫荧光检测大鼠脑组织中HIF-1α以及BNIP3的表达;通过透射电镜对海马区域自噬小体的数量进行观察;Western blot检测脑组织线粒体中自噬(LC3B)、凋亡(Beclin-1)和HIF-1α/BNIP3信号通路相关蛋白表达。结果①模型组海马组织相较于假手术组中HIF-1α以及BNIP3荧光表达强度增强(P<0.05);按摩组海马组织相较于模型组中HIF-1α以及BNIP3荧光表达强度显著增强(P<0.05)。②与模型组相比,抑制剂组脑半球肿胀、神经功能缺损评分以及脑梗死体积水平上升(P<0.05);激动剂组脑半球肿胀、神经功能缺损评分以及脑梗死体积水平下降(P<0.05);激动剂组Beclin-1以及LC3B蛋白表达上升(P<0.05);抑制剂组Beclin-1以及LC3B蛋白表达下降(P<0.05)。③与按摩组相比,按摩+抑制剂组海马区细胞排列较为分散,细胞数量变少,出现水肿现象,组织呈现受损状态。④与模型组相比,按摩组LC3B、HIF-1α、Beclin-1以及BNIP3蛋白表达上升(P<0.05);按摩+抑制剂组LC3B、HIF-1α、Beclin-1以及BNIP3蛋白表达下降(P<0.05)。结论采用循经按摩治疗缺血性脑卒中时能通过激活HIF-1α/BNIP3信号通路,进而促进脑组织线粒体自噬改善神经功能,为后续循经按摩在缺血性脑卒中患者临床治疗中的应用提供了理论基础。
Objective To investigate the effects of meridian massage on mitochondrial autophagy in ischemic stroke rats through the hypoxia-inducible factor-1α(HIF-1α)/BCL2/adenovirus E1B 19kDa interacting protein 3(BNIP3)pathway.Methods The ischemic model was made by blocking the right middle cerebral artery(MCA)of rats with thread embolism.The ischemic stroke rats with successful modeling were divided into model group,massage group,pathway agonist group,pathway inhibitor group and massage+inhibitor group.The rats in the blank control group received routine massage at non-meridian acupoints(including Baihui,Fengfu and Renzhong).The massage group was treated with circulation massage(including Biguan,Taichong,Fenglong and Zusanli),the pathway agonist group was treated with HIF-1α/BNIP3 activator,the pathway inhibitor group was treated with HIF-1α/BNIP3 inhibitor,and the massage+inhibitor group was treated with combination of circulation massage and HIF-1α/BNIP3 inhibitor.Another 10 healthy rats were selected for sham operation intervention,which only separated the blood vessels without inserting the plug.Neurological deficits were evaluated through the Zea Longa scoring system;TTC brain smear staining was used to determine the volume of cerebral infarction and the degree of hemispheric swelling in rats.The pathological changes of rat hippocampal tissue were observed by HE staining;Immunofluorescence was used to detect the expressions of HIF-1α and BNIP3 in rat brain tissue;The number of autophagosomes in the hippocampal region was observed by transmission electron microscopy.Western blot was used to detect the protein expressions related to autophagy(LC3B),apoptosis(Beclin-1),and HIF-1α/BNIP3 signaling pathways in mitochondria of brain tissue.Results①The fluorescence expression intensity of HIF-1α and BNIP3 in hippocampus of model group was increased compared with that of sham group(P<0.05);The fluorescence expression intensity of HIF-1α and BNIP3 in hippocampus of massage group was significantly increased compared with that of model group(P<0.05).②Compared with model group,cerebral hemisphere swelling,nerve function deficit score and cerebral infarction volume in inhibitor group were increased(P<0.05);The levels of cerebral hemisphere swelling,nerve function deficit score and cerebral infarction volume in agonist group were decreased(P<0.05).The protein expressions of Beclin-1 and LC3B in agonist group were increased(P<0.05).The protein expressions of Beclin-1 and LC3B in inhibitor group were decreased(P<0.05).③Compared with the massage group,the hippocampal cells in the massage+inhibitor group were more dispersed,the number of cells decreased,edema appeared,and tissue was damaged.④Compared with model group,the protein expressions of LC3B,HIF-1α,Beclin-1 and BNIP3 in massage group were increased(P<0.05);The protein expressions of LC3B,HIF-1α,Beclin-1 and BNIP3 in massage+inhibitor group were decreased(P<0.05).Conclusion In the treatment of ischemic stroke,meridian massage can activate HIF-1α/BNIP3 signaling pathway,and then promote brain mitochondrial autophagy to improve nerve function,which provides a theoretical basis for the subsequent application of meridian massage in the clinical treatment of ischemic stroke patients.
作者
魏一丁
黄海
黄菲
WEI Yiding;HUANG Hai;HUANG Fei(Hubei Provincial Hospital of Traditional Chinese Medicine/Hubei Sizhen Laboratory/Affiliated Hospital of Hubei University of Chinese Medicine,Wuhan 430074,China)
出处
《世界科学技术-中医药现代化》
北大核心
2025年第9期2699-2711,共13页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
湖北省自然科学基金项目(2023AFD170):“怡神养脑”推拿手法治疗卒中后认知障碍的临床疗效及机制探讨,负责人:黄海。
