基于GPX4-ACSL4轴探讨补阳还五汤抑制铁死亡促进脊髓损伤后神经功能恢复的作用机制  

Mechanism of Buyang Huanwutang in Inhibiting Ferroptosis and Enhancing Neurological Function Recovery After Spinal Cord Injury via GPX4-ACSL4 Axis

作　　者：许卢春 姜国正 马昱堃 宋佳伟 高誉珊 王冠龙 范娇娇 杨永栋[1] 俞兴[1] 唐向盛[3] XU Luchun;JIANG Guozheng;MA Yukun;SONG Jiawei;GAO Yushan;WANG Guanlong;FAN Jiaojiao;YANG Yongdong;YU Xing;TANG Xiangsheng(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China;School of Traditional Chinese Medicine,Beijing University of Chinese Medicine,Beijing 100029,China;China-Japan Friendship Hospital,Beijing 100029,China)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]北京中医药大学中医学院,北京100029 [3]中日友好医院,北京100029

出　　处：《中国实验方剂学杂志》2025年第5期20-30,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(82074218,81973882);国家自然科学基金青年基金项目(81804119);中央高校基本科研业务费项目(2024-JYB-JBZD-065);北京中医药大学东直门医院人才培养计划项目(DZMG-QNGG0010)。

摘　　要：目的:探讨补阳还五汤通过调控谷胱甘肽过氧化物酶4(GPX4)-长链酰基辅酶A合成酶4(ACSL4)轴抑制铁死亡,促进脊髓损伤后神经功能恢复的机制。方法:将90只大鼠随机分为假手术组、模型组、补阳还五汤低剂量组(12.5 g·kg^(-1))、补阳还五汤高剂量组(25 g·kg^(-1))和补阳还五汤+抑制剂组(25 g·kg^(-1)+5 g·kg^(-1)RSL3)。通过allen法构建脊髓损伤模型。术后7 d和28 d取材,通过巴索-比蒂-布雷斯纳汉(BBB)评分观察大鼠运动功能,苏木素-伊红、尼氏和坚牢蓝染色观察脊髓组织学形态,透射电镜观察线粒体微观结构,免疫荧光染色观察神经元特异性核蛋白(NeuN)阳性细胞数量和髓磷脂碱性蛋白(MBP)、GPX4、ACSL4荧光强度,实时荧光定量聚合酶链式反应(Real-time PCR)检测GPX4和ACSL4 mRNA表达,酶联免疫吸附测定法(ELISA)检测活性氧(ROS)、丙二醛(MDA)、谷胱甘肽(GSH)和超氧化物歧化酶(SOD)水平,比色法检测脊髓组织铁含量。结果:与假手术组比较,模型组大鼠BBB评分显著降低(P<0.01),脊髓组织出现严重病理损伤和严重线粒体微观形态破坏;模型组NeuN阳性细胞数量显著降低(P<0.01),MBP、GPX4荧光强度显著降低(P<0.01),GSH、SOD水平显著下降(P<0.01)及GPX4 mRNA相对表达水平显著下调(P<0.01);模型组大鼠ROS、MDA、组织铁含量显著上调(P<0.01),ACSL4荧光强度和ACSL4 mRNA相对表达水平显著升高(P<0.01)。与模型组及补阳还五汤+抑制剂组比较,补阳还五汤组BBB评分明显升高(P<0.05,P<0.01)。补阳还五汤组较模型组及补阳还五汤+抑制剂组具有更轻的脊髓组织病理损伤,更少的水肿和炎性细胞浸润,更多的神经元存活,更加完整的髓鞘结构。此外,补阳还五汤组线粒体微观形态改善更为明显。与模型组及补阳还五汤+抑制剂组比较,补阳还五汤组NeuN阳性细胞数量和MBP荧光强度明显增加(P<0.05,P<0.01)。同时,补阳还五汤较模型组及补阳还五汤+抑�Objective:To explore the mechanism by which Buyang Huanwutang regulates the glutathione peroxidase 4(GPX4)-acyl-CoA synthetase long-chain family member 4(ACSL4)axis to inhibit ferroptosis and promote neurological functional recovery after spinal cord injury(SCI).Methods:Ninety rats were randomly divided into five groups:sham operation group,model group,low-dose Buyang Huanwutang group(12.5 g·kg^(-1)),high-dose Buyang Huanwutang group(25 g·kg^(-1)),and Buyang Huanwutang+inhibitor group(25 g·kg^(-1)+5 g·kg^(-1)RSL3).The SCI model was established by using the allen method.Tissue was collected on the 7th and 28th days after operation.Motor function was assessed by using the Basso-Beattie-Bresnahan(BBB)scale.Hematoxylin-eosin(HE),Nissl,and Luxol fast blue(LFB)staining were performed to observe spinal cord histopathology.Transmission electron microscopy was used to examine mitochondrial ultrastructure.Immunofluorescence staining was used to detect the number of NeuN-positive cells and the fluorescence intensity of myelin basic protein(MBP),GPX4,and ACSL4.Realtime fluorescent quantitative polymerase chain reaction(Real-time PCR)was used to analyze the mRNA expression of GPX4 and ACSL4.Enzyme linked immunosorbent assay(ELISA)was performed to measure the levels of reactive oxygen species(ROS),malondialdehyde(MDA),glutathione(GSH),and superoxide dismutase(SOD).Colorimetric assays were used to determine the iron content in spinal cord tissue.Results:Compared to the sham operation group,the model group exhibited significantly reduced BBB scores(P<0.01),severe pathological damage in spinal cord tissue,and marked mitochondrial ultrastructural disruption.In addition,the model group showed a decrease in the number of NeuN-positive cells(P<0.01),reduced fluorescence intensity of MBP and GPX4(P<0.01),lower levels of GSH and SOD(P<0.01),and downregulated mRNA expression of GPX4(P<0.01).Moreover,compared to the sham operation group,the model group had elevated levels of ROS,MDA,and tissue iron content(P<0.01),along with increase

关 键 词：脊髓损伤 补阳还五汤 铁死亡 谷胱甘肽过氧化物酶4(GPX4)-长链酰基辅酶A合成酶4(ACSL4)轴 氧化应激 

分 类 号：R285[医药卫生—中药学] R289[医药卫生—中医学] R259