南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (2): 199-205.doi: 10.12122/j.issn.1673-4254.2023.02.06

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连翘酯苷B抑制小鼠脑缺血/再灌注引起的氧化应激损伤：基于激活AMPK/DAF-16/FOXO3通路

陈 兴，王开万，储德海，朱 羽，张文兵，曹慧萍，谢文宇，鲁传豪，李 侠   

  1. 汉滨区第三人民医院重症医学科，陕西 安康 725000；空军军医大学西京医院神经外科，陕西 西安 710032
  • 出版日期:2023-02-20 发布日期:2023-03-16

Forsythiaside B inhibits cerebral ischemia/reperfusion-induced oxidative stress injury in mice via the AMPK/DAF-16/FOXO3 pathway

CHEN Xing, WANG Kaiwan, CHU Dehai, ZHU Yu, ZHANG Wenbing, CAO Huiping, XIE Wenyu, LU Chuanhao, LI Xia   

  1. Department of Critical Care Medicine, Hanbin District Third People's Hospital, Ankang 725000, China; Department of Neurosurgery, Xijing Hospital, Air Force Medical University, Xi'an 710032, China
  • Online:2023-02-20 Published:2023-03-16

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摘要： 目的 研究连翘酯苷B（FB）对脑缺血/再灌注（I/R）引起的氧化应激损伤的保护作用及作用机制。方法 将90只C57BL/6小鼠随机分为 5 组，假手术组（Sham），模型组（model），FB 低、中、高剂量组（10、20、40 mg/kg），采用大脑中动脉阻塞（MCAO）2 h，再灌注24 h，制备脑I/R模型。试剂盒检测小鼠脑组织ROS、MDA、PCO、8-OhdG、SOD、GSTα4、CAT、GPx水平，Western blot 检测小鼠脑组织 AMPK、P-AMPK、DAF-16、FOXO3、P-FOXO3 蛋白表达。在此基础上，采用 AMPK 抑制剂Compound C（CC）验证FB是否通过AMPK发挥抑制氧化应激损伤作用，将36只C57BL/6小鼠随机分为，Sham组、model组、FB（40 mg/kg）组、FB（40 mg/kg）+CC（10 mg/kg）组。TTC染色检测脑梗死体积，试剂盒检测ROS、SOD水平，Western blot检测小鼠脑组织AMPK、P-AMPK、DAF-16、FOXO3、P-FOXO3蛋白表达。结果 与model组比较，FB可降低小鼠脑组织ROS、MDA、PCO、8-OHdG水平，升高小鼠脑组织中抗氧化酶SOD、GSTα4、CAT、GPx活性，提高AMPK、FOXO3磷酸化及DAF-16蛋白表达水平（P<0.01）。与FB组比较，FB+CC组AMPK和FOXO3磷酸化水平降低，DAF-1表达减少，脑梗死体积增加，ROS水平升高，SOD水平降低（P<0.01）。结论 FB能够抑制I/R引起的脑组织氧化应激损伤，作用机制是通过促进AMPK磷酸化、其下游DAF-16蛋白表达及FOXO3磷酸化，促进抗氧化酶的表达，清除过量ROS，从而发挥治疗缺血性脑卒中的作用。

关键词: 连翘酯苷B；脑缺血/再灌注；氧化应激；AMPK/DAF-16/FOXO3通路

Abstract: Objective To study the protective effect of forsythiaside B (FB) against cerebral oxidative stress injury induced by cerebral ischemia/reperfusion (I/R) in mice and explore the underlying mechanism. Methods Ninety C57BL/6 mice were randomized into sham-operated group, middle cerebral artery occlusion (MCAO) model group, and low-, medium and high-dose (10, 20, and 40 mg/kg, respectively) FB groups. The expression levels of MDA, ROS, PCO, 8-OHdG, SOD, GSTα4, CAT and GPx in the brain tissue of the mice were detected using commercial kits, and those of AMPK, P-AMPK, DAF-16, FOXO3 and P-FOXO3 were detected with Western blotting. Compound C (CC), an AMPK inhibitor, was used to verify the role of the AMPK pathway in mediating the therapeutic effect of FB. In another 36 C57BL/6 mice randomized into 4 sham-operated group, MCAO model group, FB (40 mg/kg) treatment group, FB+CC (10 mg/kg) treatment group, TTC staining was used to examine the volume of cerebral infarcts, and the levels of ROS and SOD in the brain were detected; the changes in the protein expressions of AMPK, P-AMPK, DAF-16, FOXO3 and P-FOXO3 in the brain tissue were detected using Western blotting. Results In mice with cerebral IR injury, treatment with FB significantly reduced the levels of ROS, MDA, PCO and 8-OHdG, increased the activities of antioxidant enzymes SOD, GSTα4, CAT and GPx, and enhanced phosphorylation of AMPK and FOXO3 and DAF-16 protein expression in the brain tissue (P<0.01). Compared with FB treatment alone, the combined treatment with FB and CC significantly reduced phosphorylation of AMPK and FOXO3, lowered expression of DAF-16 and SOD activity, and increased cerebral infarction volume and ROS level in the brain tissue of the mice (P<0.01). Conclusion FB inhibits oxidative stress injury caused by cerebral I/R in mice possibly by enhancing AMPK phosphorylation, promoting the downstream DAF-16 protein expression and FOXO3 phosphorylation, increasing the expression of antioxidant enzymes, and reducing ROS level in the brain tissue.

Key words: forsythiaside B; cerebral ischemia/reperfusion; oxidative stress; AMPK/DAF-16/FOXO3 pathway