Study on the Effect of Artemisia Annua Extract on Glutamate Induced Ferroptosis in Ischemic Stroke Rats

WANG Shuran; CHEN Xiaoying; WANG Yan; GAO Yuankai; XU Qing; ZHAO Zhangdi; YE Yifan; XU Minhao; ZHANG Wenyan; XU Wenhui; HU Sumin

Abstract：Objective To investigate the mechanism of intervention by Artemisia annua extract on ischemic stroke in rats based on the glutamate-ferroptosis-related pathway.Methods A total of 105 male SD rats were randomly divided into sham operation group,model group,ginkgo biloba extract group(50 mg/kg),and Artemisia annua L.extract groups(12.5 mg/kg,25 mg/kg,50 mg/kg,100 mg/kg).An embolic stroke model was established using the middle cerebral artery occlusion.Preventive medication was administered by gavage for 6 days before modeling,and the model was conducted 30 minutes of administration on the 6th day.24 h after modeling,the Zea Longa neurological deficit score was assessed,and the 2,3,5-triphenyltetrazolium chloride(TTC)staining method was used to detect the infarct volume in the rat brain.Hematoxylin-eosin(HE)staining and Nissl staining were performed to observe neuronal morphological changes.Colorimetric methods were used to measure the content of ferrous iron(Fe2+),malondialdehyde(MDA),reduced glutathione(GSH),and superoxide dismutase(SOD)in rat brain tissue.Western blot was used to detect the protein expression of solute carrier family 7 member 11(SLC7A11,xCT),glutathione peroxidase 4(GPX4),and ferritin heavy chain 1(FTH1).Real-time quantitative PCR(qPCR)was employed to measure the mRNA expression of SLC7A11,GPX4,excitatory amino acid transporter 1(EAAT1),EAAT2,glutamine synthetase(GS),and phosphate-activated glutaminase(PAG).Results Compared to the sham operation group,the model group rats showed significantly increased neurological deficit scores,brain infarct volume,and Fe2+and MDA content in brain tissue(P<0.05).Neurons in the hippocampus exhibited shrinkage and loss of dendritic structure,and GSH and SOD content decreased in model group rats(P<0.05).The protein expression of SLC7A11,GPX4,and FTH1,as well as the mRNA expression of SLC7A11,GPX4,and EAAT2,were significantly decreased(P<0.05),while PAG mRNA expression was significantly increased(P<0.05).The expression level of PAG mRNA significantly increased(P<0.05).Compared to the model group,all treatment groups showed reduced neurological deficit scores,brain infarct volume,and Fe2+and MDA content in brain tissue.Neurons in the hippocampus appeared relatively intact,and pathological damage was significantly alleviated.GSH and SOD levels increased,and the protein expression levels of SLC7A11,GPX4 and FTH1 increased to varying degrees(P<0.05).Additionally,the ginkgo biloba extract group showed increased GPX4 mRNA expression(P<0.05).All Artemisia annua extract dose groups exhibited upregulated mRNA expression of SLC7A11,GPX4,and EAAT2,while PAG mRNA expression was downregulated(P<0.05).Conclusion Artemisia annua L.extract can improve neurological function and pathological damage in rats with ischemic stroke,and its mechanism may be related to the inhibition of glutamate-induced neuronal ferroptosis.