基于Nrf2/SLC7A11/GPX4信号通路探讨失笑散对脑缺血再灌注损伤大鼠神经功能及铁死亡的影响OA

Objective:To investigate whether Shixiaosan can improve neurological function and inhibit ferroptosis in rats with cerebral ischemia-reperfusion injury(CIRI)by regulating the nuclear factor E2-related factor 2(Nrf2)/solute carrier family 7 member 11(SLC7A11)/glutathione peroxidase 4(GPX4)pathway.Methods:A rat model of CIRI was established using the intraluminal filament method.Briefly,cervical blood vessels were separated,branches of the external carotid artery were ligated,and the common carotid artery and internal carotid artery were clamped.A nylon filament was inserted through the opening of the external carotid artery to the origin of the middle cerebral artery to block blood flow and induce cerebral ischemia.After 60-120 min of ischemia,the filament was withdrawn to restore blood flow,and the external carotid artery incision was ligated.The rats were divided into a CIRI group,a Shixiaosan low-dose(-L)group(intragastric administration of 1.26 g·kg-1 Shixiaosan),a Shixiaosan high-dose(-H)group(intragastric administration of 2.52 g·kg-1 Shixiaosan),a donepezil hydrochloride tablet(DON)group(intragastric administration of 0.45 mg·kg-1 DON),and a Shixiaosan-H+Nrf2 inhibitor(ML385)group(intragastric administration of 2.52 g·kg-1 Shixiaosan combined with intraperitoneal injection of 30 mg·kg-1 ML385).An additional 12 rats underwent cervical artery separation followed by incision suturing and served as the control group.Equal volumes of double-distilled water were administered to the CIRI and control groups.Neurological function impairment was assessed using the modified Garcia JH score.Magnetic resonance imaging was used to determine the cerebral infarct volume ratio.Hematoxylin-eosin(HE)staining and Prussian blue staining were performed to observe neuronal injury and iron accumulation in the ischemic penumbra,respectively.Transmission electron microscopy was used to examine the ultrastructure of neuronal mitochondria in the ischemic penumbra.Commercial kits were used to measure ferrous iron(Fe2+),malondialdehyde(MDA),reduced glutathione(GSH)content,and reactive oxygen species(ROS)activity in the ischemic penumbra.The BODIPY(581/591)C11 fluorescent probe was used to detect intracellular lipid peroxidation levels.Western blot was performed to detect protein expression levels of Nrf2,SLC7A11,GPX4,transferrin receptor 1(TFRC),ferritin heavy chain(FHC),and ferritin light chain(FLC)in the ischemic penumbra.Results:Compared with the control group,the CIRI group exhibited neuronal injury in the ischemic penumbra,characterized by reduced neuron numbers,nucleolar shrinkage,and interstitial edema.Marked iron accumulation was observed in the tissue.Neuronal mitochondria showed atrophy and rupture,with reduced mitochondrial cristae and increased membrane density.The cerebral infarct volume ratio,Fe2+content,MDA content,ROS activity,and lipid peroxidation levels were increased,whereas the modified Garcia JH score,GSH content,and protein expression levels of Nrf2,SLC7A11,GPX4,FHC,and FLC were decreased,and TFRC protein expression was increased(P＜0.05).Compared with the CIRI group,the Shixiaosan-L group,Shixiaosan-H group,and DON group showed attenuated neuronal injury in the ischemic penumbra,reduced iron accumulation,alleviated mitochondrial damage,decreased cerebral infarct volume ratio,Fe2+and MDA contents,ROS activity,and lipid peroxidation levels,as well as increased modified Garcia JH scores,GSH content,and protein expression levels of Nrf2,SLC7A11,GPX4,FHC,and FLC,while TFRC protein expression was decreased(P＜0.05).The magnitude of changes in all indicators was greater in the Shixiaosan-H group than in the Shixiaosan-L group(P＜0.05).Compared with the Shixiaosan-H group,all measured indicators in the Shixiaosan-H+ML385 group showed opposite trends(P＜0.05).Conclusion:Shixiaosan may inhibit ferroptosis and restore neurological function in rats with CIRI by activating the Nrf2/SLC7A11/GPX4 pathway.