基于铁死亡机制比较补气活血法与行气活血法对缺血性脑卒中小鼠的脑保护作用OA

Objective:To investigate the differences in the neuroprotective effects of two distinct therapeutic approaches-Buqi Huoxue Method(tonifying qi and promoting blood circulation)versus Xingqi Huoxue Method(regulating qi and promoting blood circulation)-on mice with ischemic stroke,based on the ferroptosis mechanism.Methods:Sixty male C57BL/6N mice were randomly divided into a sham surgery group(Sham),a surgical model group(dMCAO),Buqi Huoxue group[Bu Yang Huan Wu Decoction(BYHWD)],and Xingqi Huoxue group[Tong Qiao Huo Xue Decoction(TQHXD)],with 15 mice in each group.Except for the Sham group,the other three groups underwent surgical procedures to establish a dMCAO mouse model.The BYHWD group were administered 18.564 g/kg of BYHWD via gavage,while the TQHXD group received 6.006 g/kg of TQHXD.The Sham and dMCAO groups were administered an equal volume of saline via gavage,for 7 consecutive days.Behavioral assessments were conducted on days 1,3,5,and 7 after surgery.Relevant parameters were measured after 7 days:Evans blue staining used to observe blood-brain barrier permeability;hematoxylin-eosin(HE)and Nissl staining to examine the morphological structure of brain tissue;immunohistochemical staining to assess the expression of microtubule-associated protein 2(MAP2)and neuron-specific nuclear protein(NeuN)in the brain;Prussian blue staining to assess iron content in brain tissue;immunohistochemical staining to assess the expression of related proteins in the brain,including Hepcidin,transferrin receptor 1(TfR1),ferritin light chain(FTL),4-hydroxy-2-nonenal(4-HNE),lipoxygenase(12-LOX),glutathione peroxidase(GPX4),ionized calcium-binding adaptor molecule 1(Iba-1),(CD86),(CD206),interleukin-6(IL-6),tumor necrosis factor-α (TNF-α),and transforming growth factor-β1(TGF-β1);and Western blot analysis to examine the expression of the relevant proteins TfR1,FTL,4-HNE,12-LOX,GPX4,IL-6,TNF-α,and TGF-β1 in the brain.Results:Compared with the Sham group,the dMCAO group showed reduced grip strength and shorter hanging time(P＜0.05);increased blood-brain barrier permeability and greater dye leakage(P＜0.05);neuronal cell atrophy and disorganized arrangement,accompanied by decreased MAP2 and NeuN;and increased brain iron content;upregulated mouse iron metabolism and mortality-related markers TfR1,FTL,4-HNE,12-LOX,and hepcidin(P＜0.05),decreased GPX4(P＜0.05);increased Iba-1-positive microglia with an amoeboid morphology;elevated M1 microglial marker CD86,decreased M2 microglial marker CD206;upregulated IL-6 and TNF-α(P＜0.05),and downregulated TGF-β1 expression(P＜0.05).Compared with the dMCAO group,the BYHWD group showed increased forelimb and hindlimb grip strength and prolonged hanging time in mice on postoperative 1,3,5,and 7(P＜0.05);the TQHXD group showed enhanced limbs grip strength and prolonged hanging time on postoperative day 7;both the BYHWD and TQHXD groups demonstrated improved blood-brain barrier permeability and reduced dye leakage(P＜0.05);as well as ameliorated histopathological morphology and elevated expression of MAP2 and NeuN;and decreased brain iron content;in the BYHWD group,TfR1,FTL,4-HNE,12-LOX,and hepcidin decreased(P＜0.05),while GPX4 increased(P＜0.05);in the TQHXD group,FTL and 12-LOX decreased;In both the BYHWD and TQHXD groups,Iba-1 and CD86 decreased,while CD206 increased;in both the BYHWD and TQHXD groups,IL-6 and TNF-α decreased(P＜0.05),and in the BYHWD group,TGF-β1 increased(P＜0.05).Compared with the TQHXD group,the BYHWD group showed superior recovery in left forelimb grip strength on day 1,left forelimb and hindlimb grip strength on day 3,and left forelimb grip strength on day 7;hanging time was significantly longer(P＜0.05);dye leakage was significantly reduced(P＜0.05);4-HNE increased,while GPX4 decreased(P＜0.05);the decrease in IL-6 expression was more significant(P＜0.05).Conclusion:Both Buqi Huoxue and Xingqi Huoxue methods exert neuroprotective effects to varying degrees in mice with ischemic stroke,and their mechanisms are associated with the inhibition of ferroptosis,with the effect of tonifying qi and promoting blood circulation being more significant.