Mechanism Of Buyang Huanwu Decoction Against Cerebral Ischemia-reperfusion Injury Through Ferroptosis Pathway

With the change of people’s lifestyle and the aging of the population,cerebrovascular disease（cerebrovascular disease;CVD）has become one of the three major diseases in the world,including ischemic stroke（cerebral ischemic stroke;CIS）has consistently accounted for a high proportion of all CVD patients,although there are many drugs used clinically in the prevention,detection and treatment of CVD,and cerebral ischemia/reperfusion injury in the acute onset and cerebral ischemia/reperfusion injury;CIRI)has made some progress in related treatments,but the question of how to better identify effective and affordable drugs also needs to be solved.Traditional Chinese medicine in the motherland has a history of nearly 5,000 years,and has rich clinical experience in the treatment of CVD,and traditional Chinese medicine compound has the advantages of good efficacy and economy,and can be widely used in clinical practice;However,the specific treatment mechanism of Chinese medicine compound is not completely clear,and how to study and explain the treatment mechanism of Chinese medicine compound through modern technical means has become the focus of current research.Buyang Huanwu decoction is a traditional Chinese medicine formula for the treatment of stroke with qi deficiency and blood stasis,which is composed of astragalus,angelica,red peony,earth dragon,Chuanxiong,peach kernel and safflower,and its effect of invigorating qi,activating blood and channeling is very effective in the treatment of stroke with qi deficiency and blood stasis.The previous research of this laboratory also confirmed that the decoction can treat stroke and reduce reperfusion damage after stroke through multiple ways such as apoptosis and autophagy.Ferroptosis is a new type of cell death proposed in 2012.Many studies have shown that ferroptosis is closely related to the occurrence and development of CIRI,and studies have shown that the main components of single herbs such as astragalus,angelica and safflower can play a certain therapeutic effect on CIRI by inhibiting ferroptosis.However,whether Buyang Huanwu Decoction can resist CIRI through ferroptosis pathway is still unclear.In this study,SD rats and HT22 cells were used as research objects to explore whether Buyang Huanwu Decoction can resist CIRI based on ferroptosis pathway in vivo and in vitro.The study is divided into three parts:1.Effect of cerebral blood flow reduction on middle cerebral artery embolization/reperfusion（MCAO/R）model in rats;2.Study on Buyang Huanwu Decoction anti-cerebral ischemia-reperfusion injury by regulating brain iron metabolism in rats and inhibiting ferroptosis;3.Study on Buyang Huanwu Decoction reducing the damage of HT22 cells in OGD/R model by regulating iron ion metabolism and inhibiting ferroptosis.Part One:Effect of cerebral blood flow reduction on middle cerebral artery occlusion/reperfusion model（MCAO/R）in ratsObjective:To investigate the effect of cerebral blood flow changes on the preparation of MCAO/R model in rats.Methods:Twenty-five healthy male SD rats with a body weight of 250 g～280 g were randomly divided into 5 groups:Group A（decreased cerebral blood flow by 10%-20%）,Group B（decreased cerebral blood flow by 21%-30%）,group C（decreased cerebral blood flow by 31%-40%）,group D（decreased cerebral blood flow by 41%-50%）and group E（decreased cerebral blood flow by 51%-60%）.The MCAO/R models of all animals were prepared by modified thread peg method and were monitored by laser speckle perfusion imager.The success rate of the model was observed by neurological function score at 24 h after reperfusion.72 h after reperfusion,the death rate after infarction was calculated,the change of body weight was observed and recorded,and the infarct volume was detected by TTC staining.Results:The post-infarction mortality rate was 40%（2）in group E and 0 in group A,B,C and D.Rats in group A had normal neurological function scores and no cerebral infarction,so the rats in group A were used as the basic control group.Neurological function scores:Compared with group A,neurological function scores of rats in group B had no significant difference（P>0.05）,while neurological function scores of rats in groups C,D and E were significantly increased（P<0.01）.There was no significant difference between groups C and D（P>0.05）,and group E was higher than group D（P<0.05）.Changes in body weight:there was no significant difference between group A and group B（P>0.05）,and the weight of group C,D and E decreased significantly compared with group A（P<0.01）,and the order of changes was E>D>C>B;The body weight of each group was significantly decreased compared with the previous group（P<0.05）.Cerebral infarction volume:Compared with group A,cerebral infarction lesions in groups B,C,D and E were obvious（P<0.01）,and the order of infarct volume was E>D>C>B;The volume of cerebral infarction in each group was more significant than that in the previous group（P<0.05）.Summary:During the preparation of MCAO/R model in rats,the percentage of cerebral blood flow in ischemic side decreased by 41%to 50%was the best,and the MCAO/R model had high success rate,good repeatability and stability.Part Two:Effect of Buyang Huanwu Decoction on cerebral ischemia-reperfusion injury by regulating iron metabolism in ratsObjective:To investigate the mechanism of Buyang Huanwu Decoction against CIRI by regulating brain iron metabolism and inhibiting ferroptosis in ratsMethods:Method:Sixty clean grade male SD rats were randomly divided into 4 groups:Sham group（Sham group）,model group（MCAO/R group）,Buyang Huanwu tang group（BYHWD group）and positive control group（DFO group）,with15 rats in each group.Except Sham group,MCAO/R model was established for 2 h of ischemia and 72 h of reperfusion.The BYHWD group and DFO group were treated with Buyang Huanwu Decoction and desferramine mesylate respectively,and the other two groups were treated with the same amount of normal saline.72 h after reperfusion,all rats were evaluated by Zea longa neurofunctional score and adhesive removal test for neurological impairment and somatosensory impairment.Infarct volume was detected by TTC staining.HE staining and Nissl staining were used to observe the tissue morphology and neurons.Observe iron deposition using Prussian blue staining;Using transmission electron microscopy to observe the structure of neurons and mitochondria;Enzyme linked immunosorbent assay（Elisa method）was used to detect MDA,GSH,and tissue iron content.The protein expression levels of Tf R1,DMT1,FPN1 and GPX4 were detected by Western Blot.The expression level of GPX4 was detected by immunofluorescence staining.Results:Sham group rats had no obvious neurological deficit,somatosensory injury and brain tissue infarction.Tissue staining showed that the brain tissue structure was tight,the neurons were normal in morphology and abundant in quantity,Nissl bodies were more common,and no obvious brain iron deposition was observed.Transmission electron microscopy showed that the mitochondrial structure of neurons was normal,and the mitochondrial cristae and outer membrane were intact.The results of enzyme-linked immunosorbent assay showed that the iron content and serum MDA level in Sham group were lower,and the GSH level was higher.The results of Western blot and immunofluorescence experiments showed that the expression of FPN1 and GPX4 protein in the brain tissue of Sham group was rich,and the expression of Tf R1 and DMT1 was low.Compared with the Sham group,the neurological deficits and somatosensory damage of the MCAO/R group were obvious,and the neurological function score was increased.Brain infarct volume increased significantly;tissue staining showed that the structure of brain tissue was loose,the arrangement of neurons was disordered,the number of neurons was significantly reduced,the edema of neurons was obvious,vacuoles,nuclear pyknosis and other pathological phenomena were common,Nissl bodies were significantly reduced,and the amount of brain iron deposition was significantly increased.Transmission electron microscopy showed obvious swelling of mitochondria,dissolution of matrix,reduction of cristae,rupture and disintegration of outer membrane in some severe cases.The results of enzyme-linked immunosorbent assay showed that the iron content and serum MDA level in MCAO/R group were significantly increased（P<0.01）,and GSH level was significantly decreased（P<0.01）.The results of Western blot and immunofluorescence experiments showed that the expression of FPN1 and GPX4 protein in MCAO/R group was significantly decreased（P<0.05）,and the expression of Tf R1 and DMT1 protein was significantly increased（P<0.05）.Compared with MCAO/R group,BYHWD group and DFO group rats neurological deficits and somatosensory injury significantly reduced,neurological function score decreased;brain infarct volume decreased significantly;tissue staining showed that the brain tissue structure was relatively compact,the number of neurons was significantly increased,neuronal edema was significantly reduced,vacuolation,nuclear pyknosis and other pathological phenomena were significantly reduced,Nissl bodies were significantly reduced,brain iron deposition was significantly increased.Transmission electron microscopy showed mild swelling of mitochondria in neurons,more mitochondrial cristae,intact outer membrane,and significantly reduced damage.The results of enzyme-linked immunosorbent assay showed that the tissue iron content and serum MDA level in BYHWD group and DFO group were significantly decreased（P<0.01）,and the GSH level was significantly increased（P<0.01）.The results of Western blot and immunofluorescence experiments showed that the expression of FPN1 and GPX4 in brain tissue of BYHWD group and DFO group increased（P<0.05）,and the expression of Tf R1 and DMT1 decreased（P<0.05）.Compared with DFO group,there were no significant differences in neurological deficits,somatosensory impairment,infarct volume,tissue staining and electron microscopy in BYHWD group（P>0.05）.The results of enzyme-linked immunosorbent assay showed that there was no significant difference in tissue iron content and GSH level in BYHWD group（P>0.05）,and MDA level was significantly decreased（P<0.05）.The results of Western blot and immunofluorescence experiments showed that there was no significant difference in the expression of FPN1,Tf R1 and DMT1 protein in brain tissue of BYHWD group（P>0.05）,and the expression of GPX4protein was significantly increased（P<0.05）.Summary:Buyang Huanwu Decoction can reduce the expression of proteins Tf R1and DMT1 in brain tissue,enhance the expression of FPN1 and GPX4,reduce the iron ion content in the brain tissue of the penumbra area on the ischemia-reperfusion side of rats,inhibit the reactive oxygen species produced by excessive iron participation in the Fenton reaction and the subsequent accumulation of toxic lipid peroxides causing ferroptosis,alleviate CIRI,and promote the recovery of neural function in brain tissue.Part Three:Study on reducing oxygen-sugar deprivation/reoxygen-sugar HT22 cell damage by regulating iron metabolism with Buyang Huanwu DecoctionObjective:To explore the mechanism of Buyang Huanwu Decoction alleviates HT22 cell damage in OGD/R model by regulating iron ion metabolism and inhibiting ferroptosis.Methods:Through the establishment of OGD/R model,HT22 cells were randomly divided into:normal group（Control）,model group（OGD/R）,model+Buyang Huanwu Decoction containing serum group（1%BYS,3%BYS,5%BYS,7%BYS,10%BYS）,model+blank serum group（1%CS,3%CS,5%CS,7%CS,10%CS）.The optimal concentration of Buyang Huanwu Decoction containing serum on the protective effect of OGD/R injury model of HT22 cells was investigated by CCK-8 method and detection of LDH leakage rate.HT22 cells were divided into control group（Control）,model group（OGD/R）,model+BYHWDS group（OGD/R+BYHWDS）and model+DFO group（OGD/R+DFO）.The contents of MDA and GSH in each group were detected by Elisa method.The content of ROS in each group was detected by flow cytometry.The content of Fe2+in each group was detected by fluorescence probe.The expression levels of iron homeostasis-related proteins Tf R1,FPN1,DMT1 and GPX4 in each group were detected by Western Blot.The expression levels of Tf R1,FPN1,DMT1and GPX4 were detected by immunofluorescence staining.Results:The results of CCK-8 method and LDH leakage rate showed that low concentration Buyang Huanwu Decoction-containing serum could enhance the viability of hypoxia/hypoglycemia/reoxygenation HT22 cells to varying degrees,reduce LDH leakage rate,and protect neurons.Among them,3%Buyang Huanwu Decoction-containing serum had the best therapeutic effect.Therefore,this experiment selected 3%Buyang Huanwu Decoction-containing serum for subsequent experimental research.The GSH content of neurons in the Control group was higher,and the contents of ROS,MDA and Fe2+were lower.Western blotting showed that Tf R1 and DMT1 proteins were less expressed,while FPN1 and GPX4 proteins were abundantly expressed.Immunofluorescence experiments showed that the average fluorescence intensity of Tf R1 and DMT1 proteins was weak,and the average fluorescence intensity of FPN1 and GPX4 proteins was strong.Compared with the Control group,the GSH content of neurons in the OGD/R group was significantly decreased（P<0.01）,and the contents of ROS,MDA and Fe2+were significantly increased（P<0.01）.Western blotting showed that the expression levels of Tf R1 and DMT1 were significantly increased（P<0.05）,and the expression levels of FPN1 and GPX4 were significantly decreased（P<0.05）.Immunofluorescence assay showed that the average fluorescence intensity of Tf R1 and DMT1 protein was significantly increased（P<0.05）,and the average fluorescence intensity of FPN1 and GPX4 protein was significantly decreased（P<0.05）.Compared with OGD/R group,the contents of GSH in BYHWDS group and DFO group were significantly increased,while the contents of ROS,MDA and Fe2+were significantly decreased（P<0.01）.Western blotting showed that the expression levels of Tf R1 and DMT1 were significantly decreased（P<0.05）,and the expression levels of FPN1 and GPX4 were significantly increased（P<0.05）Immunofluorescence assay showed that the average fluorescence intensity of Tf R1 and DMT1 protein expression was significantly weaker（P<0.05）,and the average fluorescence intensity of FPN1 and GPX4 protein expression was significantly stronger（P<0.05）.Compared with the DFO group,the content of Fe2+in the neurons of the BYHWDS group was significantly decreased（P<0.01）,the content of MDA was decreased（P<0.05）,and the contents of GSH and ROS were not significantly different（P>0.05）.Western blotting showed that there was no significant difference in the expression levels of Tf R1,DMT1,FPN1 and GPX4 proteins（P>0.05）.Immunofluorescence assay showed that there was no significant difference in the average fluorescence intensity of Tf R1,DMT1,FPN1 and GPX4 proteins（P>0.05）.Summary:Buyang Huanwu Decoction can reduce the expression levels of proteins Tf R1 and DMT1 in hypoxic hypoglycemic/reoxygenated HT22 cells,enhance the expression of FPN1 and GPX4,reduce the content of Fe²⁺in cells,reduce the production of ROS and MDA in cells,thereby inhibiting ferroptosis,alleviating damage to HT22 cells in OGD/R models,and promoting the recovery of brain tissue neural function.Conclusion:1.During the preparation of MCAO/R model in rats,the percentage of cerebral blood flow in ischemic side decreased by 41%to 50%was the best,and the MCAO/R model had high success rate,good repeatability and stability.2.Buyang Huanwu Decoction can reduce the expression of proteins Tf R1 and DMT1 in brain tissue,enhance the expression of FPN1 and GPX4,reduce the iron ion content in the brain tissue of the penumbra area on the ischemia-reperfusion side of rats,inhibit the reactive oxygen species produced by excessive iron participation in the Fenton reaction and the subsequent accumulation of toxic lipid peroxides causing ferroptosis,alleviate CIRI,and promote the recovery of neural function in brain tissue.3.Buyang Huanwu Decoction can reduce the expression levels of proteins Tf R1 and DMT1 in hypoxic hypoglycemic/reoxygenated HT22 cells,enhance the expression of FPN1 and GPX4,reduce the content of Fe²⁺in cells,reduce the production of ROS and MDA in cells,thereby inhibiting ferroptosis,alleviating damage to HT22 cells in OGD/R models,and promoting the recovery of brain tissue neural function.