Study On The Mechanism Of Action Of Buyang Huanwu Decoction-containing Plasma In Improving OGD/R Injury In PC12 Cell

Objective:The aim of this study is to observe the effect of Buyang Huanwu Decoction containing plasma on the repair of neuronal injury in ischemic stroke,and to explore the mechanism of its repair effect from the perspective of iron homeostasis.Methods:1.18 SPF SD male rats were randomly divided into 6 groups,and were given Buyang Huanwu Decoction at very low,low,medium and high doses,deferiprone tablets and the same amount of0.9%normal saline by gavage,1 m L/100 g,once a day,respectively.The median dose of Buyang Huanwu Decoction was 25.8 g/kg（based on the daily dose of crude drugs for adults of60 kg,the dose for rats was converted to body surface area,which was twice the clinical equivalent dose for humans）.The concentration ratio of very low,low,medium and high dose was 0.5:1:2:4.After continuous gavage for 7 days,blood samples were collected from the abdominal aorta 1 hour after the last administration,anticoagulant with 3.8%sodium citrate（1:9）,centrifuged at 4℃and 3000 r/min for 15 min to remove the supernatant,filtered and sterilized through a 0.22μm well screen,and stored at-80℃for later use.The plasma of rats in each drug administration group was mixed with the same amount of plasma as drug-containing plasma,and the plasma of rats in the blank group was mixed with the same amount of plasma as blank plasma.2.The cell culture medium was changed to the sugar-free medium with hypoxia mixture（94%N₂+5%CO₂+1%O₂）for 30 min.Then the cells were immediately placed in an airtight chamber with hypoxia mixture and cultured at 37℃with saturated humidity.Drug intervention was performed 6 h after OGD injury.3.After normal culture,the cells were divided into normal group,model group,blank plasma group,Buyang Huanwu Decoction containing plasma group（very low,low,medium and high dose）,a total of 7 groups.The model was induced by Oxygen-glucose deprivation（OGD）in a three-gas incubator.After modeling,the cells in each group were treated with the same volume of medium containing 10%plasma.After 24 hours of continuous culture,the cell viability was detected to screen the optimal drug concentration.After the optimal concentration of Buyang Huanwu Decoction was determined,the cells were divided into 4 groups:normal group,model group,optimal plasma containing Buyang Huanwu Decoction group and deferiprone group.The OGD model was established by using a three-gas incubator.The normal group and the model group were cultured with basal medium containing 10%blank plasma,and the optimal drug-containing plasma group of Buyang Huanwu Decoction and deferiidone group were cultured with basal medium containing 10%respective drugs,respectively.After 24 hours of drug intervention,the indexes were detected.4.The cell morphology of each group was observed,and the damage repair of the cell model after drug intervention was compared.CCK-8 was used to detect the cell viability,and ROS assay was used to compare the cell damage among groups.Superoxide dismutase（SOD）and malondialdehyde（MDA）were used to determine the degree of oxidative damage.The expression levels of iron metabolism transporters TF,HEP,TFR and FPN were detected by Western blot.Results:1.Compared with the normal group,the cell viability of the model group was significantly decreased（P<0.01）.Compared with the model group,the cell viability of blank plasma group had no significant difference,while the cell viability of each dose of plasma containing Buyang Huanwu Decoction group was significantly increased（P<0.05）.Compared with the low dose group,the cell viability of the medium dose and high dose drug containing plasma groups decreased significantly（P<0.01）,and the cell viability of the very low dose group showed a downward trend,and there was no statistically significant difference.2.In the normal group,the morphology of the cells was regular,two or more levels,the cell body was large and obvious,and the dendrites were entangled with each other.Compared with the normal group,the cells in the model group were significantly damaged,the number of cells was significantly reduced,the morphology was disorderly,the length of dendrites was significantly sparse,and the cell viability was significantly reduced（P<0.01）.Compared with the model group,the low-dose group and deferiprone group had significantly improved cell damage,significantly increased cell number,obvious regular morphology,increased dendrites,and significantly increased cell viability（P<0.01）.3.Compared with the normal group,the cell damage in the model group was obvious,the content of ROS increased sharply（P<0.01）,the activity of SOD decreased significantly（P<0.01）,the peroxide damage was obvious,and the content of MDA increased significantly（P<0.01）.Compared with the model group,the content of ROS in the low dose group and deferiprone group decreased significantly（P<0.01）,the activity of SOD increased significantly（P<0.05）,and the content of MDA decreased significantly（P<0.01）.There was no significant difference between low dose group and deferiprone group（P>0.05）.4.Compared with the normal group,the protein expression levels of TF,TFR and HEP in the model group were significantly increased（P<0.01）,and the protein expression level of FPN was significantly decreased（P<0.01）.Compared with the model group,the relative expression levels of TF,TFR and HEP proteins in the low dose group and deferiprone group were decreased（P<0.05）,and the expression level of FPN protein was increased（P<0.01）.There was no significant difference between low dose group and deferiprone group（P>0.05）.5.Compared with the normal group,the OD value of iron fluorescence in the model group was significantly increased（P<0.01）.Compared with the model group,the low dose group and deferiprone group had varying degrees of decreased OD value of iron fluorescence（P<0.01）.There was no significant difference between low dose group and deferiprone group（P>0.05）.6.Compared with the normal group,the apoptotic fluorescence area in the model group was significantly increased,the area was extensive,the fluorescence was obvious,and the apoptotic index was significantly increased（P<0.01）.Compared with the model group,the apoptosis fluorescence and apoptosis index in the low dose group and deferiprone group were significantly decreased（P<0.01）,and the difference was statistically significant.There was no significant difference between low dose group and deferiprone group（P>0.05）.Conclusion:1.Buyang Huanwu Decoction can improve the neuronal damage caused by ischemic stroke.2.Buyang Huanwu Decoction may play a role in improving cerebral ischemic neuronal injury by regulating iron metabolism-related proteins,inhibiting the relative expression of TF,TFR and HEP,reducing iron ion input,promoting the production of FPN,promoting iron deposition and efflux after neuronal injury,and increasing the antioxidant capacity of cells.