Exploring The Brain Protective Mechanism Of Di Dang Decoction On Acute Hemorrhagic Stroke Rats Via Quantitative Proteomics And Targeted Proteomics

Objective:Studies have shown that Chinese medicine has a positive effect on hemorrhagic stroke.Professor Ren Jixue put forward the method of"breaking blood and removing blood stasis"in the treatment of hemorrhagic apoplexy,and the clinical effect is remarkable.As a classical prescription for acute cerebral hemorrhage and apoplexy,Di Dang Decoction’s mechanism of action is still unclear.The aim of this study was to explore the mechanism of Di Dang Decoction（DDD）to protect the brain of acute cerebral hemorrhage and stroke rats by quantitative proteomics and targeted proteomics methods.Methods:First,the main active components of Di Dang Decoction were determined by HPLC-HRMS analysis.Then 135 healthy Sprague-Dawley（SD）rats were randomly divided into five groups:Control group（n=27）,Model group（n=27）,DDD low-dose group（n=27）,DDD medium-dose（n=27）and DDD high-dose group（n=27）,.They were given intragastric administration for 1,3 and 7 days respectively.The Control group and Model group were given equal dose of normal saline intragastric administration.The low,medium and high doses of Di Dang decoction were given 0.15625g/m L,0.3125g/m L and 0.625g/m L,respectively.The rat model of acute hemorrhagic stroke was induced by injecting autogenous blood into caudate nucleus.The neurologic deficit was evaluated by modified neurological deficit Score（m NSS Score）,hematoxylin-eosin staining（HE）,albumin concentration to assess the degree of BBB injury,and water content to assess the damage of brain tissue.Set the up-regulated differential protein threshold to>1.2 and down-regulated differential protein threshold to<1/1.2.Principal component analysis and Pearson correlation coefficient were used to test biological repeatability.The cerebral cortex of rats was extracted for quantitative proteomics detection.Quantitative identification of target proteins using targeted proteomics.Results:1.The main components of Di Dang Decoction:Gallic acid,Amygdalin,Sennoside B,Rhubarb glycoside 8-glucoside,Sennoside A,Emodin,Chrysophanol,Aloe Emodin,Rhein.2.MNSS Score showed that compared with the Model group（10.89±1.45）,the improvement of neurological deficit was most significant in the DDD high-dose at 7 days（6.22±1.20）,with statistical significance（P<0.05）.3.Hematoxylin and Eosin（HE）staining revealed obvious histopathological changes of cerebral hemorrhage in the Model group.The histopathological changes of the DDD low-dose,DDD medium-dose and DDD high-dose groups were significantly improved on the 3rd and 7th day,and the histopathological changes were most significant in the DDD high-dose group on the 7th day.4.The increased albumin concentration caused by hemorrhagic stroke was significantly decreased in the high-dose group of Di Dang Decoction for 7^th day,indicating that Di Dang Decoction could reduce the damage of BBB（blood-brain barrier）caused by cerebral hemorrhage,and the high-dose group of Di Dang Decoction itself had no effect on the integrity of blood-brain barrier,with statistical significance（P<0.05）.5.The results of brain water content showed that:Model group’s water content was higher than the Control group obviously,and used for DDD low-dose,DDD medium-dose and DDD high-dose after 1 day,3 days,7 days can reduce hemorrhagic stroke caused by higher areas of the brain water content,meantime the 7 days high dose group improve the most significant,that is worth when decoction high dose group of cerebral edema caused by hemorrhagic stroke has the very good improvement,the results were statistically significant（P<0.05）.6.The results of principal component analysis and Pearson correlation coefficient showed that protein quantification was consistent with biological repeatability test,and the results were statistically significant（P<0.05）.7.Quantitative proteomics results showed that compared with the Control group,S100a8 and S100a9 in the Model group were increased 3.316 times（P=0.014244）and 4.957times（P=0.000063）.It was decreased 0.614 times（P=0.033702）and 0.506 times（P=0.000024）,respectively.Compared with the Control group,Col1a1 and Col1a2 were decreased 0.677 times（P=0.000025）and 0.598 times（P=0.005657）in the Model group.Increased 1.319 times（P=0.000184）and 1.348 times（P=0.014097）,respectively,and the results were statistically significant（P<0.05）.8.By targeting proteomics results:The results of S100a8（P=0.033702）S100a9（P=0.000024）and Col1a1（P=0.000184）Col1a2（P=0.014097）were consistent with the trend of quantitative proteomics results,and the results were statistically significant（P<0.05）.Conclusions:In the high-dose group of Di Dang Decoction for 7 days,the neurological defects and brain histopathological changes of acute hemorrhagic stroke rats were significantly improved,and the integrity of blood-brain barrier was maintained and the edema of brain tissue was reduced in hemorrhagic stroke rats.It may be that down-regulation of S100a8,S100a9 and up-regulation of Col1a1,Col1a2 play a protective role in acute hemorrhagic stroke rats.