A Metabonomic Study On The Protective Effect Of Mongolian Medicine Eerdun Wurile In CIRI Rats Brain

Objective:To simulate the process of ischemic stroke in human body by successfully establishing the rat middle cerebral artery focal cerebral ischemia—reperfusion（MCAO/R）model,and to observe the changes of endogenous metabolites in the plasma of rats with cerebral ischemia-reperfusion injury after the intervention of Erdun Wurile,and to reveal the pathogenesis of CIRI,and the preventive effect and mechanism of Eerdun Wurile.Methods:（1）Seventy two SPF grade SD male rats were randomly divided into sham operation group,model group and Mongolian medicine Eerdun Wurile low,middle and high groups,and 12 rats in each group were in the positive control group.The sham-operated group and model group were given sodium carboxymethylcellulose（CMC-Na）by gavage,while the low,middle and high groups of Mongolian medicine were given Mongolian medicine Eerdun Wurile by gavage for 7 days,and the drug volume was 10 m L/kg.One hour after intragastric administration on the seventh day,the MCAO/R rat model was established in the model group,the low,middle and high groups of Mongolian drug Eerdun Wurile,and the positive control group.The rats in the sham operation group were only isolated from the right common carotid artery without thread insertion and embolism treatment.The effect of different doses of Mongolian drug Eerdun Wurile on the cerebral infarction volume of CIRI rats was observed by TTC staining;HE staining was used to observe the effect of different doses of Eerdun Wurile on the histopathology of rat cortex.（2）Collect the serum samples of rats in each group,and analyze the plasma of rats IL-6 and TNF-αby immunohistochemistry.（3）Through LC-MS metabolomics analysis,the plasma endogenous metabolites of experimental animals in each group were compared,and the interaction and influence mechanism between such substances were further explored.Pattern recognition analysis of data was carried out by means of multivariate statistical analysis,and the differences in metabolic profiles between different groups were compared;Chromatographic conditions:the column was ACQUITY UPLC（?）HSS T3（2.1×150 mm,1.8μm）with a flow rate of 0.25 m L/min.Elution mode:gradient elution,column temperature at40℃,injection volume of 2μL.In the positive ion mode,the mobile phase was 0.1%formic acid acetonitrile（B2）and 0.1%formic acid water（A2）.In the negative ion mode,the mobile phase consisted of acetonitrile（B3）and 5 m M ammonium formate water（A3）Ms conditions were as follows:positive ion spray voltage was 3.50 k V,negative ion spray voltage was-2.50k V,sheath gas was 30 arb,auxiliary gas was 10 arb.The capillary temperature was 325℃,the first stage full scanning was performed with a resolution of 70000,the first stage ion scanning range was m/z 100-1000,and the second stage lysis was performed with HCD,the collision energy was 30 e V,the second stage resolution was 17500,and the first 10 ions were cleaved.（4）Compared with the sham operation group,the changes of endogenous metabolites in the serum of rats in the model group were observed,and the metabolic disorders caused by ischemic stroke were analyzed;（5）Compared with the model group,the metabolic changes in the plasma samples of rats in the intervention group were analyzed under the action of Mongolian drug Eerdun Wurile;（6）Based on the KEGG database,the use of variable importance projection（VIP）value combined with T test to screen the metabolites with different expression under the intervention of Mongolian drug Eerdun Wurile and conduct metabolic pathway enrichment analysis to study the effect of Mongolian drug Eerdun Wurile on the metabolic disorder of ischemic stroke and specific metabolic pathway.Results:（1）The cerebral infarction in the model group was evident when compared to the sham operation group;Eerdun Wurile’s prevention of cerebral infarction in rats was significantly less than in the model group.;（2）HE results showed that compared with the sham operation group,the cortical neurons in the model group had significant changes,including pyknosis and hyperchromatic nuclei;The pyknosis and nuclear hyperchromia of cortical neurons in rats treated with low,middle and high doses of Mongolian drug Eerdun Wurile were alleviated;Compared with the low dose Eerdun Wurile,the high dose Eerdun Wurile has a more obvious reduction effect.（3）Compared with rat serum levels of IL-6 and TNF-αlevels:The model group exhibited a significant rise in serum levels of IL-6 and TNF-αwhen compared to the sham operation group（P<0.01）;conversely,the Eerdun Wurile middle and high groups experienced a significant decrease in these two hormones（P<0.01）.（P<0.01）.（4）Based on LC-MS metabonomics platform,metabolite profile analysis was carried out according to PCA,OPLS-DA and other pattern recognition analysis methods.The results showed that the quality of model establishment was good,and the metabolic profile of each group was well distinguished;（5）Compared with the model group,the model group down-regulated oxaloacetic acid,Isocitric acid,Dodecanoic acid,Panthenol,Palmitic acid,Linoleic acid,Sphingosine,Inositol,Lipoxygen A4,Prostaglandin J_2,and up-regulated S-adenosyl-L-methionine,succinic acid,N（pi）-methyl-L-histine,（15Z）-tetracarbenoic acid,D-gluconate-1,5-lactone,etc.（6）After administration of Mongolian medicine Eerdun Wurile,Isocitric acid,Inositol,Aconitic acid,Lipoxygen A4 and Prostaglandin J₂were up-regulated（P<0.05）.Conclusion:（1）The high,middle and low doses of Mongolian medicine Eerdun Wurile have the protective and therapeutic effects on CIRI model rats,and the high dose has the best effect on CIRI model rats,while the low dose group has the worse effect.（2）Mongolian medicine Eerdun Wurile can up-regulate Isocitrate,N-acetylserotonin,Indole-3-acetaldehyde,Uric acid,Lipoxylate A4,Acetylcholine,and Prostaglandin J₂and down-regulate Hypoxanthine and Glutaric acid,indicating that Mongolian medicine Eerdun Wurile can reduce inflammatory reaction and cell necrosis.Regulate energy metabolism,amino acid metabolism,lipid metabolism disorders to play a brain protective role.The above results were consistent with the traditional therapeutic effect of Eerdun Wurile in the treatment of“Bai Mai”injury.