Study On The Inhibition Of Inflammatory Response After Subarachnoid Hemorrhage In Mice By Aloe-emodin, A Component Of Sanhua Decoction

Purpose: Etiologies of subarachnoid hemorrhage(SAH)include traumatic and spontaneous SAH,in which 75-80% of spontaneous SAH is caused by intracranial ruptured aneurysms.Although SAH only accounts for 5% of stroke,it is one of the neurological diseases that seriously threaten human health due to its high mortality rate and the average age of the disease,which is only 55 years old.Globally,the reported rates are higher in Japan and Finland,lower in South and Central America,and tends to be higher in women than men(1.24 times higher than men).Previous researchers believed that the cerebral vasospasm(CVS)is the main reason for the prognosis of SAH,but as the clinical study results of endothelial receptor antagonist Clazosentan(Conscious-2)were announced,the pathophysiological process of SAH have re-evaluated again.At present,researchers believe that early brain injury(EBI)played a key role in the pathophysiological process of SAH,which is the main cause of poor prognosis and also a critical time window for the treatment of SAH.In the period of EBI,SAH can induce many pathophysiological processes,including inflammatory response,nerve cell apoptosis,blood-brain barrier destruction and oxidative stress.Therefore,the research focus has turned to these pathophysiological processes of EBI.In traditional Chinese medicine,the symptoms caused by SAH are classified under the category of stroke and severe headache.Sanhua Decoction was first recorded in Liu Wan-su's writing Collections for Life-Saving from Plain Questions through its Pathomechanism Qi-Tonifying.The book contains a detailed description of stroke and the included formulas which are used to treat stroke are still widely used today.Sanhua Decoction is also one of the100 classical formulas in the “Catalogue of Ancient Famous Classical Formulas(the First Batch)” compiled by the State Administration of Traditional Chinese Medicine and the State Administration of Medical Products,and is recommended for the treatment of stroke.Network pharmacology,as one of the means of interpretation of the pharmacological action of Chinese medical,is based on the analysis and integration of the known natural chemical components contained in TCM formulas and their corresponding potential action targets.The purpose of Network pharmacology is to find information on the interaction among the candidate targets of the natural chemical components and the targets of disease,and then build a molecular network of the disease targets-drug targets.Finally,the topological characteristics of the network are analyzed and these drug targets are enriched and interpreted.Because Sanhua Decoction contains many chemical components and the pathophysiological mechanism of SAH is complex,also the number of disease targets involved is large,so network pharmacology is applied to screen the drug components of Sanhua Decoction and the main pathway for the treatment of SAH,in order to explain the mechanism of action,and eventually verify it in animal models.Finally,Aloe-emodin(AE),a natural anthraquinone compound,was screened out of Sanhua Decoction.Current studies have shown that anthraquinone compounds have various pharmacological effects such as alleviating cerebral ischemia/reperfusion injury,inhibiting inflammation,antiviral and anti-tumor,but there is still a lack of research reports on the therapeutic effect of AE in SAH.The purpose of this study is to investigate the role and mechanism of Sanhua Decoction and AE in the EBI of SAH.Methods: The first part of the experiment was to analyze the pharmacological effects of Sanhua Decoction by using network pharmacology.Firstly,the SAH disease database was constructed.The relevant targets of SAH disease were searched by using CTD database,DRUGBANK database,TTD database and DISGENET database.Secondly,the chemical composition of each single herb of Sanhua Decoction was searched in the TCMSP database,and the oral bioavailability and drug-like value of the corresponding drug components were sorted out.After that,Pub Chem database searched the Smiles structure formula of chemical components of each herb,and input the searched Smiles structure formula to Swinstargetprediction database and SEA database to find out the corresponding targets of each component of Sanhua Decoction and build the database.And the STRING database and Cytoscope software were used to obtain the protein-protein interaction data between SAH targets and drug targets,and the image was visualized and output.Finally,Genemania database and Metascape database were used to enrich and analyze the drug component targets of Sanhua Decoction.The second part of the experiment is to verify the results of network pharmacological analysis of Sanhua Decoction.Sanhua decoction was prepared with granule preparations of rhubarb,notopterygium root,fructus Aurantii Immaturus and cortex magnoliae officinalis,and the dosage of the drug was calculated.Specified pathogen free(SPF)mice of male C57BL/6strain were given intragastric administration twice a day.After 5 days of intragastric administration,SAH mouse model was established.The animal model was performed by internal carotid artery puncture,and the severity of SAH model was evaluated,mice with a score ?7 were excluded from the study.Mice were randomly divided into Sham group,Vehicle group,0.5×SHD,1×SHD and 2×SHD groups.The therapeutic effect of Sanhua Decoction on SAH model mice was determined by comparing the modified Garcia neurological function score,balance beam score,brain water content,expression of inflammatory cytokines TNF-? and IL-10 in SAH model group and drug treatment group before and after intragastric administration intervention.The result of network pharmacological analysis suggested that AE,a component of Sanhua Decoction,could inhibit the inflammatory response.Therefore,the third part of the experiment was to evaluate the efficacy of AE in the treatment of SAH animal models.AE was administered intraperitoneally,and the time points for evaluation were 24 hours and 72 hours after the modeling of SAH.The experimental subjects were male SPF mice of C57BL/6strain,which were divided into Sham group,Vehicle group,AE(10mg/kg)group,AE(20mg/kg)group and AE(40mg/kg)group at the 24 h time point after SAH.At 72 hours after SAH,they were divided into Sham group,Vehicle group and AE group(20mg/kg).The therapeutic effect of AE on SAH model mice was confirmed by comparing the modified Garcia neurological function score,balance beam score,brain water content,apoptosis of neurons,expression of tight junction protein ZO-1,Occludin,Iba-1,NF-?B and PKA/CREB pathway related proteins in disease model group and drug treatment group before and after AE intervention.Results:1.The results of network pharmacological analysis of Sanhua Decoction showed that a total of 334 targets with strong correlation with SAH were found in CTD database,DRUGBank database,TTD database and Disgenet database.A total of 55 drug components of Sanhua Decoction were selected from TCMSP database according to oral bioavailability and drug-like properties.Rhubarb has 16 chemical components;fructus Aurantii Immaturus has22 medicinal components;cortex magnoliae officinalis has 2 chemical components and there were 15 medicinal components among notopterygium root.A total of 127 targets were identified for drug components.And a total of 21 key targets of Sanhua Tang in the treatment of SAH were obtained by Merge plugin of Cytoscope software,among which ADORA1,ADORA2 A,NOS2 and PTGS2 were inflammatory targets.After the drug-targets of Sanhua Decoction were enriched and analyzed in Genemania database according to their functions,14 targets related to inflammation were obtained,including ALOX15,ADORA1,ADORA2 a,ADORA3,AOX1,CXCR1,KIT,NOS2,NOX4,NR1H3,PIK3 cg,PLA2G2A,PTGS2,and SYK.There were 7 targets involved in calcium ion transport,including DRD4,HTR2 B,OPRM,MYLK,Ca MK2 B,PLA2G1B and F2.There were 5 cell activation targets related to immune response,namely RORA,RORC,KIT,SYK and RIK3 CG.There were 7 targets related to protein kinase activity,including PLA2 GIB,INSR,CDK1,KIT,PKN1,ALK and DRD4.There were 4 targets involved in glial cell differentiation,namely F2,CDK1,CDK5 and CDK6.There are three targets associated with iron ion binding,namely FTO,ALOX5 and ALOX15.There are 5 targets involved in inflammatory cell activation,namely: PIK3 CG,SYK,KIT,RORC and RORA.The targets involved in inflammatory cell-mediated immune response are Kit,SYK,PIK3 CG,OIA2G1B and NOS2.There are 4 targets related to autophagy,which are AKT1,DAPK1,MAPT and HTR2 B.There were 9 targets involved in nerve regeneration,namely AKT1,F2,CAMK2 B,ACHE,OPRM1,MAPT,CDK1,CDK5,and CDK5R1.There were 6 targets involved in neuronal projection development,namely CAMK2 B,ACHE,AKT1,MAPT,CDK5 and CDK5R1.2.The verification results of network pharmacological analysis of Sanhua Decoction in mouse SAH model indicated that Sanhua Decoction could alleviate the neurological damage in the acute phase of SAH.Compared with the Vehicle group,1×SHD and 2×SHD groups improved the modified Garcia neurological function and balance beam test score,reduced the content of brain water,decreased the expression of inflammatory cytokine TNF-? and increased the expression of IL-10.3.Animal experiments proved that AE,the component of Sanhua Decoction,can reduce the nerve function injury of SAH: Compared with Vehicle group,the modified Garcia neurological function score at 24 and 72 h after SAH was improved in the AE group(20mg/kg).The balance beam test scores of AE(20mg/kg)and AE(40mg/kg)groups after 24 hours SAH were also improved.AE(20 mg/kg)group improved the balance beam experiment rating of 72 hours after SAH too.And the content of brain water decreased at 24 and 72 h after the intervention of AE.After 24 hours SAH,Western blot results showed that Cleaved caspase-3 and Cleaved PARP expression decreased in the AE(20mg/kg)and AE(40mg/kg)groups compared to the Vehicle group.TUNEL staining also indicated a decrease in the number of TUNEL positive neurons after AE intervention.After 24 hours of SAH,the results of the Western blot showed that after the treatment of AE,AE(10mg/kg)group,AE(20mg/kg)group and AE(40mg/kg)group were increased the expression of ZO-1 and Occludin but decreased the expression of Iba-1.The double-labeling immunofluorescence results showed that the number of Iba-1 and TNF-? double labeled cells decreased but the number of Iba-1and Arg-1 double labeled cells increased.After SAH treatment,the expression of NF-?B in cytoplasm increased,while the expression of NF-?B in nucleus decreased,and the expression of p-I?B? and p-IKK?/? decreased.Conclusions:1.Among the selected targets of pharmaceutical ingredients of Sanhua Decoction,14 targets were related to inflammation.And among the core targets,ADORA1,ADORA2 a,NOS2 and PTGS2 were related to inflammation.2.Sanhua Decoction alleviates the inflammatory response after SAH model by reducing TNF-? and increasing the expression of IL-10,which verifies the results of network pharmacological.3.AE,one of the components of Sanhua Decoction,inhibited the activation of microglia cells and promoted the transformation of microglia from M1 phenotype to M2 phenotype through NF-?B and PKA/CREB pathways,thereby alleviating the inflammatory response in early brain injury of SAH.