Chemical Composition Analysis And Protective Mechanism Of Shuanghe Xingshen Decoction In The Treatment Of Ischemic Stroke By Regulating TNF Signaling Pathway

Background:Stroke is an important cause of death and disability among adults in China,especially ischemic stroke,the incidence and prevalence of which are increasing year by year,with a consequent increase in mortality and per capita medical costs.Currently,ischemic stroke has become a serious threat to people’s physical and mental health,adversely affecting the quality of life.Therefore,the prevention and treatment of ischemic stroke have become an important issue that needs to be addressed.Western medicine treats ischemic stroke mainly by reducing disability and recurrence.On the one hand,it treats by intravenous thrombolysis and mechanical embolization,but it has a clear time window for treatment.On the other hand,relapses are mainly reduced by antiplatelet,antihypertensive and lipid-lowering treatments,but there are still problems of drug resistance and poor compliance.Chinese medicine has the advantages of no time window,low drug resistance,broad indications,low cost,wide audience,and multi-target and multi-pathway onset of action in the treatment of ischemic stroke.It was found that phlegm and blood stasis play an important role in the pathogenesis of ischemic stroke.Therefore,this study was carried out by using Shuanghe Xingshen Decoction(SHXSD),which is a modification of Shuanghe Decoction,to further clarify the therapeutic effect and mechanism of action of SHXSD in improving ischemic stroke.Aim:1.Characterization of the chemical composition of Shuanghe Xingshen Decoction(SHXSD)using ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-QTOF/MS).2.Screening of the core components of SHXSD for ischemic stroke(IS)based on UHPLC-QTOF/MS results,combined with network pharmacology and molecular docking techniques to analyze the mechanism of action of SHXSD.3.To verify the neuroprotective effect of SHXSD on IS rats by animal experiments and to validate the core targets with high docking scores from the screened molecules at the protein level.Materials and methods:1.Thermal reflux extraction was used to obtain the samples of SHXSD to be tested.The UHPLC-QTOF/MS technique was used to characterize the chemical profile of SHXSD,and the molecular mass information of the excimer ion peaks provided by high-resolution mass spectrometry was used to identify and resolve the chemical components of the compound.2.The active ingredients of SHXSD were screened using Calculation of molecular properties and bioactivity score database by the Lipinski’s rule of five.The Swiss Target Prediction database was used to predict the targets of action of the active ingredients.The OMIM database,Gene Cards database,Dis Ge NET database and Drug Bank database were used to obtain ischemic stroke-related aims.Venn diagram analysis was used to obtain the intersection targets of drug genes and disease genes.Screen of core clusters of SHXSD for ischemic stroke by protein interaction analysis(PPI)and MCODE plug-in analysis of Cytoscape 3.9.0 software.Predict the mechanism of action of core clusters using KEGG pathway enrichment analysis and GO functional enrichment analysis.Molecular docking techniques were used to assess the binding interaction between the active ingredient and the core target in the core cluster.3.The ischemic stroke rat model was constructed using the Middle Cerebral Artery Occlusion(MCAO)model method.The modeled rats were subjected to LONGA behavioral score and TTC staining to initially evaluate the success of the model,and a LONGA score of 1 to 3 was considered successful.60 SD rats were randomly divided into four groups(n=15)Sham-operated(Sham)group: 14 days of continuous saline(1ml/100g),MCAO group: 14 days of continuous saline(1 ml/100g),MCAO+SHXSD group: 14 days of continuous SHXSD(1 ml/100g),and ischemic stroke model +edaravone(MCAO+EDA)group: EDA(3mg/1kg)was injected intraperitoneally for 14 days after modeling.The modified neurological deficit score(mNSS)was recorded on days 1,3,7 and 14 after modeling,to further validate the therapeutic effect of SHXSD on ischemic stroke.To evaluate the protective effect of SHXSD on neurological damage in hippocampal and cortical brain tissues in ischemic stroke by observing morphological changes in hippocampal and cortical brain tissues of rats through HE staining and Nissler staining.Validation of key targets predicted by network pharmacology analysis and molecular docking analysis by protein blotting(Western Blot,Wb)was performed to analyze the mechanism of action of SHXSD for IS.Results:1.572 chemical constituents of SHXSD were identified by UHPLC-QTOF/MS technique,and the main constituents of SHXSD were flavonoids,esters,organic acids,phenylpropanoids,and terpenoids.2.Based on the 572 chemical components obtained by mass spectrometry,265 active components were screened using the Lipinski class V principle,and 1294 targets of the 265 active components were obtained from the drug prediction target database.The disease target database was used to predict 4880 targets related to ischemic stroke.The intersection targets of drug-acting targets and disease-related targets were obtained by Venn diagram analysis,and a total of 816.Combined with PPI analysis and Cytoscape 3.9.0 software’s MCODE plug-in analysis,20 key clusters were obtained,and the score ranking of key clusters was obtained based on gene tightness correlation calculation to screen the core clusters,and the genes of the obtained core clusters were screened for 182 core components,76 core therapeutic targets and 20 related signaling pathways using KEGG pathway enrichment and GO enrichment analysis.The molecular docking results showed that MMP9,MMP3 and p-IKBα were relatively more strongly bound to the core components of SHXSD.3.Longa score and TTC staining showed that the neurological function score was reduced and the cerebral infarct area was reduced in the MCAO+SHXSD and MCAO+EDA groups compared with the MCAO group,and SHXSD had a good therapeutic effect for the acute phase of IS.The results of mNSS scores showed that on days 1,3,7 and 14 after modeling,the neurological deficits of rats in the MCAO group were significantly impaired,and the neurological function scores of rats in the MCAO+SHXSD and MCAO+EDA groups were significantly lower than those in the MCAO group,and the neurological function was significantly better.4.HE staining results showed that in the cortical and hippocampal tissues of the Sham group,the cells were normal in structure,regular in arrangement,with intact nuclei and clear borders.In the MCAO group,the neurons in the ischemic hemispheric cortical and hippocampal tissues of rats were atrophied,with deep-stained nuclei and obvious interstitial space.Compared with the MCAO group,the MCAO+SHXSD and MCAO+EDA groups had fewer interstitial vacuoles in brain tissue,less fixed atrophy,and significantly less brain tissue damage,with statistically significant differences(p <0.05).Nissler staining results showed that the pyramidal neurons in the Sham group showed blue staining,normal cell morphology,tight and regular arrangement,and dark blue coarse and dense granules of Nissler vesicles.The MCAO group,the MCAO+SHXSD group and the MCAO+EDA group showed a significant decrease in niosomes compared with the Sham group;the number of niosomes increased in the MCAO+SHXSD group and the MCAO+EDA group compared with the MCAO group,and the difference was statistically significant(p < 0.05).Western blot experiments showed that compared with the Sham group,the protein expression of MMP9 and MMP3 in the MCAO groupwas significantly increased and the expression of p-IKBαwas significantly decreased,while the protein expression of MMP9 and MMP3 was down-regulated(p<0.05)and p-IKBα was up-regulated in the MCAO+SHXSD and MCAO+EDA groups.Conclusions:1.UHPLC-QTOF/MS technology can be used to identify the chemical components in SHXSD and reveal the material basis of its efficacy.2.Network pharmacology analysis can be used to describe the active ingredients and overall mechanism of action of SHXSD in the treatment of ischemic stroke from the perspective of ‘multiple components-multiple targets-multiple pathways’.3.SHXSD can improve the neurological damage caused by ischemic stroke,and its protective mechanism is related to the regulation of inflammatory response mediated by MMP9,MMP3 and p-IKBα.