| Objective1.To detect the components of Miaoyao Tongqiao Huashuan Decoction by non-target metabolomics,and to explore the possible-mechanism-of Miaoyao Tongqiao Huashuan Decoction on cerebral ischemia-reperfusion injury based on network pharmacology research methods.2.To observe the clinical efficacy of Miaoyao Tongqiao Huashuan Decoction on acute ischemic stroke patients with wind-phlegm stasis after thrombolytic reperfusion injury and the changes of inflammation-related mechanism indicators.3.To explore the inflammatory mechanism of Miaoyao Tongqiao Huashuan Decoction on blood-brain barrier after thrombolysis reperfusion injury in acute ischemic stroke rat model.Methods1.Network pharmacology research partThe results of non-target metabolomics were input into the Chinese Medicine database(YaTCM)to obtain the drug components,and the active ingredients were screened according to the absorption rate and drug-like(OB≥30%,DL≥0.18).The action targets of drug active ingredients were obtained from Pharmaceutical Chemistry database(ChEMBL)and Traditional Chinese Medicine Immuno-oncology database(TCMIO),and a data set of drug active ingredients action targets was established.GeneCards(?):The Human Gene Database and the Human Gene and Gene Phenotype Database(OMIM)were used to analyze the disease targets,and the genes and corresponding protein targets related to cerebral ischemia-reperfusion injury were obtained to establish a disease target dataset.The intersection of the above two data sets was used to analyze the target of drug active ingredients and disease targets by Gene Ontology(GO)database and Kyoto Encyclopedia of genes and Genomes(KEGG)Pathway database and KEGG Pathwa analysis.At the same time,the protein-protein interaction(PPI)in the STRING database Homo sapiens(human)was used to construct the network interaction map of target proteins and the network interaction map of active ingredient-target-pathway.2.Clinical researchAccording to the inclusion and exclusion criteria,patients with acute ischemic stroke of wind-phallus-stasis type were randomly divided into western medicine group and miao medicine+western medicine group.The western medicine group was treated with conventional western medicine,and the miao medicine+western medicine group was treated with conventional western medicine combined with Miaoyao Tongqiao Huashuan Decoction for 14 days.The relevant clinical data were collected,and the baseline data of general conditions(age,weight,course of disease,gender,past history,personal history)and baseline data of efficacy indicators(NIHSS score of neurological deficit degree,Barthel score of daily living ability,mRS Score of disability degree,TCM syndrome score)before treatment were observed.The therapeutic indexes(NIHSS,Barthel,mRS And TCM syndrome scores)at different time points before and after treatment were observed in the two groups,and the serum Tumor necrosis factor-α(TNF-α)and Interleukin-6(IL-6)were detected by ELISA.IL-6),Nuclear Factor-KB(NF-κB),Matrix metalloproteinase-9(MMP-9),Aquaporin Protein-4(aquaporin protein-4,Nf-κb),and MMP-9.AQP4)levels,and the clinical efficacy,inflammation related indicators and safety of the two groups were evaluated after treatment.3.Experimental research partSPF SD male rats were randomly divided into sham operation group,model group,thrombolysis group,and miao medicine+thrombolysis group.The rat autologous thromboembolism model was prepared.After 14 days of corresponding treatment,the neurobehavioral score,brain water content,cerebral infarction volume,cerebral hemorrhage transformation and blood-brain barrier permeability were measured.ELISA was used to detect the changes of IL-1β,IL-6,TNF-α levels in brain tissue of each group.WB was used to detect the mitogen-activated protein kinase kinase 4(Recombina)in brain tissue of each group nt Mitogen Activated Protein Kinase Kinase 4,MAP2K4),MMP-9,NF-κB and AQP4.Results1.Network pharmacology research partAfter screening,95 active ingredients of Miaoyao Tongqiao Huashuan Decoction were obtained,corresponding to 1567 targets,and 496 disease targets of cerebral ischemia-reperfusion injury,and 214 common targets were obtained after intersection.The main targets of Miaoyao Tongqiao Huashuan Decoction,such as MAPT,GBA and XBP1,are related to inflammatory mechanisms,including IL,TNF-α and MMP-9,AQP-4,mitogen-activated protein kinase(MAPK)and NF-κB.The biological process(BP)of Miaoyao Tongqiao Huashuan Decoction on cerebral ischemia-reperfusion injury had 3192 entries,mainly related to cellular immune response,oxidative stress,active peptides and serine metabolism.There are 85 pathways of its action,mainly including lipid and atherosclerosis pathways,neurotrophin signaling pathways,sphingolipids signaling pathways,etc.,and it has a certain correlation with inflammatory response,apoptosis and calcium signaling pathways.2.Clinical researchA total of 100 patients were enrolled,and 95 patients completed this study,including 47 patients in the western medicine group and 48 patients in the miao medicine+western medicine group,except for 5 patients who dropped out or were excluded.The baseline data of general conditions and efficacy indicators showed that the two groups were comparable before treatment(P>0.05).The results of efficacy measures were analyzed as follows:(1)There was no significant difference in NIHSS score between the two groups immediately after thrombolysis and 24 hours after thrombolysis(P>0.05).14 days after thrombolysis,compared with the western medicine group,the NIHSS score of miao medicine+western medicine group decreased,and the difference was statistically significant(P<0.05).(2)There was no significant difference in Barthel score 24 hours after thrombolysis between the two groups(P>0.05).Compared with the western medicine group,the Barthel score of the miao medicine+western medicine group increased 14 days after thrombolysis,and the difference was statistically significant(P<0.01).(3)There was no significant difference in mRS Scores between the two groups at 24 hours and 14 days after thrombolysis(P>0.05).(4)According to the analysis of TCM syndrome score,compared with the western medicine group,the TCM syndrome score of miao medicine+western medicine group decreased 14 days after thrombolysis,and the difference was statistically significant(P<0.01).(5)Compared with the western medicine group,the total effective rate of the miao medicine+western medicine group increased 14 days after thrombolysis,and the difference was statistically significant(P<0.01).(6)ELISA results:There was no significant difference in the levels of TNF-α,IL-6,MMP-9,AQP4 and NF-κB between the two groups at 24 hours after thrombolysis(P>0.05).14 days after thrombolysis,compared with the western medicine group,the levels of the above indicators in the miao medicine+western medicine group were decreased,and the differences were statistically significant(P<0.01 or P<0.05).(7)From the analysis of safety evaluation,during the treatment,there were no significant differences in the levels of blood routine,liver and kidney function related indicators between the two groups at 24 hours and 14 days after thrombolysis.The D-D level of the miao medicine+western medicine group was lower than that of the western medicine group 24 hours after thrombolysis,and the Fg level was higher than that of the western medicine group 14 days after thrombolysis.In the western medicine group,there was 1 case of minor nasal hemorrhage and 1 case of intracranial hemorrhagic transformation(both occurred within 24 hours after thrombolysis),which were considered to be related to the adverse reactions of rt-PA.No related adverse reactions and adverse events were found in the miao medicine+western medicine group.3.Animal studies100 rats were randomly divided into sham operation group,model group,thrombolysis group and miao medicine+thrombolysis group,with 25 rats in each group.The number of dead rats in each group and the mortality rate were:sham operation group,no death,the mortality rate was 0%;There were 2 deaths in the model group,with a mortality rate of 8%.Three rats died in the thrombolysis group,with a mortality rate of 12%.In the miao medicine+thrombolysis group,4 rats died,with a mortality rate of 16%.There was no difference in body weight between the groups before treatment,which was comparable.The results of efficacy measures were analyzed as follows:(1)Compared with the sham-operation group,the neurobehavioral scores of the model group,the thrombolysis group,and the miao medicine+thrombolysis group were significantly increased before treatment,indicating that the model was successfully established in each group(P<0.01),and there were no significant differences among the other groups(P>0.05).Compared with the model group,the scores of thrombolysis group and miao medicine+thrombolysis group decreased significantly at 24 hours and 14 days after treatment(P<0.05 or P<0.01).Compared with the thrombolysis group,the scores of the miao medicine+thrombolysis group decreased 24 hours after treatment and 14 days after treatment(P>0.05).(2)Compared with the sham-operation group,the brain water content of the model group,the thrombolysis group,and the miao medicine+thrombolysis group were significantly increased(P<0.01),and there was no significant difference among the other groups(P>0.05).Compared with the model group,the thrombolysis group and the miao medicine+thrombolysis group had a significant reduction in brain water content at 24 hours and 14 days after treatment(P<0.01).Compared with the thrombolysis group,the brain water content of the miao medicine+thrombolysis group decreased significantly 24 hours after treatment and 14 days after treatment(P<0.01).(3)Compared with the model group,there was no significant difference in the volume of cerebral infarction before treatment in the thrombolysis group and the miao medicine+thrombolysis group(P>0.05).The volume of cerebral infarction was significantly reduced 24 hours and 14 days after treatment in the thrombolysis group and the miao medicine+thrombolysis group(P<0.01).Compared with the thrombolysis group,there was no significant difference in the cerebral infarction volume before treatment and 24 hours after treatment in the miao medicine+thrombolysis group(P>0.05),but the cerebral infarction volume was significantly lower 14 days after treatment than the thrombolysis group(P<0.01).(4)In the analysis of hemorrhagic transformation,compared with the sham-operation group,the hemoglobin concentration in the model group,the thrombolysis group,and the miao medicine+thrombolysis group was significantly increased before treatment(P<0.01),and there was no difference among the other groups(P>0.05).Compared with the model group,the thrombolysis group and the miao medicine+thrombolysis group had a significant reduction in cerebral hemoglobin concentration at 24 hours and 14 days after treatment(P<0.01).Compared with the thrombolysis group,the cerebral hemoglobin concentration in the miao medicine+thrombolysis group decreased significantly 24 hours after treatment and 14 days after treatment(P<0.05).(5)BBB permeability analysis:compared with the sham-operation group,the EB concentration in the model group,thrombolysis group,and miao medicine+thrombolysis group was significantly increased before treatment(P<0.01),and there was no significant difference among the other groups(P>0.05).Compared with the model group,the concentration of EB in the thrombolytic group and the miao medicine+thrombolytic group decreased significantly 24 hours and 14 days after treatment(P<0.01).Compared with the thrombolytic group,the EB concentration of the vaccine plus thrombolytic group decreased 24 hours after treatment and 14 days after treatment(P>0.05).(6)Analysis of ELISA resultsCompared with the sham-operation group,the IL-1β level in the model group,the thrombolysis group,and the miao medicine+thrombolysis group was significantly increased before treatment(P<0.01),and there was no significant difference among the other groups(P>0.05).Compared with the model group,the levels of IL-1β in the thrombolysis group and the miao medicine+thrombolysis group were significantly decreased 24 hours and 14 days after treatment(P<0.01).Compared with the thrombolysis group,there was no significant difference in IL-1 β level at 24 hours after treatment in the vaccine plus thrombolysis group(P>0.05).IL-1 β levels were significantly decreased in the miao medicine+thrombolysis group 14 days after treatment(P>0.05).Compared with the sham-operation group,the levels of IL-6 and TNF-α in the model group,the thrombolysis group,and the miao medicine+thrombolysis group were significantly increased before treatment(P<0.01),and there was no significant difference among the other groups(P>0.05).Compared with the model group,the levels of IL-6 and TNF-α in the thrombolysis group and the miao medicine+thrombolysis group were significantly decreased 24 hours and 14 days after treatment(P<0.01).Compared with the thrombolysis group,the levels of IL-6 and TNF-α in the miao medicine+thrombolysis group decreased 24 hours and 14 days after treatment(P>0.05).(7)Analysis of WB detection resultsCompared with the sham-operation group,the expression of MAP2K4,MMP-9 and NF-κB protein in the model group,thrombolysis group and miao medicine+thrombolysis group were significantly increased before treatment(P<0.01),and there were no significant differences among the other groups(P>0.05).0.05).Compared with the model group,the protein expressions of the above indexes in the thrombolysis group and the miao medicine+thrombolysis group were significantly decreased at 24 hours and 14 days after treatment(P<0.01).Compared with the thrombolysis group,there was no significant difference in the protein expression of the above indicators in the miao medicine+thrombolysis group at 24 hours after treatment(P>0.05),but the protein expression of the above indicators in the miao medicine+thrombolysis group was significantly decreased at 14 days after treatment(P<0.05).AQP-4 protein expression analysis showed that compared with the sham-operation group,the expression of AQP-4 protein in the model group,the thrombolysis group,and the miao medicine+thrombolysis group before treatment was significantly increased(P<0.01),and there was no significant difference among the other groups(P>0.05).Compared with the model group,the expression of AQP-4 protein in the thrombolysis group and the miao medicine+thrombolysis group was significantly decreased at 24 hours and 14 days after treatment(P<0.01).Compared with the thrombolysis group,the expression of AQP-4 protein in the miao medicine+thrombolysis group decreased 24 hours after treatment and 14 days after treatment(P>0.05).Conclusion1.The mechanism of Miaoyao Tongqiao Huashuan Decoction in the intervention of cerebral ischemia-reperfusion injury may be achieved by directly or indirectly acting on multiple inflammatory signaling pathways,which has certain research value.2.The conventional western medicine therapy,Miaoyao Tongqiao Huashuan Decoction combined with conventional western medicine therapy,these two kinds of treatments are effective methods in the clinical treatment of acute ischemic stroke,it can improve the wind resistance type thrombolysis in patients with phlegm and blood stasis after brain ischemia-reperfusion injury of nerve function defect and degree of disability,can reduce the performance of syndromes and improve the ability of daily life,and safety and reliable.3.After 14 days of treatment,Miaoyao Tongqiao Huashuan Decoction combined with conventional western medicine therapy was superior to conventional western medicine therapy alone in improving the degree of neurological deficit after cerebral ischemia-reperfusion injury,reducing TCM syndrome manifestations,and improving neurological function and activities of daily living in patients with acute ischemic stroke of wind-phsputum stasis type.4.The levels of TNF-α,IL-6,NF-κB,MMP-9 and AQP-4 were down-regulated by conventional western medicine therapy,Miaoyao Tongqiao Huayshuan Decoction combined with conventional western medicine therapy.After 14 days of treatment,the down-regulation effect of Miaoyao Tongqiao Huashuan Decoction combined with conventional western medicine therapy was superior to conventional western medicine therapy was better than that of conventional western medicine alone.5.Miaoyao Tongqiao Huashuan Decoction can improve the degree of neurological deficit,reduce brain edema,reduce the volume of cerebral infarction,and reduce cerebral hemorrhagic transformation after cerebral ischemia-reperfusion injury in model rats.6.Miaoyao Tongqiao Huashuan Decoction can down-regulate the level of IL-β and the protein expression of MAP2K4,MMP-9 and NF-κB in the brain tissue of the model rats after thrombolysis.7.In addition to the basic treatment of conventional western medicine,Miaoyao Tongqiao Huashuan Decoction can improve the neurological deficit after thrombolysis cerebral ischemia-reperfusion injury in patients with acute ischemic stroke of wind-phlegm stasis type,and promote the repair of brain tissue and nerve tissue.The mechanism may be:Miaoyao Tongqiao Huashuan Decoction can regulate the changes of TNF-α,IL-6,NF-κB,IL-β,MMP-9,AQP4 and MAP2K4 to inhibit the inflammatory response after thrombolysis cerebral ischemia-reperfusion injury,reduce the damage of blood-brain barrier,reduce brain edema,reduce the volume of cerebral infarction and cerebral hemorrhage transformation. |
