| Background:With the change of life pace and the rapid arrival of the aging society,the epidemic trend of coronary heart disease combined with anxiety is becoming increasingly severe.Anxiety is an independent risk factor for the occurrence and development of coronary heart disease,which seriously affects its long-term prognosis.Our team has been working on the clinical and basic research of Chaihujialonggumuli decoction in the treatment of cardiovascular diseases combined with mental disorders.It has been confirmed that Chaihujialonggumuli decoction can significantly improve angina pectoris and anxiety in patients.However,it is not clear whether the blood components and their targets of Chaihujialonggumuli decoction are directly related to the inflammatory response induced by NLRP3/Caspase-1 pathway.Therefore,this study observed the correlation between the degree of TCM syndrome and inflammatory reaction and anxiety state in patients with coronary heart disease combined with anxiety.The blood components of Chaihujialonggumuli decoction were determined by high performance liquid chromatography.The core targets and action pathways of Chaihujialonggumuli Decoction were explored by integrated pharmacology and deep neural networks-quantitative structure-activity model(DNN-QSAR).To verify the partial intervention mechanism of Chaihujialonggumuli decoction on myocardial infarction complicated with anxiety by regulating NLRP3/Caspase-1 pathway through cell experiments,in order to provide evidence and support for different aspects of traditional Chinese medicine classic prescriptions and make up for the lack of treatment methods for coronary heart disease combined with anxiety.Objective:1.Through clinical research to clarify the correlation between the degree of TCM syndrome and inflammatory reaction,blood lipid level and anxiety state in patients with coronary heart disease combined with anxiety,and to further explore the internal logical relationship between the degree of TCM syndrome and inflammatory reaction and anxiety state in patients with coronary heart disease combined with anxiety.2.Through experimental study to clarify the blood components of Chaihujialonggumuli decoction and its effective material basis in vivo;At the same time,based on the blood components,combined with integrative pharmacology and DNN-QSAR learning model,the core targets and pathways of Chaihujialonggumuli decoction in the treatment of myocardial infarction with anxiety were deeply explored.To verify the intervention mechanism of Chaihujialonggumuli Decoction on inflammatory response after myocardial infarction by regulating NLRP3/Caspase-1 pathway at the cellular level.Methods:1.Study 1:A total of 120 patients with coronary heart disease and anxiety diagnosed by western medicine were selected.The TCM syndrome scale was used to quantify the degree of TCM syndrome,and the Pittsburgh sleep Quality Index(PSQI)was used to quantify the degree of sleep.Hamilton Anxiety scale(HAMA)score and symptom Checklist-90(SCL-90)score were used to quantify the degree of anxiety.The levels of Hs-CRP,TC and TG were detected by biochemical analyzer,and the level of IL-6 was detected by enzyme-linked immunosorbent assay.The correlation between TCM syndrome degree score and inflammatory factors levels of IL-6 and Hs-CRP,and the correlation between TCM syndrome score and TC and TG levels were analyzed.The correlation between TCM syndrome degree score and HAMA score,PSQI score and SCL-90 score;Correlation between the levels of IL-6 and Hs-CRP inflammatory factors and HAMA score,PSQI score and SCL-90 score.2.Study 2:The database of chemical components of Chaihujialonggumuli decoction was constructed based on our team’s previous research and literature retrieval.The components were deduced by using UPLC-LTQ-Orbitrap-MS/MS technology and Xcalibur software to compare the exact molecular weight,retention time and secondary characteristic fragment ions of the samples,and then compared with some standards for verification.According to the pharmacological strategy of biological prescription analysis,the active components of Chaihujialonggumuli decoction that can be absorbed into the blood of rats were qualitatively analyzed.3.Study 3:In this study,based on the blood components of Chaihujialonggumuli decoction confirmed by UHPLC-LTQ-Orbitrap-MS/MS,the key targets and core components of Chaihujialonggumuli decoction in the treatment of myocardial infarction with anxiety were screened by integrated pharmacology,and the PPI network was constructed and the key pathways were analyzed.In order to clarify the function of the intersection targets of Chaihujialonggumuli decoction and myocardial infarction with anxiety,GO enrichment analysis was performed on the intersection targets,and KEGG enrichment analysis was performed on the pathways.Then the ExCAPE-DB database was used to query the structural information and pIC50 values of the compounds corresponding to the core targets,and the Pytorch deep learning framework was used to construct the best DNN-QSAR model of Chaihujialonggumuli decoction in the treatment of myocardial infarction with anxiety.The pIC50 of its core components was verified by DNN-QSAR training model,and the effective components of ChaihujiaLongguMuli decoction in the treatment of myocardial infarction with anxiety were quantitatively analyzed.4.Study 4:H9c2 cardiomyocytes were cultured in vitro and randomly divided into three groups:model group,Chinese medicine group and SDF-1α group.The activity of H9c2 cardiomyocytes was detected by CCK-8 method.Western Blot was used to detect the expression of inflammatory proteins NLRP3,Caspase-1 and ASC related to the pyroptosis mechanism in the border zone of myocardial infarction.RT-qPCR was used to detect the expression of inflammatory genes NLRP3,Caspase-1,ASC and IL-1β in the border zone of myocardial infarction.Elisa was used to detect the content of IL-1β.The morphological changes of pyroptosis in H9c2 cells were detected by Tunel method.Results:1.Study 1:There was a significant positive correlation between TCM syndrome score and serum IL-6 and Hs-CRP levels in Coronary Heart Disease Complicated with Anxiety of Heart and Gallbladder Stasis-Heat Syndrome(r=0.836,P<0.01;r=0.945,P<0.01),and positively correlated with serum TC and TG levels(r=0.807,P<0.01;r=0.774,P<0.01).There was a significant positive correlation between the TCM syndrome degree score of CHD combined with anxiety and the scores of HAMA scale,PSQI scale and anxiety factor score of SCL-90 scale(r=0.880,P<0.01;r=0.804,P<0.01;r=0.841,P<0.01).The IL-6 and Hs-CRP levels were positively correlated with the scores of HAMA,PSQI and SCL-90(r=0.8 76,P<0.01;r=0.831,P<0.01;r=0.816,P<0.01)and(r=0.893,P<0.01;r=0.879,P<0,01;r=0.897,P<0.01).2.Study 2:Combined with the research results of previous teams,UPLC-LTQ-OrbitrapMS/MS technology and Xcalibur software,this study identified that Chaihujialonggumuli decoction mainly includes flavonoids,amino acids,organic acids,anthraquinones,alkaloids,benzylpropanoids,amides,and other compounds.Fifty-three prototype components were detected,mainly includes flavonoids,organic acids,amino acids,and anthraquinones.3.Study 3:A total of 448 targets of Chaihujialonggumuli decoction were identified according to the criteria in the Swiss Target Prediction database.A total of 83 intersections with myocardial infarction targets and anxiety targets were found in the Gene-Cards database.After screening,Chaihujialonggumuli decoction and myocardial infarction complicated with anxiety disease have 25 common targets.R-studio/R 3.6.3 was used to analyze the intersection of the target data of Chaihujialonggumuli decoction into blood and the target data of myocardial infarction with anxiety.The analysis results showedNLRP3,IL1β,IL18,CASP1,GSDMD,CXCL8,AKT1,ESR1,PPARG,TTR,PIK3CA,JAK2,BRAF,COMT,TLR4,MAPK1,CYP2C19,TLR9,ALDH2,PON1,and ACE,TXNIP,MYD88,P2RX7 and NLRP6,may be the most important targets of Chaihujialonggumuli decoction in the treatment of myocardial infarction with anxiety.GO enrichment analysis identified 84 cellular components,186 molecular functions,and 1989 biological processes.KEGG enrichment analysis identified 188 pathways,which were related to inflammation,oxidative stress,lipid and atherosclerosis,cytokine receptor interaction,etc.The DNN-QSAR models of the four core targets were constructed,and the pIC50 of the core components were verified.The results showed that the compounds of the core components were consistent with the compounds of Chaihujialonggumuli Decoction confirmed by UPLC-LTQ-Orbitrap-MS/MS technology.4.Study 4:CCK-8 results showed that the OD value of other SDF-1α concentrations was significantly lower than that of 100 ng/ml SDF-lα(P<0.01),indicating that H9c2 cardiomyocytes had the highest activity and proliferation rate under the intervention of 100 ng/ml SDF-1α.WB results showed that:Compared with the model group,the protein expression levels of NLRP3,Caspase-1 and A SC in the SDF-1α group were decreased,and the difference was statistically significant(P<0.01).Compared with the model group,the protein expression levels of NLRP3,Caspase-1 and ASC in the traditional Chinese medicine group were significantly decreased,and the difference was statistically significant(P<0.01).RTqPCR results showed that:Compared with the model group,the expression levels of NLRP3 mRNA,Caspase-1 mRNA,ASC mRNA and IL-1β mRNA in H9c2 cardiomyocytes in the SDF1α group were significantly decreased(P<0.01).The expressions of NLRP3 mRNA,Caspase1 mRNA,ASC mRNA and IL-1β mRNA in H9c2 cardiomyocytes in the Chinese medicine group were significantly decreased(P<0.01).ELISA results showed that compared with the model group,the levels of IL-1β in peripheral blood of the SDF-1α group and the Chinese medicine group were significantly decreased(P<0.01).Compared with the SDF-1α group,the level of IL-1α in peripheral blood of the Chinese medicine group was significantly decreased(P<0.01).The results of Tunel assay showed that:In the model group,the number of pyroptosis cells reached the peak,and the number of Tunel positive cells decreased in the SDF-1α group.After the intervention of serum containing the drug,only a few pyroptosis cells could be seen under the microscope,and the number of pyroptosis was significantly reduced(P<0.01).Conclusions and significance:1.Study 1:The present study found that there was a significant correlation between the degree of TCM syndromes and inflammation,blood lipid levels,and anxiety in patients with CHD and anxiety.With the increase of the degree of TCM syndrome,the inflammation,blood lipid related indicators and the degree of anxiety also increased.The above indicators can be combined to determine the occurrence of coronary heart disease combined with anxiety in clinical practice.In-depth study of the correlation between the degree of TCM syndrome and inflammatory reaction of coronary heart disease combined with anxiety and the related risk factors of the disease can provide reliable clinical basis for predicting the occurrence,treatment and prognosis of coronary heart disease combined with anxiety.2.Study 2:Based on the previous research results of our team,a total of 53 prototype components were identified by UHPLC-LTQ-Orbitrap-MS/MS technology combined with Xcalibur software,and the effective material basis of Chaihujialonggumuli decoction in vivo was confirmed,which provided a theoretical basis for the quality control and clinical application of traditional Chinese medicine compound.Study 3:The integrative pharmacological analysis predicted that Chaihujialonggumuli decoction could treat anxiety in MI patients through inflammatory response and oxidative stress.Through DNN-QSAR model prediction,deep machine learning was further performed on Chaihujialonggumuli decoction in the treatment of myocardial infarction with anxiety,so as to explore the effective components of the classic formula,and lay the foundation for subsequent in-depth research in animal experiments,cell experiments and molecular biology.Study 4:Chaihujialonggumuli Decoction can inhibit the inflammatory response related to pyroptosis in cardiomyocytes by regulating the NLRP3/Caspase-1 pathway. |
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