| Purpose:To predict the therapeutic target of improving cardiac function by using network pharmacology method.Further animal experiments were conducted to verify the effect of the prescription to replenish qi and activate blood circulation on improving cardiac function,and transcriptome methods were used to clarify and verify the mechanism of the prescription to protect the cardiac function of the model rats with qi deficiency and blood stasis.Through the systematic study of this prescription,more healthy diet options and ideas were provided for the clinical treatment of cardiac dysfunction.Material and method:The active components and their corresponding molecular targets of the prescription were obtained from the pharmacological database and analysis platform of Chinese medicine system(TCMSP).Then,the target proteins related to cardiac dysfunction were obtained from Disgenet database.After the intersection of the two,the target of Buqi Huoxue Formula for the treatment of chronic cardiac dysfunction was obtained.David database was used to enrich the functions of the above targets,and the KEGG and GO enrichment information was obtained.Based on this,the potential mechanism of this prescription in the treatment of chronic cardiac insufficiency was predicted.A total of 180 SPF SD rats with half male and half female were randomly divided into 6 groups: control group,model group,positive drug group,and Buqi Huoxue prescription high,medium and low groups.Except for the blank group,the model was made by the combined stimulation method of hunger,fatigue and cold,and drug intervention was also given.The experiment lasted for60 days.The changes of body weight,heart shape,hemorheology and blood items were measured during the experiment.At the same time,the contents of CK,CK-MB,α-HBD,LDH1 and AST2 in serum of rats were analyzed during treatment.Finally,we obtained the improvement of the pathological morphology and blood index of rats by the formula of invigorating qi and activating blood circulation.In order to further obtain the mechanism of reinforcing qi and activating blood formula to improve the syndrome of rats with qi deficiency and blood stasis.In this study,transcriptome sequencing was performed on the heart tissues of rats in the blank group,model group,high-dose drug group and positive control group to obtain the differentially expressed genes in each group.After functional enrichment of the above genes,the potential mechanism of BQHUO formula to improve cardiac insufficiency of Qi-deficiency and blood-stasis syndrome was further determined from the perspectives of signal pathway(KEGG)and biological function(GO).Validate the results obtained from network pharmacology and provide potential therapeutic targets for further mechanism studies.Results:1.Network pharmacology:From the TCMSP database,141 active components of the prescription for replenishing qi and promoting blood circulation,a total of 2035 potential therapeutic targets and 289 unique molecular targets were obtained,and a total of 86 public targets were overlapping with the targets of inner dysfunction in the disease database.After analyzing and visualizing the protein interaction relationships of the above targets(using the STRING database),the regulatory networks of the above targets were obtained.Among them,there were 9 core targets with high centrality,such as MAPK14,Fos,etc.After functional enrichment of the above targets,it was speculated that the prescription of invigorating qi and activating blood circulation might pass the effect.Signaling pathways such as HIF-1 signaling pathway and VEGF signaling pathway improve cardiac dysfunction.2.Zoological experiments:According to the results,during the study,except for the blank group,the body weight of the other groups of rats showed a significant downward trend(P<0.01),under drug intervention,the body weight of rats in the BQHUOXUE formula group was higher than that in the model group,however,there was no statistical difference among the three groups(P > 0.05).3.Tissue blood flow results showed:Compared with the blank group,the renal and tail blood flow in the model group decreased significantly before administration,but increased gradually after administration(P < 0.01 or P<0.05).Renal blood flow: Compared with the blank group,renal blood flow in the model group decreased significantly on day 20,40 and 60(P<0.05);On day 20,there was no significant difference in renal blood flow between the positive control group and the medium and low dose group of BQHUOXUE formula compared with the model group,but the renal blood flow in the high dose group of BQHUOXUE formula was significantly increased compared with the model group(P < 0.01).Compared with model group,renal blood flow of positive control group and BQHUO formula at 40 and 60 d was significantly increased(P<0.05).Liver blood flow: Compared with the blank group,liver blood flow of 20 d model group showed an increasing trend(P>0.05),there was no significant difference in liver blood flow between the positive drug group and the high,middle and low dose Buqi Huoxue Formula group compared with the model group(P>0.05),there was no statistical significance;Compared with blank group,liver blood flow of model group was significantly increased at 40 d and 60d(P<0.01),the liver blood flow of rats in the high,medium and low dose groups of invigorating qi and promoting blood circulation and the positive control group were significantly decreased compared with the model group(P<0.01).Tail blood flow: Tail blood flow of model group was significantly decreased compared with blank group on day 20,40 and 60(P<0.01),the high,middle and low dose groups(except the medium dose group of40 d and 60d)and the positive control group were significantly higher than the model group(P<0.05).4.The results of hemorheology showed:When whole blood viscosity was 20 days,compared with model group,blood viscosity of high dose prescription group and blank group increased significantly at high and low shear rate(P<0.05);Compared with blank group,model group at 40 d and 60 d decreased significantly(P<0.05),at 40 d,the high shear rate in each administration group was significantly higher than that in the model group(P<0.05),60 d positive control group and high dose group were significantly higher than model group at each shear frequency(P<0.05),the low-dose formula group was significantly higher than model group at high shear rate(P<0.05).Plasma viscosity: After 20 days,there was no significant difference in plasma viscosity among the groups(P>0.05),compared with blank group,model group was significantly improved on 40 and 60 days(P<0.05),the prescriptions in high,middle and low dose groups were significantly decreased compared with model group(P<0.05).The hemorheology was improved in the drug group,and the improvement was more obvious with the increase of the dose.5.The four results of coagulation showed that:At day 20,there was no significant difference in indicators among all groups(P>0.05);At 40 d,compared with the blank group,PT in the model group was significantly prolonged(P<0.01),the content of FIB decreased significantly(P<0.05);Compared with the model group,the PT in the high-dose group of invigorating qi and activating blood circulation significantly shortened(P<0.05).Compared with the blank group,PT and APTT were significantly prolonged in the 60 d model group(P<0.01),the content of FIB decreased significantly(P<0.01);Compared with model group,PT and APTT were significantly shortened in positive drug group and high,middle and low dose group of Buqi Huoxue Formula(P<0.05),FIB was significantly increased except for the low-dose group of invigorating qi and promoting blood circulation and the positive control group(P<0.05).6.Echocardiogram results showed that:Compared with blank group,EF,FS and SV of model group on 40 and 60 days were significantly decreased(P<0.01).Compared with model group,EF,FS and SV of high dose group and positive drug group were significantly increased at 40 days(P<0.01),EF in low-dose group increased significantly(P<0.05);Compared with model group,EF,FS and SV of high,middle and low dose prescription group and positive drug group were significantly increased at 60d(P<0.01).The effect of cardiac function was significantly improved in the drug group.7.Myocardial enzyme spectrum of biochemical indexes showed:At 40 d and 60 d,the contents of CK,CK-MB,α-HBD,LDH1 and AST2 in model group were significantly increased compared with blank group(P<0.01).Compared with model group,the contents of CK,CK-MB,α-HBD,LDH1 and AST2 in positive control group and prescription high-dose group were significantly decreased on 40 d and 60d(P<0.05);The contents of CK,α-HBD and AST2 in low-dose group decreased significantly(P<0.05),the contents of CK and CK-MB were significantly decreased on day 60(P<0.05).The model group had a tendency towards myocardial infarction,and the drug group effectively alleviated the necrosis of myocardial cells.8.HE staining results showed that:The myocardial tissue of blank group was normal structure,the lines of myocardial cells were close and clear,and the cell structure was complete.No vascular dilation or infiltration of inflammatory cells were observed in the interstitium.In the model group,myocardial cells were disordered in arrangement and obvious fibrosis in the space.The damage degree of myocardial cells in the low-dose group was significantly reduced compared with the model group,and the cell structure was generally orderly with a small amount of fibrosis changes,but there was no significant formation of large fibrous lesions.The degree of myocardial fibrosis was further reduced in the middle and high dose groups,and no significant fibrosis was observed.9.Transcriptome sequencing results showed that:Compared with the blank group,there were 547 differentially regulated genes in the model group,156 of which were up-regulated and 391 of which were down-regulated.A total of 192 differentially regulated genes were found in model group and prescription group,with 48up-regulated genes and 144 down-regulated genes.Compared with the positive control group,160 differential genes were regulated in the model group,40 of which were up-regulated and120 of which were down-regulated.P< was obtained after enrichment analysis of differential regulatory genes between model group and drug group.There are 27 signal pathways of 0.05,and the intersection targets will be obtained by targeting with network pharmacological prediction targets and regulatory differential targets,a total of 3 targets,namely FOS,FASN and SPPZ1.Among them,Fos was the core prediction target,and 9signaling pathways were obtained by intersecting the core KEGG pathway predicted by network pharmacology with the signal pathways enriched by regulatory differential genes,of which 4 pathways were involved by Fos.It is indicated by TNF signaling pathway,Measles(Measles),and Estrogen signaling pathway,among which Estrogen signaling pathway is closely related to cardiac dysfunction.Conclusion: In this study,induced by many factors such as cold,fatigue and hunger,the model rats of Qi deficiency and blood stasis under low coagulation state were successfully reconstructed,and the model rats were excavated through the changes of blood flow in the model rats,and the cardiac insufficiency model of rats caused by Qi deficiency and blood stasis was successfully found by the increase of myocardial enzyme spectrum indexes.Under the effect of the prescription of invigorating qi and activating blood,the indexes of coagulation function,hemorheology,tissue blood flow,heart function,myocardial enzyme and so on in the model rats with qi deficiency and blood stasis were significantly improved,which proved that the prescription had good pharmacodynamic effect.The results of transcriptome sequencing were compared with the previous network pharmacological prediction results,which proved that the predicted targets FOS,FASN and SPP1 played an important regulatory role in the protection of cardiomyocytes.It is further speculated that the prescription of invigorating qi and activating blood regulates the synthesis of Estrogen by regulating the expression of signaling proteins such as FOS and KRT14 on the Estrogen signaling pathway,so as to achieve the purpose of improving cardiac function.The regulation of FASN increases the number of new lipids.By regulating Toll-like Receptor Signaling Pathway under the action of FOS and SPP1,the inflammatory response caused by the main functional immune response can be induced,in which CCL5 is significantly down-regulated and the inflammatory damage of cardiomyocytes can be reduced.Together,they can improve cardiac insufficiency. |
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