Study On The Protective Mechanism Of Xuefu Zhuyu Decoction On Ischemic Myocardium And Clinical Observation On The Treatment Of Stable Angina Pectoris Based On Network Pharmacology

Objective:1、Explore the mechanism of protective effect of Xue Fu Zhu Yu Decoction on ischemic myocardium through animal and cellular experiments。2.To observe the clinical efficacy of Xue Fu Zhu Yu Decoction on stable angina pectoris（Qi stagnation and blood stasis type）,and to evaluate its effectiveness and safety;and to determine the serum levels of ROS,SOD and MDA in patients。Methods1.Experimental studies1.1Network pharmacology:The active ingredients of Xue Fu Zhu Yu Decoction were screened by TCMSP,TCMID and BATMAN-TCM databases,matched with the predicted disease targets from Drug Bank,Gene Card,OMIM and Disgenet databases to obtain key genes,and then GO and KEGG analyses were performed to predict the mechanism of action of Xue Fu Zhu Yu Decoction.Finally,animal and cellular experiments were applied to validate the predicted mechanism.1.2 Animal experiment:40 clean grade female C57BL/6 mice were used for the experiment and randomly divided into 4 groups:control group（Control group）,model group（ISO group）,model group+XFZY low dose group（ISO+XFZY-Low Dose）,model group+XFZY high dose group（ISO+XFZY-High Dose）,where the control group was given Physiological saline 5mg/kg/d and isoprenaline 5mg/kg/d in the model group,applied for 14days.The model group+XFZY low dose group（ISO+XFZY-Low Dose）and the model group+XFZY high dose group（ISO+XFZY-High Dose）were given 10g/kg and 20g/kg of Xue Fu Zhu Yu Decoction,Respectively,by gavage twice daily,while the control group and the model group were given equal amounts of saline by gavage.The cardiomyocyte morphology was observed by HE staining;apoptosis of cardiomyocytes was observed by TUNEL staining;cTnI,CK,CK-MB,LDH,SOD,MDA and cGMP were measured by ELASA method;Bax,Bcl,MDA and cGMP were measured by western blot for Bax,Bcl-2,cleaved-Caspase-3,PKG protein expression;PCR for Bax,Bcl-2,cleaved-Caspase-3expression.1.3 Preparation of drug-containing serum:5 female C57BL/6 mice were fed normally for 3 days and then gavaged for 10 days with Xue Fu Zhu Yu Decoction,the equivalent dose of the drug in mice was equivalent to 0.0025 times that in humans according to the human/animal body surface area conversion ratio table.g/kg was administered by gavage.The supernatant was removed by centrifugation at 3000 r/min for 15 min and inactivated in a water bath at 56°C for 30 min.The blood was filtered through a 0.22μm microporous membrane to remove bacteria and stored at-20°C.1.4 Cellular experiments:Cells were cultured under anaerobic conditions（94%N₂,5%CO₂and 1%O₂）and maintained at 37°C for 6 hours,and maintained at 37°C for 6 hours.Reoxygenation conditions were 95%O₂,5%CO₂for 6 hours.The cell concentration was adjusted to 1×10 5cells/m L and inoculated in 96-well plates with 100μl per well for 24h.H9C2 cardiomyocytes were randomly divided into 4 groups:blank group:H9C2 cell group;model group:OGD/R group;low dose group:OGD/R group+5%drug-containing serum group;high dose group:OGD/R group+15%drug-containing serum group;CCK-8 was used to detect cell activity;TUNEL method was used to detect apoptosis in cardiac muscle tissue and calculate the apoptosis rate of cardiac muscle cells;flow cytometry was used to detect apoptosis and ROS content;ELASA method was used to detect LDH,SOD,MDA,cGMP content;western blot was used to detect Bax,Bcl-2,cleaved-Caspase-3,PKG1 The protein expression of Bax,Bcl-2,cleaved-Caspase-3 and PKG1 was detected by western blot.2.Clinical research:Sixty-two patients with stable angina pectoris who attended the outpatient clinic of the Third Department of Cardiovascular Medicine at the First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine between January 2022and July 2022 and were suffering from Qi stagnation and blood stasis.The patients were randomly divided into 31 cases in the control group and 31 cases in the treatment group.The control group was treated according to the standard Western medical protocol;the treatment group was given Xue Fu Zhu Yu Decoction on the basis of the control group for a duration of 4 weeks,with one patient in each group being excluded due to failure to follow the prescribed requirements for medication.The actual completion was 30 cases.The TCM symptom score and angina pectoris score were compared before and after treatment,and the changes of ROS,MDA and SOD levels in the serum of the patients were detected before and after treatment,and the safety of the drugs was monitored.Results1.Network pharmacology:We obtained 80 target genes corresponding to 179 active ingredients of Blood Mansions and Stasis Removal Tang from TCMSP,TCMID and BATMAN-TCM databases;6273 disease targets from four databases,namely Drug Bank,Gene Card,OMIM and Disgenet;after combining the two,we obtained intersecting target genes 64.PPI network construction and topology analysis after String database showed that there were ESR1,PTGS2,NR3C1,GSK3B,PPARG,MAOB,ADRB2,NOS3,MAPK14,MAOA,AR,ADRA2A,PTGS1,ACHE,DRD1,ESR2,OPRM1,DPP4.GO analysis showed that the biological processes that affect the biological function based on P value include:circulatory vascular function,regulation of microtubule size,regulation of vascular size,regulation of microtubule size,etc;the cellular component CC（Celluar component）includes Molecular function（MF）includes:dopamine binding,steroid hormone receptor activation,G protein-coupled receptor activity,nuclear receptor activity,transcription factor activity,neurotransmitter receptor activity,etc;KEGG analysis identified neuroactive ligand-receptor interactions,calcium signalling pathways,cGMP-PKG signalling pathways and other pathways.2.Animal experiment2.1 HE staining:The cardiomyocytes in the blank group were long,neatly arranged and appeared blue-purple after HE staining,and the cytoplasm appeared pink.Compared with the blank group,the myocardial cells in the model group were thicker,more disorganized,with looser interstices and vacuole formation,and inflammatory cell infiltration could be seen in the intercellular matrix.After treatment with Xue Fu Zhu Yu Decoction,both the low-dose group and the high-dose group showed improvement in the above pathological manifestations compared with the model group.2.2 TUNEL:Apoptosis index in the blank group was:6.06±0.88%;apoptosis index in the model group was:33.90±2.22%;apoptosis index in the low-dose group was:24.68±0.68%;apoptosis index in the high-dose group was:20.80±0,61%;apoptosis index in the model group increased significantly compared with the blank group（P<0.05）;apoptosis index in the low-dose and high-dose groups decreased significantly compared with the model group（P<0.05）.The apoptosis index of the model group was significantly increased compared with that of the blank group（P<0.05）;compared with that of the model group,the apoptosis index of the low-dose and high-dose groups was significantly reduced（P<0.05）.2.3 ELISA:cTnI,CK,CK-MB,LDH,MDA levels were significantly higher in the model group compared with the blank group（P<0.05）;SOD,cGMP levels were significantly lower（P<0.05）;cTnI,CK,CK-MB,LDH,MDA levels were significantly lower in the low and high dose groups compared with the model group（P<0.05）;SOD and cGMP levels were significantly higher in the low and high dose groups（P<0.05）2.4 Westen blot:Bax and Cleaved-caspase3 protein expression in myocardial tissues of the model group was significantly increased compared with the blank group（P<0.05）;Bcl-2 and PKG1 protein expression was significantly decreased compared with the model group（P<0.05）;Bax and Cleaved-caspase-3 protein were significantly increased in the low and high dose groups compared with the model group（P<0.05）;Bcl-2 and PKG1 were significantly increased compared with the model group（P<0.05）.were significantly lower（P<0.05）;Bcl-2 and PKG1 were significantly higher（P<0.05）.2.5 RT-PCR:Bax gene expression in myocardial tissue was significantly higher in the model group compared with the blank group（P<0.05）;Bcl-2 gene expression was significantly lower（P<0.05）;Bcl-2 gene expression was significantly higher in the high-dose group compared with the model group（P<0.05）,but not statistically significant in the low-dose group（P>0.05）;Bax gene expression was significantly lower in the low-dose and high-dose groups compared with the model group（P<0.05）.Bax gene expression was significantly decreased in both the low-dose and high-dose groups compared with the model group（P<0.05）;caspase-3 gene expression was not significantly different between the groups（P>0.05）;3.Cellular experiment3.1 CCK-8 assay cell activity:H9c2 cells in the blank group were densely grown in a long shuttle shape against the wall;cell morphology in the model group changed significantly,with irregular morphology visible under the microscope;cell density and morphology in the drug-containing serum group improved;cell activity in the blank group was 0.65±0.10,in the model group:0.40±0.08,in the low-dose group:0.53±0.07,in the high-dose group:0.62±0.1.The cell activity in the model group decreased significantly compared with the blank group（P<0.05）;cell activity in the low-dose and high-dose groups recovered significantly compared with the model group（P<0.05）;3.2 Apoptosis detection by flow cytometry:apoptotic cells were significantly higher in the model group compared with the blank group（P<0.05）;apoptosis was significantly reduced in the low and high dose groups compared with the model group（P<0.05）;3.3 ROS content by flow cytometry:intracellular ROS content was significantly higher in the model group compared to the blank group（P<0.05）;ROS content was significantly lower in the low and high dose groups compared to the model group（P<0.05）,and3.4 TUNEL experiment:apoptosis of H9c2 cells was significantly increased in the model group compared with the blank group（P<0.05）;apoptosis was significantly improved in the low-dose and high-dose groups compared with the model group（P<0.05）;3.5 ELISA:LDH and MDA levels were significantly increased in the model group compared with the blank group（P<0.05）;SOD and cGMP levels were significantly decreased（P<0.05）;LDH and MDA levels were significantly decreased in the low-dose and high-dose groups compared with the model group（P<0.05）;SOD and cGMP levels were significantly increased（P<0.05）3.6 Westen blot:compared with the blank group,the expression of Bax and Cleaved-caspase-3 in myocardial tissue of rats in the model group was significantly increased（P<0.05）;the expression of Bcl-2 and PKG1 was significantly decreased（P<0.05）;compared with the model group,the expression of Bax and Cleaved-caspase-3 in the low and high dose groups was significantly decreased（P<0.05）;compared with the model group,the expression of Bax and Cleaved-caspase-3 in the low and high dose groups was significantly decreased（P<0.05）.3 protein expression was significantly lower（P<0.05）;Bcl-2 and PKG1 protein expression was significantly higher（P<0.05）.3.7 RT-PCR:Bax gene expression in myocardial tissue was significantly higher in the model group compared with the blank group（P<0.05）;Bcl-2 gene expression was significantly lower（P<0.05）;Bcl-2 gene expression was significantly higher in the low-dose group and the high-dose group compared with the model group（P<0.05）;Bax gene expression was significantly lower in both groups（P<0.05）;caspase-3 gene expression was not significantly different between the groups（P>0.05）;4.Clinical studies4.1 Chinese medicine symptoms:The Chinese medicine symptoms included chest pain,chest tightness,shortness of breath,palpitations and fullness in the chest.There was no statistically significant difference in the TCM symptom scores between the control group and the treatment group before treatment（P>0.05）;the TCM symptom scores in the treatment group decreased significantly compared with the control group after the administration of Xue Fu Zhu Yu Decoction（P<0.05）;the effective rates of the TCM symptoms in the control group and the treatment group were 70%and 96.7%respectively,which were statistically significant（P<0.05）;4.2 Efficacy of angina pectoris:The evaluation of the efficacy of angina pectoris included four aspects:the amount of anginal activity triggered,the frequency of angina pectoris,the duration of angina pectoris and the dosage of nitroglycerin.There was no statistical significance in the points of angina symptoms before treatment in the two groups（P>0.05）;the points of angina activity,frequency of angina and duration of angina in the treatment group were significantly lower than those in the control group after the administration of Xue Fu Zhu Yu Decoction（P<0.05）;however,there was no statistical significance in the comparison of nitroglycerin dosage between the two groups（P>0.05）.4.3 Serum levels of ROS,SOD and MDA:The levels of ROS,SOD and MDA in the serum of patients in both groups before treatment were not statistically significant（P>0.05）;the levels of ROS and MDA of patients in the treatment group were significantly lower than those in the blank group after the application of Xue Fu Zhu Yu Decoction（P<0.05）and the levels of SOD were significantly higher than those in the blank group（P<0.05）.Conclusion1.The network pharmacological analysis revealed that Xue Fu Zhu Yu Decoction may affect cardiomyocyte apoptosis by regulating the cGMP-PKG signaling pathway2.Xue Fu Zhu Yu Decoction can reduce cardiomyocyte injury caused by chronic ischemia;3.Xue Fu Zhu Yu Decoction reduced cardiomyocyte apoptosis by inhibiting Bax,Cleaved-caspase-3 and increasing Bcl-2 protein expression,the mechanism of which may be related to the regulation of cGMP-PKG signaling pathway.4.Xue Fu Zhu Yu Decoction can improve the efficacy of TCM symptoms in patients with stable angina pectoris;increase the activity tolerance of patients with stable angina pectoris,reduce the frequency of angina pectoris and the duration of angina pectoris attacks,and has a good safety profile.5.Xue Fu Zhu Yu Decoction can reduce ROS and MDA levels and increase SOD levels in the serum of patients with stable angina pectoris.