| Coronary artery disease(CAD)is a serious cardiovascular disease that threatens human health and lives worldwide,with mortality rates increasing every year.Acute coronary syndrome(ACS)is a serious type of CAD and a major cause of death in patients with coronary artery disease,with almost half of all deaths occurring after ACS.The formation and rupture of plaque in the walls of coronary vessels is the pathological basis for the development and progression of ACS.Acute coronary syndrome belongs to the category of "chest paralysis" in Chinese medicine.Stasis and toxicity are important pathological mechanisms of coronary artery disease,and "stasis and toxicity"is a pathological condition that includes both "blood stasis" and "toxicity".On the one hand,"stasis and toxicity" is a pathological state that includes the coexistence of "blood stasis" and "toxicity",where "toxicity" is defined as a more harmful disease evil;on the other hand,it refers to blood stasis that breeds toxicity over time.Animal models are an important vehicle for the in-depth study of disease mechanisms.Compared to the single-factor "disease" models used in Western medicine,the combined disease and evidence models of Chinese medicine better reflect the characteristics of the "evidence" of Chinese medicine and are relatively closer to clinical practice,making them an important tool for the study of the foundations of Chinese medicine and the development of new drugs.There are various ways to establish animal models of coronary artery disease based on different TCM evidence types,but so far there are few studies on animal models of coronary artery disease with stasis and toxin interconnection.Our team has successfully established the rat model of coronary heart disease with Qi deficiency and blood stasis,and the small pig model of coronary heart disease with phlegm and blood stasis,and has rich experience in the research and operation of the animal models of disease and evidence combination.In this experiment,we will try to establish a model of acute coronary syndrome by combining coronary artery ligation with lipopolysaccharide(LPS)injection for the first time,based on the biological characterization of stasis and toxin interconnection evidence summarized by the clinical study of the project group.This formula is a representative formula for the clinical treatment of ACS.It is derived from the formula of the Chinese medicine master Chen Keji,who has experience in the treatment of acute myocardial ischaemia-"Gui Ji Tong Yu Tang",which has the effect of benefiting Qi,resolving turbidity,activating blood circulation and detoxifying.Therefore,in this study,the formula was used to test the reliability of animal models and to investigate the mechanism of action of this formula in activating blood circulation and detoxifying the blood to protect against ACS.Part Ⅰ.Establishment of a rat model of acute coronary syndrome with stasis and toxin interconnection1.ObjectivesBased on the pathophysiological characteristics of ACS and the biological characteristics of clinical ACS,we compared three modelling methods,namely singlefactor modelling,toxin followed by stasis modelling and stasis followed by toxin modelling,at different modelling times,to explore the optimal modelling method for the rat model of acute coronary syndrome with stasis and toxin.2.MethodsThe SD male rats were randomly divided into four groups:(1)sham-operated group;(2)single-factor modeling group,where the coronary arteries were ligated alone;(3)toxicity followed by stasis modeling group,where the coronary arteries were ligated 24 h after intraperitoneal injection of lipopolysaccharide(LPS);(4)stasis followed by toxicity modeling group,where the coronary arteries were ligated 10 min after intraperitoneal injection of LPS.The evaluation indexes were set based on the "Clinical Diagnostic Criteria of Stasis and Toxicity in Ischemic Cardiovascular Disease".After 3 h,24 h and 3 d of modeling,the rats were observed for general status,tongue and pulse signs and other TCM symptoms,and myocardial ischemia was monitored by ECG,cardiac function was detected by cardiac ultrasound,myocardial ischemia and infarct area was detected by myocardial Evans blue/TTC staining,myocardial enzymes CK,CK-MB,LDH and troponin T were detected to evaluate the degree of myocardial injury,platelet aggregation rate,coagulation rate,and myocardial blood flow were detected.Platelet aggregation rate,four coagulation tests(APTT,PT,TT,FIB)and blood rheology were used to assess the level of blood stasis.The level of leukocytes and serum inflammatory factors IL-1β and IL-6 were measured to assess the degree of "toxicity"and hematoxylin-eosin(HE)staining was used to observe the morphology of the myocardium.3.ResultsAfter 3 h of modelling,the pulse amplitude,tongue R,G and B values of the rats in the one-factor modelling group decreased significantly compared with those in the sham-operated group,the colour of the sublingual veins deepened,the ST segment elevated,the cardiac function decreased,the levels of serum inflammatory factors IL1β and IL-6,cardiac enzymes CK,LDH and troponin T increased significantly,and the total number of white blood cells and myocardial infarct area increased significantly.HE staining showed that the myocardium of the sham-operated group was neatly arranged,and there was a small amount of inflammatory cell infiltration in the interstitial space of the myocardium of each model group,but the difference between the sham-operated and sham-operated groups was not significant.After 24 h of modeling,the pulse amplitude,tongue R,G,B values and cardiac function of the rats in the one-factor modeling group decreased significantly compared with those in the sham-operated group,and the sublingual complex grew and became darker.levels were increased.Compared to the one-factor modeling group,the pulse amplitude and lingual R,G and B values decreased,the sublingual veins became darker and longer,the cardiac EF and FS decreased,and the cTnT level increased in the prestasis and post-stasis modeling groups.The results showed that the myocardium of the sham-operated group was neatly arranged with few slight gaps,while the myocardial fibres of each model group were loosely arranged with mild breaks and a large number of inflammatory cell infiltrations were present;the inflammatory cell infiltrations were more pronounced in the toxicity followed by stasis modeling group and the stasis followed by toxicity modeling group than in the one-factor modeling group.After 3 d of modelling,compared with the sham-operated group,the one-factor modelling group showed a significant decrease in pulse amplitude,a decrease in tongue R,G and B values,a thickening and growth of the sublingual ligament,a decrease in cardiac function,an increase in cTnT content,an increase in fibrinogen content,an increase in platelet aggregation rate and whole blood viscosity,and an increase in serum inflammatory factors IL-1β and IL-6 levels;compared with the one-factor modelling group,the sublingual ligament in the first-virus followed by stasis modelling group and the first-stasis followed by toxicity modelling group showed a significant increase in inflammatory cell infiltration.In addition,the pulse amplitude,tongue R,G and B values,cardiac EF and FS scores decreased significantly and the area of myocardial infarction increased significantly in the first-stasis-followed-by-poison modeling group.HE staining showed that myocardial fibres were better arranged in the sham-operated group,while myocardial fibroblasts and fibroblasts were proliferated and neutrophils were widely distributed in each model group.4.SummaryFrom the perspective of the duration of modelling,the whole blood viscosity and platelet aggregation rate of the models prepared by the three modelling methods did not change significantly after 3 h of modelling compared to the sham-operated group at low,medium and high shear rates,which were not fully consistent with the characteristics of blood stasis.After 24 h of modeling,compared with the sham-operated group and the other two model groups,the rats in the stasis followed by toxicity modeling group showed a significant decrease in cardiac function and a significant increase in serum inflammatory factors and platelet aggregation rate.After 3 d of modeling,the serum myocardial enzyme levels in the three model groups were not statistically different from those in the sham-operated group,indicating that the disease started to move from the acute phase to the recovery phase after 3 d of modeling,which was inconsistent with the pathological characteristics of acute myocardial ischemia and the sudden onset of the symptoms of stasis and toxicity.Therefore,the rat model established by using the method of stasis before toxicity(LPS injection 10 min after coronary artery ligation)and modelling for 24 h is more consistent with the symptoms and characteristics of acute coronary syndrome with stasis and toxicity.Part II Effect of LPS injection dose on the rat model of acute coronary syndrome with stasis and toxin interconnection1.ObjectiveTo observe the effect of LPS injection dose on the establishment of acute coronary syndrome model based on the screening of LPS moulding method after coronary artery ligation,and to explore the optimal moulding dose.2.MethodsSD male rats were randomly divided into 5 groups:(1)sham-operated group(2)one-factor modelling group(3)LPS small dose group(2.5 mg/kg),(4)LPS medium dose group(5 mg/kg)and(5)LPS large dose group(10 mg/kg).The one-factor modelling group was modelled by ligating the left anterior descending branch of the coronary artery,and the LPS small,medium and large dose groups were modelled by ligating the left anterior descending branch of the coronary artery for 10 min followed by intraperitoneal injection of different doses of LPS.After 24 h of moulding,the lingual,pulse and caudal perfusion of each group were observed.The cardiac function of each group was evaluated using small animal cardiac ultrasound,the area of myocardial infarction was detected by TTC staining,the serum cTnT level and the levels of inflammatory factors hypersensitive C-reactive protein(Hs-CRP),LPS,IL-1βand IL-6 were measured,and the blood routine,coagulation five items,platelet aggregation rate,and The coagulation evaluation indexes such as blood rheology,HE staining to observe the morphological changes of myocardial tissue.3.ResultsAll the indexes in the sham-operated group were at normal levels.Compared with the sham-operated group,the pulse amplitude,tongue RGB value and caudal perfusion were reduced in the one-factor modelling group and the LPS small,medium and large dose groups,the sublingual complex was dilated,cardiac function was decreased,platelet aggregation rate and whole blood viscosity,fibrinogen content,serum cTnT and inflammatory factors IL-1β,Hs-CRP and LPS content were increased,and myocardial infarct area was increased,in addition the LPS small In addition,serum IL6 levels increased significantly in the LPS small,medium and large dose groups,prothrombin and prothrombin time shortened and prothrombin activity increased,whereas no significant differences were seen in the serum IL-6 levels compared to the prothrombin,prothrombin time,prothrombin activity and sham operation groups in the one-factor modelling group.Compared to the one-factor modelling group,the LPS small dose group showed significantly lower pulse amplitude and higher serum IL-1β levels,with no statistical differences seen in the analysis of other index test results;the LPS medium and high dose groups showed significantly lower pulse amplitude,lingual RGB values,caudal perfusion,left ventricular ejection fraction and shortening fraction,growth and purple darkness of the sublingual complex,platelet aggregation rate,serum cTnT,IL-1β,IL6,and LPS levels increased,and the area of myocardial infarction increased significantly.HE staining showed that the myocardial fibers in the sham-operated group were neatly aligned,the myocardial fiber gap was widened in each model group,there was a large number of inflammatory cell infiltration,and the extent of inflammation was cumulatively increased in the LPS medium-and high-dose groups compared with the one-factor modeling group and the LPS low-dose group.4.SummaryIn the LPS small dose group,there was no significant difference in the results of the indicators compared with the single-factor modeling group,while the level of decline in cardiac function,the degree of "blood stasis" and the release of inflammatory factors in the LPS medium and large dose groups were more severe than those in the single-factor modeling group and the LPS small dose group.The lesions in the LPS medium-dose group and the LPS high-dose group were similar.Therefore,the model of ACS was prepared by injecting LPS medium dose(5mg/kg)after coronary artery ligation.Part Ⅲ:Reliability evaluation of the rat model of acute coronary syndrome and study on the mechanism of activating blood circulation and detoxification to protect ACS with Qing Xin Xie Yu formulaSection I.Validation of the reliability of the rat model of acute coronary syndrome with stasis and toxin interjunction by using formula to test the evidence1.ObjectiveTo validate the reliability of the rat model of acute coronary syndrome with the evidence of stasis and toxicity.2.MethodsThe rat model of acute coronary syndrome with mutual junction of blood stasis and toxins established in the first two parts of this experiment was used to verify the reliability of the model by measuring the evidence with prescriptions.The model was evaluated by the pharmacodynamic results,using blood-activating and detoxifying drugs with the right evidence and drugs to warm the middle and dispel cold with the wrong evidence.The experiment was divided into 6 groups:(1)sham-operated group;(2)model group;(3)Si-wei-tang group(formula evidence-less group),given 4.32 g/kg of Si-wei-tang;(4)Qing-xin-liquor-erasing formula high-dose group(formula evidence-appropriate group),given 3.96 g/kg of Qing-xin-liquor-erasing formula;(5)Qing-xin-liquor-erasing formula small-dose group,given 1.98 g/kg of Qing-xin-liquorerasing formula;(6)positive drug group,given Tegretol Prophylaxis was given for 5 days.The tongue,pulse,cardiac function,myocardial infarction area,serum cTnT content,observation of caudal perfusion,detection of platelet aggregation rate,blood rheology,blood routine,observation of myocardial histomorphology using HE staining and observation of myocardial ultrastructure by electron microscopy were performed in each group.The accuracy of the model was evaluated comprehensively.3.ResultsThe rats in the sham-operated group had a light red tongue,thin white coating and a smooth and strong pulse,and all the indexes were at normal levels.Compared with the sham-operated group,the pulse amplitude,tongue RGB value and caudal blood perfusion of the model rats were significantly lower,the tongue was purple and dark,the sublingual veins were purple and dilated,the cardiac function was significantly decreased,the erythrocyte volume distribution fraction,platelet count,platelet ratio,platelet aggregation rate and whole blood viscosity,and serum cTnT content were significantly increased,and the area of myocardial infarction was significantly larger.Compared with the model group,the pulse amplitude of both the Qing Xin Jie Yu Fang High Dose Group and the Si Nei Tang Group were significantly increased,but the tongue performance of the rats in the Si Nei Tang Group was not significantly different from that of the model group.The tongue of rats in the Qing Xin Xie Yu Fang High Dose Group was light red,the RGB value of the tongue surface increased,the sublingual veins shortened and became lighter in colour,tending to the level of the sham-operated group,and the caudal blood perfusion was significantly increased.The heart rate,left ventricular ejection fraction,short-axis shortening rate,cardiac output and output per beat of rats in the large and small dose groups of Qing Xin Jie Yu Fang and the positive drug group were all significantly increased compared with the model group,while the Four Reverse Tang group only increased the heart rate and cardiac output,with no significant improvement in other cardiac function indexes.The whole blood viscosity and serum cTnT level of all the administered groups decreased significantly compared with the model group.In addition,the large and small dose groups and the positive drug group of Qing Xin Jie Yu Fang could significantly reduce the volume distribution fraction of red blood cells,platelet count,platelet ratio and platelet aggregation rate,and decrease the area of myocardial infarction,while these indexes of Si Wei Tang did not improve significantly.The HE staining results showed that the myocardial fibres in the sham-operated group were more neatly arranged,while the myocardial fibres in the model group were disordered and the fibre gap was significantly widened,accompanied by a large number of inflammatory cells infiltration and focal myocardial necrosis;compared with the model group,all the administered groups could reduce the extent of myocardial injury and the number of inflammatory cells.Electron microscopic observation showed that the myocardial fibres in the sham-operated group were neatly arranged and the mitochondrial structure was intact,while the myocardial fibres in the model group were disordered and dissolved,and the mitochondria were swollen;compared with the model group,the myocardial fibres in the Qing Xin Jie Yu Fang high-dose group(the corresponding group of formulae)and the positive drug group of Qing Xin Jie Yu Fang small-dose group were relatively neatly arranged,while the myocardial fibres in the Si Wei Tang group(the group of formulae that should not be used)were severely dissolved and worse than the model group.4.SummaryThe reliability of the model was judged according to the principle of "using the right formula for the right evidence" in Chinese medicine.The high-dose group of the formula Qing Xin Xie Yu can improve the cardiac function,reduce the area of myocardial infarction,reduce blood stasis and tissue neutrophil distribution in the model of ACS,showing the efficacy of activating blood and detoxifying toxins;while the group of Si Wei Tang,which is a traditional Chinese medicine for treating myocardial ischemia,has the effect of warming the middle of the body and dispersing cold,although it can improve the cardiac function of the model to a certain extent,the effect of activating blood is poor and blood stasis is still The therapeutic effect on the rats in the model of acute coronary syndrome with stasis and toxin interlocking evidence was obviously inferior to that of the corresponding group in the formula,indicating that the medicine was not right for the evidence.In this study,the method of injecting LPS 10 min after coronary artery ligation proved that the model of acute coronary syndrome with stasis and toxicity is basically reliable.Section 2:Transcriptomics-based study on the potential genes of Qingxin Xieyu formula for the treatment of acute coronary syndromes1.ObjectiveTo identify potential gene targets for the treatment of acute coronary syndrome with Qingxin Xieyu formula by transcriptome sequencing.2.MethodsMyocardium from the sham-operated group,the model group and the high-dose group of Qing Xin Jie Yu Fang were subjected to transcriptome sequencing,and the sequencing results were analyzed by clustering,differential gene expression,GO function and KEGG enrichment pathway.The results were used to analyse the biological function changes and the pathways affected by the occurrence of the acute coronary syndrome,and to speculate on the potential biological processes and mechanisms of the intervention of the Qing Xin Xie Yu formula.The key signaling pathways and potential gene targets of the Qing Xin Xie Yu formula in the regulation of the acute coronary syndrome were identified by comparing the mRNA genes between the Qing Xin Xie Yu formula and the model group.3.ResultsA total of 1585 genes were up-regulated and 1469 genes were down-regulated in the model group compared with the sham-operated group,while 204 genes were upregulated and 102 genes were down-regulated in the Qingxin Xieyu Fang group compared with the model group.The results of KEGG enrichment pathway analysis showed that the genes in the myocardial tissue of rats in the model group were mainly enriched in the TNF signaling pathway,chemokine signaling pathway and IL-7 signaling pathway,which were related to the formation of neutrophil outer traps and hematopoietic cell lineage.The difference genes between the Qing Xin Jie Yu group and the model group were mainly enriched in immune response and inflammation related signaling pathways.Seven highly expressed genes were identified in the Qing Xin Jie Yu Fang group:Ly6e,CD14,TLR4,NFKBIZ,REL,RELA and RELB,which corresponded to the Ly6e/CD14/TLR4/NF-Bp65 signaling pathway.4.SummaryThe transcriptomic results suggest that the Qing Xin Jie Yu formula mainly affects the immune response and inflammation-related pathways to improve the stasis and toxin interactions in acute coronary syndrome.Qing Xin Jie Yu Fang may regulate the expression of Ly6e,CD14,TLR4,NFKBIZ,REL,RELA,RELB and other genes,presumably acting through the Ly6e/CD14/TLR4/NF-κBp65 signaling pathway.Section 3:Study on the mechanism of action of Qingxin relieving stasis formula in the treatment of acute coronary syndrome with stasis and toxin interconnection evidence1.ObjectiveTo investigate the mechanism of action of Qing Xin Jie Yu Fang in the treatment of acute coronary syndrome based on the results of transcriptomics,and to verify whether it acts through regulating the Ly6e/CD14/TLR4/NF-κBp65 signaling pathway.2.MethodsThe serum and myocardial tissues of rats in the sham-operated group,the model group,the high-dose group of Qing Xin Jie Yu Fang,the low-dose group of Qing Xin Jie Yu Fang,and the positive drug group reserved in the first section of the experiment were examined.The mRNA levels of Ly6e,CD14,TLR4,NFKBIZ,REL,RELA and RELB,which are genes with significant differential expression and high expression between the transcriptomic screening group and the model group,were detected by qRT-PCR,and the expression of CD 14 protein in myocardial tissue was detected by immunohistochemistry.pNF-κBp65,NF-κBp65 expression levels.3.ResultsCompared with the sham-operated group,the serum LPS,IL-1β,IL-6,IL-18 and TNF-α levels were significantly increased in the model group;while compared with the model group,both the large and small dose groups of Qing Xin Jie Yu Fang and the positive drug group could significantly reduce the serum LPS,IL-1β,IL-6,IL-18 and TNF-α levels.The qRT-PCR results showed that compared with the sham-operated group,the model group significantly up-regulated the mRNA levels of immune and inflammationrelated genes Ly6e,CD 14,TLR4,NFKBIZ,REL,RELA and RELB;compared with the model group,Qing Xin Jie Yu Fang could regulate the mRNA of Ly6e to normal levels,reduce the mRNA expression levels of REL,and have a significant effect on The mRNA expression of CD 14,TLR4,NFKBIZ,RELA and RELB also showed a downregulation trend.Immunohistochemical results showed that the expression level of CD 14 protein was significantly higher in the model group compared with the shamoperated group;compared with the model group,the expression intensity of CD 14 protein could be reduced in the high-dose group,the low-dose group and the positive drug group of Qing Xin Jie Yu Fang.WB assay showed that the expression of Ly6e,CD 14,TLR4 and p-NF-κB p65 protein were significantly up-regulated in the myocardial tissue of the model group compared with the sham-operated group.Compared with the model group,the high dose and positive drug of Qing Xin Jie Yu Fang could significantly down-regulate the expression levels of Ly6e,CD 14,TLR4 and p-NF-κB p65,and the small dose of Qing Xin Jie Yu Fang could reduce the expression of Ly6e,TLR4 and CD14,and had a tendency to reduce the expression of p-NF-κB p65 protein.4.SummaryThe mechanism of action of Qing Xin Xie Yu Fang in the treatment of ACS is related to the regulation of Ly6e/CD14/TLR4 signaling pathway,reduction of NF-κB p65 phosphorylation and reduction of inflammatory factor release.ConclusionA rat model of ACS with both acute coronary syndrome and stasis-toxic intercrossing syndrome could be successfully prepared by intraperitoneal injection of LPS(5 mg/kg)for 24 h after coronary ligation of the rat heart.The formula can reduce the platelet ratio and aggregation rate,modulate the Ly6e/CD14/TLR4 inflammatory signaling pathway,decrease the level of NF-κB p65 phosphorylation and reduce the release of various serum inflammatory factors,thus exerting the effect of blood circulation and detoxification. |
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