| BackgroundChronic obstructive pulmonary disease(COPD)is a common disease characterized by the persistence of airflow limitation that seriously endangers human health and has become the third leading cause of death worldwide.Acute exacerbation of chronic obstructive pulmonary disease(AECOPD)is an important cause of death in COPD patients,hypoxia and infection aggravate vascular endothelial injury,aggravate blood hypercoagulability,easily complicated by cardiovascular and cerebrovascular diseases,pulmonary arteries,lower extremity deep veins and other multiple sites of thrombosis,increasing mortality.Studies have shown that the incidence of AECOPD with venous thromboembolism is 3.9%～12.59%,and the incidence of lower extremity deep venous thrombosis is 19%～29%.Faced with the high incidence of thrombotic events in AECOPD,early intervention of hypercoagulable state to reduce the occurrence of thrombotic events is an important clinical problem to be solved urgently.Hypercoagulable state has not yet reached the indications for the application of antithrombotic therapy,and there is a lack of clinical evidence-based evidence to prevent and treat AECOPD hypercoagulable state to reduce the occurrence of thrombotic events.At present,the Global Initiative for Chronic Obstructive Pulmonary Disease(GOLD)and the Chinese expert consensus on the diagnosis and treatment of AECOPD only recommend that anticoagulant therapy should be considered for AECOPD patients who are bedridden,polycythemia,dehydration and hospitalized for COVID-19,while the adverse reactions of traditional anticoagulants and the accessibility,price and other factors of new anticoagulants restrict the clinical application to a certain extent,resulting in serious lack of prevention and treatment of AECOPD hypercoagulable state and the lack of safe and effective methods to truncate the disease.Modern medicine says that hypercoagulable state mostly belongs to the category of "blood stasis" in traditional Chinese medicine(TCM),and blood-activating and stasis-dissipating drugs have a certain effect in improving blood hypercoagulability.However,hypercoagulable state is used as a secondary pathological change in the process of AECOPD,and the pathogenesis changes of underlying diseases determine the occurrence and development of hypercoagulable state.TCM believes that phlegm is the main pathogenic factor and pathological product of AECOPD,and the formation of blood stasis is closely related to phlegm,phlegm turbidity stagnation and heat,phlegm heat injury Jin blocking collaterals caused by poor blood flow and blood stasis.Under the enlightenment of Zhu’s viewpoint of "self-depression into accumulation,self-accumulation into phlegm,phlegm with blood stasis,and then into nest sac",combined with the relationship between phlegm and blood stasis in the process of AECOPD disease,in the early stage,phlegm-heat stasis is the pathogenesis of hypercoagulable state in AECOPD,proposes phlegm and blood stasis with the same treatment,and establishes Qingjin Huazhuo Recipe based on the principles of clearing away heat and promoting lung,phlegm and removing blood stasis.Previous clinical studies have found that Qingjin Huazhuo Recipe can significantly improve the clinical efficacy and prolong prothrombin time(PT)in AECOPD patients,suggesting that it has the effect of inhibiting the extrinsic coagulation pathway.AECOPD patients with airflow limitation aggravated,lung function ventilation indicators decreased,accompanied by hypoxemia,the body in the hypoxic environment aggravated vascular endothelial injury,activated tissue factor(TF)to initiate coagulation response,hypoxia is one of the important factors leading to AECOPD vascular endothelial injury.Hypoxia-inducible factor-1α(HIF-1α)is a key gene that plays a regulatory role in the hypoxic environment,and specifically regulates the transcription of downstream vascular endothelial growth factor(VEGF),VEGF specifically regulates endothelial cell function,and the abnormal expression of HIF-1α/VEGF pathway under hypoxic conditions leads to vascular endothelial dysfunction and promotes blood hypercoagulability.Based on the pathogenesis characteristics and previous research results of AECOPD,the scientific hypothesis is proposed that Qingjin Huazhuo Recipe with phlegm and blood stasis can improve the hypercoagulable state and prevent thrombotic events in patients with AECOPD,which may be related to the regulation of HIF-1α/VEGF pathway to protect vascular endothelial function.Because there is still a lack of efficacy evaluation and mechanism study of Qingjin Huazhuo Recipe in the intervention of hypercoagulable state in AECOPD,this study will discuss from the following three parts:Part I,through network pharmacology methods to explore the potential target and mechanism of action of Qingjin Huazhuo Recipe in the treatment of COPD,and provide a scientific basis for subsequent studies;Part Ⅱ,objectively evaluate the efficacy and safety of Qingjin Huazhuo Recipe in the intervention of hypercoagulable state in AECOPD patients with phlegm-heat stasis lung syndrome through clinical study;Part Ⅲ,based on HIF-1α/VEGF affecting vascular endothelial function,to investigate the mechanism of Qingjin Huazhuo Recipe in preventing and treating hypercoagulable state of AECOPD by carrying out animal experimental studies.The aim is to provide new ideas and methods for the prevention and treatment of hypercoagulable state of AECOPD in order to improve the clinical efficacy,reduce thrombotic events and improve the prognosis of patients.Objective1 To investigate the potential target and mechanism of Qingjin Huazhuo Recipe in the treatment of COPD,and to provide a scientific basis for subsequent studies.2 Objective To evaluate the efficacy and safety of Qingjin Huazhuo Recipe in the treatment of hypercoagulable state in patients with AECOPD of phlegm-heat stasis lung syndrome.3 Based on the effect of HIF-lα/VEGF on vascular endothelial function,this paper discusses the mechanism of Qingjin Huazhuo Recipe in preventing and treating hypercoagulable state of AECOPD,and expands the understanding of the biological basis of "treating phlegm and blood stasis at the same time".Methods1 Network Pharmacology Study MethodsThe main chemical components and targets of Qingjin Huazhuo Recipe core drugs were searched by systematic pharmacology database and analysis platform of traditional Chinese medicine.COPD related targets were obtained by TTD,CTD,DisGeNET and GeneCards databases to screen the intersection targets between drugs and diseases.Cytoscape3.7.2 was used to construct a drug-compound-target-disease network to analyze the active components of drugs that play a therapeutic role;STRING database was used to construct a protein-protein interaction relationship(PPI)network topology analysis map to analyze the core targets;DAVID database was used for gene function(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis to obtain the potential biological pathways and signaling pathways of Qingjin Huazhuo Recipe in the treatment of COPD.2 Clinical Study MethodsPatients with AECOPD who were hospitalized in the Department of Respiratory Medicine of Wangjing Hospital of China Academy of Chinese Medical Sciences from April 2021 to November 2022 were screened according to the inclusion and exclusion criteria,and those who met the conditions were included in this study.They were randomly divided into the control group and the observation group.The control group used the treatment regimen recommended by the Chinese expert consensus for the diagnosis and treatment of AECOPD(controlled oxygen therapy,bronchodilators,glucocorticoids,antibiotics,mechanical ventilation,etc.).The observation group was given Qingjin Huazhuo Recipe orally on this basis for 7 days.The general conditions,occurrence of thrombotic events and adverse events were recorded.The coagulation parameters[tissue plasminogen activator-plasminogen activator inhibitor-1 complex(t-PAIC),plasmin-antiplasmin complex(PIC),thrombomodulin(TM),thrombin-antithrombin complex(TAT),activated partial thromboplastin time(APTT),prothrombin time(PT),D-dimer],arterial blood gas analysis indicators[arterial partial pressure of oxygen(PaO2),arterial partial pressure of carbon dioxide(PaCO2)],pulmonary ventilation function indicators[forced expiratory volume in one second as a percentage of predicted value(FEV1%pred)],TCM syndrome score,safety index changes were analyzed.The efficacy and safety of Qingjin Huazhuo Recipe in intervening hypercoagulable state in AECOPD patients with phlegm-heat stasis lung syndrome were objectively evaluated.3 Experimental Study Methods8-week-old SPF SD male rats were randomly divided into control group,model group,prevention group and treatment group.Rats in the prevention group were intragastrically administered with Qingjin Huazhuo Recipe during modeling and sacrificed after the end of modeling;after the end of modeling,rats in the treatment group were intragastrically administered with Qingjin Huazhuo Recipe,and rats in the control and model groups were intragastrically administered with normal saline and sacrificed after 14 days of treatment.Record general condition,photographs of the lingual surface and sublingual venation were collected using a Nikon digital camera,and pictures were processed using Photoshop CS6 to calculate the r value of tongue color and the sublingual venation score to assess blood stasis in rats;the histopathological changes of rat lung tissues were observed by hematoxylin-eosin staining using a light microscope to assess the model and observe the effects of Qingjin Huazhuo Recipea on the structural destruction and inflammatory infiltration of rat lung tissues;the pulmonary vascular endothelium changes was observed by transmission electron microscopy,and the serum levels of nitric oxide(NO),von Willebrand factor(vWF),and TF were measured by nitrate reductase method and enzyme-linked immunosorbent assay(ELISA)to observe the effects of Qingjin Huazhuo Formula on vascular endothelium and coagulation function;the expression levels of HIF-1α and VEGF were detected from the gene and protein levels using real-time PCR and Western-blot methods to confirm that HIF-la/VEGF was an important pathway for Qingjin Huazhuo Recipe to protect vascular endothelium from hypercoagulable state.Results1 Network Pharmacology Study ResultsA total of 87 main active ingredients and 164 targets of TCM components of Qingjin Huazhuo Recipe were obtained;a total of 3051 targets of COPD were obtained,and 112 targets of drug and disease intersection were obtained.The main drugs of Qingjin Huazhuo Recipe in treating COPD are Morus alba,Ephedra sinica,Lepidium seed and Perilla frutescens,which mainly act on IL-6,TNF,MAPK,VEGF and other key targets,and are enriched in biological processes such as drug response,positive regulation of nitric oxide biosynthesis process,hypoxia response and TNF pathway,HIF-1 signaling pathway,PI3K-Akt signaling pathway and other related pathways.2 Clinical Study Results2.1 General conditionsA total of 90 patients with AECOPD who met the criteria were included,45 patients in the control group and 45 patients in the observation group.There was no significant difference in gender,age,duration of COPD,severity classification of AECOPD,and combined diseases between the two groups(P>0.05),with comparability.During the study,6 patients dropped out due to hemolysis of blood samples,and 84 patients were finally included in the statistical analysis,including 41 patients in the control group and 43 patients in the observation group.2.2 Clinical efficacy evaluation indicatorsThe levels of t-PAIC and PIC in the two groups after treatment were significantly lower than those before treatment(P<0.01),and the levels of TAT and TM in the observation group after treatment were lower than those before treatment(P<0.05),the levels of TAT and TM in the control group after treatment were not significantly different from those before treatment(P>0.05);the levels of t-PAIC and PIC in the observation group after treatment were better than those in the control group(P<0.05).PT in both groups after treatment was prolonged compared with that before treatment(P<0.05),APTT in observation group after treatment was significantly prolonged compared with that before treatment(P<0.01),D-dimer level in observation group after treatment was significantly decreased compared with that before treatment(P<0.01),APTT and D-dimer levels in the control group after treatment were not significantly different from those before treatment(P>0.05);PT and D-dimer levels in observation group after treatment were better than those in control group(P<0.05).PaO2 and FEV1%pred levels in the two groups after treatment were significantly higher than those before treatment(P<0.01),PaCO2 levels in the two groups after treatment were not significantly changed from those before treatment(P>0.05);PaO2 and FEV1%pred levels in the observation group after treatment were better than those in the control group(P<0.05).PaO2 and FEV1%pred were negatively correlated with t-PAIC,PIC and TAT,and positively correlated with PT,of which PaO2 was moderately correlated with t-PAIC,PIC and PT(R values were 0.424,0.504 and 0.521,respectively),and FEV1%pred was moderately correlated with PIC and PT(R values were 0.597 and 0.561,respectively).The TCM syndrome scores of the two groups after treatment were significantly lower than those before treatment(P<0.01);the TCM syndrome scores of the observation group after treatment were better than those of the control group(P<0.05).During the treatment period,2 cases of acute myocardial infarction occurred in the control group,accounting for 4.88%.No thrombotic event occurred in the study group.2.3 Safety MeasuresThe levels of PLT,ALT,GGT,BUN and Cre in the two groups after treatment were not significantly different from those before treatment(P>0.05),and the changes of PLT,ALT,GGT,BUN and Cre levels in the two groups after treatment were not significantly different(P>0.05).No adverse reactions occurred during treatment in both groups.3 Experimental Study Results3.1 General conditionsThe rats in the control group had good mental status,stable breathing,bright white hair,normal drinking and eating,and gradually increased body weight.In the model group,the rats were agitated,the response decreased,shortness of breath and food intake decreased in the early stage of modeling;with the prolongation of modeling time,the rats gradually became apathetic,unresponsive,the activity decreased,shortness of breath aggravated or even alar incitement,the oronasal secretions increased significantly,airway phlegm sounds were heard,the hair color was dark and dull,hair loss was severe,drinking water intake decreased,and the body weight increased slowly.Rats in the prevention and treatment groups were intermediate between the control and model groups.3.2 Tongue color,sublingual venationThe tongue color of rats in the control group was light red and moist,and the sublingual venation was normal without tortuosity;the tongue color of rats in the model group was significantly purple and dark,ecchymosis was observed,R value and r value were significantly lower than those in the control group(P<0.01),accompanied by cyanosis of lips,purple and thick sublingual venation,and the sublingual venation score was significantly higher than that in the control group(P<0.01);the tongue color R value,r value and sublingual venation score of rats in the prevention group and the treatment group were not significantly different(P>0.05),but the tongue color R value,r value and sublingual venation score of rats in the macroscopically observed treatment group were slightly darker than those of rats in the prevention group.3.3 Histopathological changes in lungThe lung tissue structure of the control group was normal,and there was little inflammatory cell infiltration;the bronchial epithelial cells of the model group were detached,the cilia collapsed,the mucous cells increased,some of them showed mucus blocking the airway,a large number of inflammatory cell infiltration was observed,and the alveolar space expanded and fused into bullae;the conditions of the prevention group and the treatment group were intermediate between the control group and the model group.3.4 Pulmonary vascular endothelial changesIn the control group,the endothelial cell structure was normal,the basement membrane was intact,the nuclear size was normal,and the organelle structure was basically normal.In the model group,the endothelial cells were slightly pyknotic,the thickness of basement membrane was uneven,locally blurred and damaged;the vascular lumen was slightly narrow and collapsed locally;the tight junction structure between endothelial cells was not obvious;the nuclei were irregular and the chromatin was pyknotic;there were few organelles,the mitochondria were slightly swollen,the matrix was locally dissolved and faded,and the cristae were broken and reduced.In the prevention group and the treatment group,the vascular endothelial cells were slightly edematous,the thickness of basement membrane was uneven and continuous;the intercellular structure was fair;the nuclei were irregular and the chromatin was uniform;the intracellular organelles were less and the structure was not obvious,and the overall situation was better than that in the model group.3.5 Serum NO,vWF,TF expression levelsCompared with the control group,the serum NO level was significantly decreased(P<0.01)and the vWF and TF levels were significantly increased(P<0.01)in the model group;compared with the model group,the serum NO level was significantly increased(P<0.01)and the vWF and TF levels were significantly decreased(P<0.01)in the prevention and treatment groups;there was no significant difference in the NO,vWF,and TF levels between the prevention and treatment groups(P>0.05).3.6 Expression levels of HIF-1α and VEGF in lung tissueCompared with the control group,the levels of HIF-1α and VEGF mRNA in the lung tissue of the model group were increased(P<0.05);compared with the model group,the levels of HIF-1α and VEGF mRNA in the lung tissue of the prevention and treatment groups were decreased(P<0.05);compared with the treatment group,the levels of HIF-1α mRNA in the prevention group were lower(P<0.05).Compared with the control group,the levels of HIF-la and VEGF protein in the lung tissue of the model group were significantly higher(P<0.01);compared with the model group,the levels of HIF-1α and VEGF protein in the lung tissue of the prevention and treatment groups were significantly lower(P<0.01);compared with the treatment group,the levels of VEGF protein in the prevention group were lower(P<0.01).Conclusion1 Qingjin Huazhuo Recipe effectively reduces the TCM syndrome score,t-PAIC,PIC and D-dimer levels,prolongs PT,and increases PaO2 and FEV1%pred levels in patients with AECOPD with phlegm-heat stasis lung syndrome,which has the effects of relieving clinical symptoms,improving hypoxia,improving blood hypercoagulability,and reducing the occurrence of thrombotic events,with high drug safety.2 Qingjin Huazhuo Recipe has the effect of preventing and treating hypercoagulable state in rats with chronic obstructive pulmonary disease(COPD)of phlegm-heat stasis and lung syndrome,which is related to the regulation of HIF-1α/VEGF pathway and has the advantage of protecting vascular endothelial function.It can effectively truncate the disease and reduce the occurrence and development of blood hypercoagulable state,reflecting the idea of "treating the disease" and "treating the disease" in traditional Chinese medicine.. |
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