Clinical And Basic Research Of Linggui Zhugan Decoction And Xiaochengqi Decoction Huacui Fang On Inflammatory Response After Cardiac Surgery

At present,cardiovascular disease has become the top disease that endangers human health and life.Cardiopulmonary bypass and off-pump cardiac surgery have become the most important and effective clinical treatments for cardiovascular diseases,especially in the acute stage.In recent years,with the development of science and technology,most of the technical barriers of cardiac surgery have been shattered,and the clinical effectivemess and prognosis of surgery have been significantly improved.However,the systemic inflammation after cardiac surgery often leads to the failure and poor prognosis.One common solution,the use of antibiotics does not work as expected,and may even result in the disorder of intestinal flora and aggravate the complications after cardiac surgery.Heart disease（coronary heart disease）belongs to the category of "chest obstruction" in traditional Chinese medicine,and its pathogenesis is "Yang Wei Yin Xuan".Traditional Chinese medicine has its unique advantages in both prevention and treatment of heart disease and anti-inflammatory.Lingguizhugan decoction combined with Xiaochengqi Decoction（LXHCF）is made up of Lingguizhugan decoction,Xiaochengqi decoction,Pingweisan Decoction and Fuzi Xiexin Decoction,which have positive effects inenhancing cardiac function,increasing cardiac output,diuretic,and enhancing gastrointestinal motilityTherefore,in this study,patients with coronary atherosclerosis,angina pectoris and myocardial infarction have been treated with off-pump coronary artery bypass grafting,patients with aortic and mitral insufficiency have been treated with on-pump heart valve replacement.The inflammatory state,differences inpatients before and after operation and the intervention of LXHCF were measured.In order to investigate the effectiveness of LXHCF on inflammatory response after cardiac surgery,we used ligation of left anterior descending coronary artery（LAD）to simulate the inflammatory model mice after cardiac surgery,and combined with 16S rDNA high-throughput sequencing to track the changes of intestinal flora in mice,analyzed the correlation between LXHCF and inflammation,and explored the mechanism of LXHCF regulating intestinal flora to prevent and treat inflammation after cardiac surgery.Furthermore,network pharmacology technology has been used to study and predict the anti-inflammatory mechanism of LXHCF.Based on clinical animal research network pharmacology prediction and 16S rDNA high-throughput sequencing of intestinal microflora,this project has carried out clinical and basic research on the effectivesnessof LXHCF on inflammatory response after cardiac surgery,which provided the basis for further research on the correlation between inflammatory response after cardiac surgery and intestinal microflora and clinical application of traditional Chinese medicine.（1）Clinical study of Linggui Zhugan Decoction and Xiaochengqi Decoction on postoperative patients with coronary heart diseaseMethod:78 patients have been divided into four groups.All patients have either coronary artery bypass grafting（off-pump）or heart valve replacement（cardiopulmonary bypass）and underwentthe same surgeon.The four groups are:1.Coronary artery bypass grafting control group（n=41）;2.Coronary artery bypass grafting experimental group（n=14）;3.Cardiac valve replacement control group（n=18）;4.Antibiotic comparison group（n=5）.In this last group,5 patients were given antibiotic therapy and LXHCF intervention after operation.The blood routine indexes including white blood cell count（WBC）,neutrophil count（neut）and neutrophil percentage（neut%）were measured before operation,1-day post-operation,before LXHCF intervention and 1 day,2 days,3 days and 4 days post intervention.Objectives are to compare the degree and state of inflammatory reaction in patients with two kinds of cardiac surgery,and to observe the effectiveness of LXHCF intervention on inflammatory reaction in patients with two kinds of cardiac surgery.The results showed that:（1）there was no significant difference in WBC,NEUT and NEUT%between the two methods before operation;There was a serious inflammatory reaction in patients after two kinds of heart surgery.The WBC,NEUT and NEUT%were significantly increased on the first day after operation,and the patients in the heart valve replacement group were worse than those in the heart bypass group.① Antibiotics alone can not limit the systemic inflammatory reaction of patients after cardiac surgery;Compared with 1 day after operation,WBC,neut and neut%in the control group were still significantly increased 3-4 days after operation（4-5 days after antibiotic treatment）.③Combined with LXHCF intervention can significantly improve the systemic inflammatory response of patients undergoing CABG;Compared with the control group,LXHCF could significantly reduce the WBC content in the whole blood on the 2nd and 3rd day,the neut content in the whole blood on the 3rd day and the neut%on the 2nd day.④ Combination with LXHCF intervention can significantly improve the systemic inflammatory response in patients undergoing heart valve replacement;Compared with the control group,the levels of WBC and neut in the whole blood of LXHCF group decreased significantly on the first day after administration,and decreased significantly from the third day after administration.（2）Anti inflammatory effect of Lingguizhugan Decoction and Xiaochengqi Decoction on inflammatory model mice after cardiac surgeryOn the basis of previous clinical studies,the left anterior descending coronary artery ligation was used to simulate the postoperative inflammation model.The modelled mice were divided into model group,low-dose LXHCF-1 group（5 g/kg）,high-dose LXHCF-h group（25 g/kg）,cefixime group（0.08 g/kg）and cefixime+LXHCF group（0.08 g/kg+LXHCF 25 g/kg）,13 rats in each group were treated for 7 days.On the 3rd and 7th day after administration,WBC,neut,neut%,mon and neut were measured by five kinds of sorting cell counter.The levels of IL-6 and CRP in plasma were measured by enzyme-linked immunosorbent assay.At the end of the experiment,H&E staining was used to observe the pathological changes of myocardial tissue.Objective to investigate the intervention effect of LXHCF on inflammatory reaction after cardiac surgery,and to provide basis for clinical medication of prevention and treatment of postoperative complications.The results showed that on the third day after operation,the model mice showed systemic inflammatory reaction,the contents of WBC,mon and neut in whole blood were significantly increased,and the contents of IL-6 and CRP in plasma were significantly increased（P<0.05,0.01）.Compared with the model group,LXHCF-1 and antibiotic+LXHCF could significantly reduce the whole blood WBC,Mon,neut and mon contents,and significantly reduce the plasma IL-6 and CRP contents（P<0.01,0.05）;On the 7th day,LXHCF-1 and LXHCF-h could significantly reduce the blood neut of model mice,while LXHCF-1,LXHCF-h and antibiotic+LXHCF could significantly reduce the plasma IL-6 of model mice;LXHCF-h,LXHCF-1 and antibiotic+LXHCF can also significantly improve the myocardial cell lysis,distortion,necrosis and inflammation.However,antibacteric had no significant effect on the above inflammatory indexes and myocarditis.（3）Effectiveness of LXHCF on intestinal flora in mice after cardiac surgeryThe changes of intestinal flora in patients after cardiac surgery are closely related to the inflammatory reaction.Based on the anti-inflammatory effect of LXHCF on inflammatory model mice,the changes of intestinal flora in mice were detected by 16S rDNA high-throughput sequencing,and its correlation with inflammation was analyzed forfurther exploration in the anti-inflammatory mechanism of LXHCF regulating intestinal flora after cardiac surgery.The results showed that the abundance of intestinal microflora in mice decreased significantly after cardiac surgery,while the abundance of Spirochaetes and Fusobacteria at the portal level increased significantly;The abundance of Prevotellaceaeucg-001 decreased significantly;7 days after operation,the relative abundance of Lactobacillus increased significantly.Compared with the model control group,antibiotic alone can further aggravate the flora disorder of the model mice.antibiotic+LXHCF can reverse the flora disorder caused by antimicrobial alone.Moreover,the abundance of intestinal flora in model mice was significantly increased after administration of LXHCF;At the phylum level,the relative abundance of Firmicutes and spirochaetes decreased significantly,while the abundance of Bacteroidetes increased significantly;The abundance of 9 genera（Alloprevotella,Escherichia-Shigella,Lactobacillus,Rikenellaceae_RC9_gut_group,Prevotella₁,Ruminococcaceae_UCG-014,Lachnospiraceae_NK4A136_group,uncul tured_Bacteroidales_bacterium,uncultured_organism）decreased significantly.The abundance of parabacterioides increased significantly.Further functional prediction analysis showed that LXHCF intervention group and antibiotic+LXHCF intervention could reverse the model mice,and antibiotic intervention alone could significantly up regulate immune system,immune system diseases related pathways and down regulate cardiovascular disease pathways.（4）Discussion on the mechanism of LXHCF in prevention and treatment of inflammatory reaction after cardiac surgery based on network pharmacologyTCMSP database was used to screen the effective active ingredients with oral bioavailability（OB≥ 30%）and drug like（DL≥ 0.18）as pharmacokinetic parameters;CTD and genecards database were used to predict the interaction between active components of LXHCF and systemic inflammatory targets;Combined with string,DAVID and KEGG database,we analyzed the key target genes,biological functions and pathways,and elaborated the mechanism of LXHCF in the treatment of systemic inflammatory response after operation.The results showed that there were 137 active components in LXHCF,234 therapeutic targets related to systemic inflammation,10 active components could interact with 28 or more targets,and 9 key targets could interact with 34 or more active components;234 targets were mapped to 117 pathways.The first 20 pathways involved inflammatory response（HIF-1 signaling pathway,TNF signaling pathway）,immune response（Toll like receptor signaling pathway,nod like receptor signaling pathway,T cell receptor signaling pathway）,hormone signaling pathway（estrogens signaling pathway,prolactin signaling pathway）Among them,tumor necrosis factor,nod like receptor,toll like receptor,T cell receptor,chemokines and other signaling pathways are directly related to anti-inflammatory immunity.To sum up,clinical studies have shown that patients with either off-pump coronary artery bypass grafting or cardiopulmonary bypass for heart valve replacement,all shown severe inflammatory reaction after operation.Antibiotics alone can not inhibit this kind of inflammatory reaction in a short time,while LXHCF can significantly inhibit the inflammatory reaction of two kinds of coronar operation.Preliminary studies have shown that LXHCF and antibiotic+LXHCF can significantly improve myocardial lesions and inflammatory injury in mice with inflammation after cardiac surgery,and its mechanism may be related to reducing the levels of inflammatory cytokines and inflammatory related cells in blood and improving intestinal flora disorder.Furthermore,based on network pharmacology,LXHCF plays an anti-inflammatory role by regulating toll like receptor,tumor necrosis factor,nod like receptor,T cell receptor,chemokines and other signaling pathways through multiple components,multiple targets and multiple pathways.This study carried out the clinical and basic research of LXHCF on the inflammatory response after cardiac surgery,and found the advantages of LXHCF and its combination with antibiotics to improve the inflammation and intestinal flora disorder after cardiac surgery,which provided the basis for the in-depth study of the correlation between inflammatory injury after cardiac surgery and intestinal flora and the clinical application of traditional Chinese medicine.