| With the rapid economic development and increasing stress,the incidence of psychocardiacology diseases is increasing year by year.Coronary artery disease with comorbid anxiety is a common clinical psychocardiacology disease,but there is a lack of safe and effective treatment measures due to its unknown mechanism.Our team’s previous clinical study confirmed that Chaihujialonggumulitang granules(Bupleurum Falcatum Dragon Bone and Oyster Shell Granule,BFG)could improve angina pectoris and anxiety in patients,but the chemical composition and specific effectiveness mechanism of this formula have not been clarified.Objectives:(1)To analyze and summarize the Chinese medical symptoms and related factors of coronary heart disease with comorbid anxiety,and to provide evidence basis for traditional Chinese medicine(TCM)treatment of coronary heart disease with comorbid anxiety.(2)To systematically evaluate the efficacy and safety of Chaihujialonggumulitang in the treatment of coronary heart disease with comorbid anxiety,and to provide evidence-based clinical practice.(3)To identify the effective chemical components of Chaihujialonggumulitang,and to lay the foundation for the study of its mechanism.(4)To explore the mechanism of Chaihujialonggumulitang in the treatment of myocardial infarction with comorbid anxiety from the Nrf2/HO-1 signaling pathway.Methods:(1)Study 1 collected clinical information and Chinese medical four diagnostic information of patients with coronary heart disease with comorbid anxiety state through crosssectional survey,explored the characteristics and distribution of Chinese medical symptoms of patients with coronary heart disease with comorbid anxiety by cluster analysis and factor analysis,and explored the correlates of patients’ anxiety degree by chi-square test and logistic regression analysis.(2)Study 2 searched Pubmed,the Cochrane Library,Web of Science,the CNKI,Wanfang,Vipshop and the biomedical literature database for publicly published clinical randomized controlled trials of Chaihujialonggumulitang for the treatment of coronary heart disease with comorbid anxiety,retrieved from date of library construction to December 2022.Meta-analysis was performed by RevMan 5.3 software,and sources of heterogeneity were sought by subgroup analysis and sensitivity analysis.(3)Study 3 prepared the sample solution of Chaihujialonggumulitang and identified its components by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry.(4)Study 4 used ligation of the left anterior descending coronary artery combined with uncertain empty bottle stimulation to establish myocardial infarction with comorbid anxiety model(i.e.complex model).The rats were divided into blank control group,sham group,myocardial infarction group,complex group,Chinese medicine group and activator group.The Chinese medicine group was gavaged with Chaihujialonggumulitang(BFG),and the activator group was injected with dimethyl fumarate(DMF)intraperitoneally once a day for 6 days.Cardiac ultrasound,cardiac enzymes,HE staining and Masson staining were used to evaluate cardiac function and myocardial pathological changes.Behavioral tests,hippocampal neurotransmitters and hippocampal histopathological staining(HE staining and Nissler staining)were used to evaluate anxiety-like behavior and hippocampal tissue pathological changes.Biochemical analysis kits were used to detect oxidative stress parameters such as MDA,SOD,CAT and GPX in myocardial tissues,serum and hippocampal tissues.RT-qPCR and Western blotting were used to detect gene and protein expression of Nrf2,Keap1,HO-1,Bax,Bcl-2 and Cleaved caspase-3 in myocardial and hippocampal tissues.Results:(1)Study 1:①Clinical information:196 patients with coronary artery disease with comorbid anxiety were included,of whom 127 were women;155 were mostly patients over 60 years old;126 were mental workers and 70 were manual workers;73 were of normal weight and 88 were overweight;64 were former smokers and 75 were alcohol drinkers.Among the previous medical history,the largest number of cases were combined with hypertension,111 cases,followed by 82 cases combined with hyperlipidemia.②TCM symptoms:The top ten clinical TCM symptoms in patients with coronary heart disease with comorbid anxiety state were:chest tightness,palpitations,chest pain,poor sleep,heart trouble,distension and pain in the ribs,impatience and irritability,dreaminess,good tai-hui,and dizziness.Tongue symptoms were more frequent with thin white moss,thick greasy moss and yellow moss;pulse symptoms were more frequent with string pulse,sunken pulse,thin pulse and slippery pulse.The clustering analysis showed that the TCM pathological elements in patients with coronary artery disease with comorbid anxiety mainly involved heart,liver,spleen,kidney,gallbladder and large intestine;the pathological elements mainly involved qi stagnation,blood stasis,fire-heat,phlegm-dampness,qi deficiency and yin deficiency;the TCM evidence types were phlegm-fire disturbing the heart,qi stagnation and blood stasis and qi-yin deficiency of heart and kidney.Factor analysis yielded that its Chinese medical evidence elements mainly involved the heart,liver,spleen,kidney,gallbladder and large intestine,and its pathological evidence elements mainly involved qi stagnation,blood stasis,fire-heat,phlegm-dampness,qi deficiency and yin deficiency,and the Chinese medical evidence types were liver-depression and spleen deficiency,phlegm-fire disturbing the heart,heart-kidney yin deficiency and qi stagnation and blood stasis.③Study of relevant factors:The results of univariate analysis showed that the degree of anxiety in patients with coronary artery disease with comorbid anxiety state was statistically different from gender,nature of occupation,body mass index,hypertension and history of hemodynamic reconstruction(P<0.05),and not from other clinical information(P>0.05).The results of multifactorial analysis showed that the level of anxiety in patients with coronary artery disease with comorbid anxiety was positively correlated with women,brain workers,overweight,obesity,hypertension and history of hematologic reconstruction(P<0.05).(2)Study 2:A total of 14 papers were included,and Meta-analysis showed that compared with the control group,the trial group could significantly improve angina efficiency(RR:1.3 3,95%CI[1.17,1.51])and electrocardiogram efficiency(RR:1.21,95%CI[1.06,1.38]),reduce angina attack frequency(MD:-1.87,95%CI[-2.17,-1.56]),chest tightness and chest pain symptom scores(MD:-2.29,95%CI[-2.56,-2.01]),SAS scale score(MD:-5.77,95%CI[7.21,-4.34])and HAMA scale score(MD:-5.63,95%CI[-6.85,-4.41]),the differences were all statistically significant(P<0.01).In addition,the test group was able to improve the TCM symptoms with no significant adverse effects.(3)Study 3:A total of 222 chemical components were identified in Chaihujialonggumulitang,mainly including 72 flavonoids,31 triterpenoids,24 organic acids,15 anthraquinones,13 saponins,12 glycosides and 12 esters,as well as other classes of chemical components.(4)Study 4:①Cardiac ultrasound showed no significant differences in ultrasound indices between the control and sham groups;compared with the sham group,LVEF and LVFS were decreased in the AMI and complex groups(P<0.05),and LVIDd and LVIDs were enlarged in the complex group(P<0.05).Compared with the complex group,LVEF and LVFS were increased,LVIDd and LVIDs were reduced in the BFG and DMF groups(P<0.05).Serum myocardial enzymes showed that serum CK-MB and cTnT levels were elevated in the AMI and complex groups compared with those in the sham group(P<0.05).Compared with the complex group,serum CK-MB and cTnT levels were reduced in the BFG and DMF groups(P<0.05).H&E staining showed that myocardial fibers in the control and sham groups were orderly arranged with clear nuclei and uniform cytoplasmic staining.Compared with the sham group,myocardial fibers in the AMI and complex groups were broken,disorganized,with inflammatory infiltration and larger infarct area(P<0.05).Compared with the complex group,myocardial fibers in the BFG and DMF groups were less broken and the infarct area was reduced(P<0.05).Masson staining showed that myocardial fibers in the control and sham groups were orderly arranged with almost no collagen fibers.Compared with the sham group,there were more collagen fibers,fiber scar formation and severe myocardial fibrosis in the AMI and complex groups(P<0.05).Compared with the complex group,collagen fibers were reduced in the BFG and DMF groups(P<0.05).②In the behavioral test,there were no significant differences between the control and sham groups.In the elevated plus-maze test,the times entering the open arm and the dwell time in the open arm were decreased in the AMI and complex groups compared with the sham group(P<0.05).The times entering the open arm and the dwell time in the open arm were increased in the BFG and DMF groups compared with the complex group(P<0.05).In the open field test,the total distance and central distance were reduced in the AMI and complex groups compared with the sham group(P<0.05).And the total movement distance and central distance were increased in the BFG and DMF groups compared with the complex group(P<0.05).There was no significant difference in hippocampal 5-HT and DA expression between the control and sham groups.Compared with the sham group,the expression of hippocampal 5-HT and DA was decreased in AMI and complex groups(P<0.05).Compared with the complex group,the expression of hippocampal 5-HT and DA was increased in the BFG and DMF groups(P<0.05).Hippocampus pathological staining showed that the neurons in the hippocampal region of the control and sham groups had normal morphology,clear contours,tight arrangement,and uniform cytoplasmic staining.Compared with the sham group,the number of neurons in the hippocampal region of the AMI and complex groups decreased,were loosely arranged,and the number of nisus vesicles decreased.Compared with the complex group,the number of neurons in the hippocampus of the BFG group increased,were relatively closely arranged and had less damage.③In the oxidative stress parameters of myocardial tissue,serum and hippocampal tissue,there were no significant differences between the control and sham groups.Compared with the sham group,the MDA content was increased,while the SOD,CAT and GPX antioxidant enzyme activities were decreased in the AMI and complex groups(P<0.05).Compared with the complex group,the MDA content was decreased,while the SOD,CAT and GPX antioxidant enzyme activities were increased in the BFG and DMF groups(P<0.05).④In the gene and protein expression of Nrf2,HO-1 and Keapl in myocardial and hippocampal tissues,there were no significant differences between the control and sham groups.Compared with the sham group,Nrf2 and Keapl expression were increased,while HO-1 expression was decreased in the AMI and complex groups(P<0.05).Compared with the complex group,Nrf2 and HO-1 expression were increased,while Keapl expression was decreased in the BFG and DMF groups(P<0.05).⑤In the gene and protein expression of Bcl-2,Bax and Cleaved caspase-3 in myocardial tissue,there was no significant difference between the control and sham groups.Compared with the sham group,Bcl-2 expression was decreased,and Bax and Cleaved caspase-3 expression were increased in the AMI and complex groups(P<0.05).Compared with the complex group,Bcl2 expression was elevated,and Bax and Cleaved caspase-3 expression was reduced in the BFG and DMF groups(P<0.05).Conclusions:(1)In patients with coronary artery disease with comorbid anxiety,the pathogenic evidence elements were mainly heart,liver and spleen,involving kidney,gallbladder and large intestine;the pathogenic evidence elements mainly included qi stagnation,blood stasis,fire-heat,phlegm-dampness,qi deficiency and yin deficiency;the TCM evidence types were mainly qi stagnation,blood stasis,phlegm-fire disturbing the heart,liver-depression,spleen deficiency and heart-kidney yin deficiency.The degree of anxiety in patients with coronary artery disease with comorbid anxiety states was positively correlated with women,brain workers,overweight,obesity,hypertension,and history of hemodynamic reconstruction.(2)Meta-analysis showed that Chaihujialonggumulitang could improve angina pectoris and anxiety in patients with coronary artery disease with comorbid anxiety,and there were no adverse effects.The main pathological elements are qi stagnation,blood stasis,phlegm and fire heat.(3)Various chemical components of Chaihujialonggumulitang are known to scavenge free radicals,inhibit oxidative stress and cell apoptosis.(4)Chaihujialonggumulitang may alleviate oxidative stress and apoptosis by activating the Nrf2/HO-1 signaling pathway in rats with myocardial infarction with comorbid anxiety,thereby improving cardiac function and anxietylike behavior. |
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