| BackgroundAs survival of oncology patients is prolonged and quality of life is improved,many tumors are gradually being treated as a chronic disease pattern over time.At the same time,studies have shown that the field of cardiovascular disease has paid a "heavy price" for this,with cardiovascular disease mortality among tumor survivors being higher than mortality from recurrence of the tumor itself.Oncology cardiology,as an emerging interdisciplinary discipline,has emerged in an era of rapid development of cardiovascular and oncology disciplines.Chinese medicine has now been proven to have definite efficacy in the treatment of oncological cardiology,and has played a positive role in the protracted battle against oncological cardiology.Our team found that myocardial damage caused by chemotherapy belongs to the core pathology of traditional Chinese medicine:"phlegm,stagnation and toxicity deficiency,toxicity damage to the heart ligaments",and our preliminary basic research found that crocin can protect cardiomyocytes by reducing oxidative stress and improving myocardial mitochondrial function.In view of the role of oxidative stress in cardiovascular diseases,the academic community has shown considerable interest in the potential role of saffron glucoside as a cardioprotective agent,especially in terms of anthracycline-induced cardiotoxicity,which deserves further study in clinical trials.However,the lack of clinical trials to verify the efficacy and safety of saffron glucoside is not conducive to the clinical promotion of saffron glucoside.Therefore,this clinical trial was designed to further clinically validate the efficacy of saffron glucoside and to explore the therapeutic significance of genetic candidate gene polymorphisms in the treatment of chemotherapy-induced cardiotoxicity.Objective1.To design a clinical trial to preliminarily evaluate the clinical efficacy and feasibility of the combined Chinese and Western medicine treatment of crocin on myocardial injury cardioprotection after anthracycline chemotherapy drugs for breast cancer,to provide highquality clinical evidence for the comprehensive treatment of oncological heart disease and to fill the gap of cardiotoxicity prevention drugs.2.To analyze the effect of saffron glucoside on cardiotoxicity of anthracyclines based on messenger ribonucleic acid(mRNA)sequencing,to integrate and discover saffron glucoside-regulated marker genes,to construct key protein interaction modules and differential gene co-expression networks,to enrich the interaction network between targets,and to analyze their core biological processes and pathways.3.To design genome-wide association studies to identify the expression of single nucleotide polymorphisms associated with anthracycline-related myocardial injury,to confirm the therapeutic significance of genetic candidate gene polymorphisms in the treatment of chemotherapy-induced cardiotoxicity,and to propose reliable genetic predictors of chemotherapy-induced cardiotoxicity.Method1.150 patients with confirmed primary breast cancer treated with anthracyclines were included in a randomized controlled trial and randomly divided into a trial group and a control group.Patients in the trial group were treated with saffron total-glucoside tablets in combination with standard chemotherapy regimen,and were given 4 saffron total-glycoside tablets orally three times a day for 6 months starting from day 1.Patients in the control group were treated with standard chemotherapy regimen.All patients were evaluated for efficacy at month 6 of treatment.The primary efficacy index was the incidence of cardiotoxic events,secondary efficacy indexes included echocardiography,electrocardiogram,brain natriuretic peptide,other indexes included quality of life evaluation scale,Chinese medicine evidence score scale and monitoring of adverse events and safety indexes.2.Peripheral venous blood was collected from 3 patients with primary breast cancer treated with saffron total glucoside tablets in combination with anthracyclines and 3 patients with breast cancer treated with anthracyclines only.Differential gene identification was performed on the extracted mRNAs from the two groups using DESeq package in R language.Network modules of protein interactions were constructed for all differential mRNAs,and the pathways of up-regulated mRNAs and down-regulated mRNAs were enriched and functionally annotated.Differential variable splicing analysis was performed on the data using rMATS software.Finally,co-expression analysis was performed and a coexpression network map was constructed using Cytoscape software to obtain the core regulatory genes.3.Peripheral blood samples were collected from 100 patients with primary breast cancer treated with anthracycline-based chemotherapeutic agents.Among them,65 patients without cardiotoxicity and 35 patients with cardiotoxicity were included to construct a DNA database of primary breast cancer patients treated with anthracycline-based chemotherapeutic drugs.Potential single nucleotide polymorphisms(SNPs)of predictive significance were identified by sequencing through DNA gene chips.Also based on the collected clinical information,corrected analysis was performed using age as a covariate,association analysis was completed using the R package and Plink to screen for significant SNPs,Manhattan and QQ plots were drawn,and LocusZoom analysis was used for selected genomic regions.Finally,the SNP loci with significant associations were annotated and enriched for susceptibility gene analysis.Result1.132 patients were finally included in the study and 18 patients were shed.There were 66 patients in each of the trial and control groups.The main efficacy index was the incidence of cardiotoxic events in both groups,which was 3.03%(2 cases)in the test group and 15.15%(10 cases)in the control group,with a statistically significant difference between the groups(P=0.015).The left ventricular ejection fraction(LVEF)after treatment was 64.88±3.98%and 63 ± 3.25%in the test and control groups,respectively,with a statistically significant difference between the groups(P=0.006).The change in LVEF from baseline in the test and control groups was-0.02±4.86%and 1.09±4.86%,respectively,showing no statistical difference(P>0.05).The comparison of LVEF before and after treatment was statistically significant in the control group(P=0.004),while no statistical difference was seen in the test group(P>0.05).The secondary indices included echocardiography,brain natriuretic peptide and electrocardiography.Among them,echocardiographic results were divided into LVEF,left ventricular end-diastolic diameter(LVEDD),left atrial diameter(LAD),right ventricular diameter(RVD),E-peak flow rate(E),A-peak flow rate(A),and E/A values,and there was no statistical difference between the test and control groups in the group comparison after treatment and between the group comparison of the difference before and after treatment(P>0.05).The E values in the control group were 82.96±16.52 cm/s and 75.13±17.29 cm/s before and after treatment,respectively.there was a statistically significant difference between the control group before and after treatment(P=0.004),while no statistical difference was observed in the test group(P>0.05).Brain natriuretic peptide values remained stable in both groups during the study period,with no statistically significant differences between and within group comparisons(P>0.05).ECG results were analyzed for heart rate,QRS,QTc,and P-R,and there were no statistical differences between the test and control groups in the between-group comparisons after treatment and between-group comparisons of the differences before and after treatment(P>0.05).The abnormal ECG results were also counted.The results showed that only 3 cases of atrioventricular block were observed in the control group,but there was no statistical difference between groups in the test group(P=0.08).These patients were one incomplete right bundle branch block,one first-degree AV block,and one ventromedial block,respectively.Only 4 patients in the control group had premature ventricular contractions,and the difference between the two groups was statistically significant(P=0.042).Ten patients in the test group had ST-T segment changes compared with 21 in the control group,with a statistically significant difference between the groups(P=0.024).A total of 8 of these patients had T-wave inversion and hypoplasia,1 patient in the test group and 7 patients in the control group,with a statistically significant difference between the two groups(P=0.029).One patient in the control group was assessed as grade Ⅱ when evaluated for NYHA classification after treatment.Anthracyclines cause a variety of arrhythmic and myocardial ischemic conditions,and patients in the trial group partially improved these abnormalities.For quality-of-life evaluation,using the FACT-B questionnaire as a reference for qualityof-life evaluation,there was no statistical difference between the test and control groups in terms of group comparison after treatment and group comparison of the difference before and after treatment(P>0.05).Of interest was the within-group comparison of functional status scores,which revealed a statistically significant within-group difference between the test and control groups before and after treatment(P=0.019;P=0.01),and anthracycline chemotherapy significantly affected the functional status of patients.In addition,withingroup comparisons of the social/family status scores and the total co-module were likewise significantly different in the control group(P=0.013;P=0.04),but no significant differences were found in the within-group comparisons of the test group(P>0.05).The Carlsbad score was used to assess the health status of the patients.There was no statistically significant difference between the two groups during the study period and in the comparison of the difference(P>0.05).The difference between the pre-and post-treatment Carlsbad scores in the test group was 88.94 ± 12.66 and 92.12±6.45,respectively(P=0.04).Patients in the trial group benefited in terms of health and tolerance of chemotherapy side effects.The TCM points before treatment were 1.23±1.4 and 1.17 ± 1.21 in the test and control groups,respectively,and after treatment were 1.2±1.42 and 1.21± 1.21,respectively,with a change from baseline of 0.03±0.17 and-0.05 ± 0.21 points,with a statistically significant difference between the two groups(P=0.026).To further analyze the characteristics of cardiotoxicity due to chemotherapy,patients were divided into cardiotoxicity group and non-cardiotoxicity group with reference to the primary endpoint event,and the distribution characteristics of TCM symptoms in patients with cardiotoxicity were summarized,i.e.patients with cardiotoxicity events were mainly characterized by distention and fullness,weakness,dry and bitter mouth,with occasional manifestations of dark complexion,palpitation and chest tightness;and patients with Qi stagnation,Qi deficiency,phlegm coagulation and blood stasis were the main characteristics of the symptoms.All patients enrolled in the trial did not have serious abnormalities in blood routine and liver and kidney function indexes,and no serious drug-related adverse reactions occurred,and the drug safety of saffron total glucoside tablets was good.2.The study obtained 16 saffron glucoside marker genes regulating tumor heart disease,of which 2 genes were up-regulated in expression(XKR3,AL592183.1)and 14 genes were down-regulated in expression(DEFA3,MT-RNR1,SLC25A39,PPBP,C9orf78,TMCC2,PF4,IGHV3-23,NRGN CLU,CA1,YBX1,FAM210B).The study also analyzed the functional pathways and interactions between differential genes and proteins,and obtained the relevant functional annotation maps of protein interaction networks and five groups of key protein interaction modules to explore the functional pathways of their differential genes found to be mainly related to oxidative stress,mitochondrial respiration,detoxification,and extracellular matrix receptor interactions.The study was also enriched with variable splicing analysis events and core regulatory genes(EGF,ALAS2,PF4,THBS1,AHSP,GYPB,CLU,PPBP,PROS1,SPARC).3.all patients with primary breast cancer included in the study were female and the cumulative dose of anthracyclines was on average 349± 92 mg/m2.at baseline,LVEF was 67.54± 5.13%and 64.29 ± 3.49%in the cardiotoxicity group(AIC group)and control group(Con group),respectively,and decreased to 57.88±4.5%and 63.31± 4.29%in the two groups at follow-up.±At baseline,LVEDD was 43.34 ± 4.15 mm and 43.92 ± 3.22 mm in the AIC and Con groups,respectively,at follow-up.The LVEDD expanded to 44.03±4.85 mm and 43.96±3.16 mm in the follow-up period,respectively,and the difference between the two groups was statistically significant,P<0.001;in addition,the LVEDD increased in both the AIC and Con groups,and the difference between the two groups was also statistically significant,P<0.05.Analysis of the 99 samples that passed the quality measurement finally revealed The 81 SNP loci showed genome-wide correlation,of which the SNPs locus with P<10-4 was the rs11619798 mutated gene(P=9.82E-5,95%confidence interval[0.09623,0.4613]).GO enrichment analysis showed that these genes were associated with cell adhesion,immune cell activation,cytoskeleton,immunoglobulin production,MHC-class Ⅱ protein complexes,purine nucleotide transmembrane transporter activity and other related functions.Conclusion1.The clinical study has shown that crocin combined with anthracycline chemotherapeutic agents for the treatment of primary breast cancer for 6 months effectively reduced the incidence of cardiotoxic events and improved patients’ quality of life and some clinical symptoms without affecting the efficacy of tumor treatment.2.The genes regulated by crocin involve multiple biological functions and signaling pathways,and the regulatory pathways of its differential genes may be related to oxidative stress,mitochondrial respiration,detoxification,and extracellular matrix receptors.3.The results of genome-wide association analysis confirmed the therapeutic significance of gene polymorphisms in the diagnosis and treatment of anthracycline-induced cardiotoxicity,and these results will contribute to early screening and individualized prevention of tumor heart disease. |
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