In this case-control study we enrolled a total number of 89 non-diabetic patients, with known DCM with NYHA (New York Heart Association) class II and III who were referred to cardiology department affiliated to Namazee hospital, Shiraz, Iran between March 2016 and February 2019. The patients participated in our study were DCM ones that were diagnosed more than 3 months before the study and received optimal medical therapy according to medical guidelines and had no history of hospitalization. Of these patients, 49 were diagnosed as ischemic ones evidenced by coronary angiography consist of advanced three-vessel coronary artery disease or thrombosis-induced transmural myocardial infarction and forty patients were included in idiopathic DCM group after ruling out the possible causes by means of history, transthoracic echocardiography and coronary angiography. Also, we enrolled 49 age and sex adjusted healthy non-diabetic controls without any disease to compare with these two groups. All patients underwent two-dimensional echocardiography using a vivid E9 system (GE, Norway) and all measurements were performed according to the latest recommendations of American Society of Echocardiography  .The left ventricular end-diastolic volume (LVEDV) and left ventricular ejection fraction (LVEF) was calculated based on Simpson’s biplane method. For homogenous sampling, the LVEDV index exceeding 100 mL/m2 and LVEF between 20-35% were considered as inclusion criteria as previously described in similar performance . The present methodology met all the rules contributed to Helsinki protocol. This study was approved by the institutional review board of Shiraz University of Medical Sciences and won the approval of the Ethical Committee (IR.SUMS.MED.REC9045.39.01.93). All the experimental methods programed in the present study were in accordance with confirmed setups used in previous studies.
Sample collection and ribonucleic acid isolation
Five-milliliter peripheral blood was collected in Ethylenediaminetetraacetic acid (EDTA)-containing tubes from each patient at the time of diagnosis prior to chemotherapy treatment and also healthy individuals. The peripheral blood mononuclear cells (PBMCs) were isolated from each patient and controls using Ficoll-hypaque density gradient centrifugation. Total RNA was extracted by TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions and guidelines as previously described briefly [15, 23, 24].
SYBR green real-time polymerase chain reaction
For the quantitative analysis of IL 1, IL27 and TNF mRNAs expression level, the SYBR Green Real-Time PCR method was performed using SYBRPremix Ex TaqTM II (Tli RNaseH Plus) (Takara, Japan) and designed primers speciﬁc for each genes in an iQ5 thermocycler (BioRad Laboratories, USA) according to the manufacturer’s instructions as previously described briefly [15,23, 24].
Data were analyzed by Statistical Package for Social Sciences (SPSS) software, version 18. The differences in the mean expression level of IL 1, IL 27 and TNF-α before and after chemotherapy as well as patients according to the comparison of NYHA class, LVEF and LVEDVI between patients with idiopathic and ischemic dilated cardiomyopathy which were compared independent t test. Also, the mean expression level of IL 1, IL 27 and TNF-α regarding laboratory data was analyzed by the Chi-square test.