Surgical management and Grouping of patients
24 children patients with non-cyanotic congenital heart disease were from the Department of Cardiothoracic surgery, the second affiliated Hospital of Wenzhou Medical University and Yuying Children's Hospital. Exclusion criteria: 1) patients with severe complications such as severe liver disease, kidney disease or lung disease affect the peripheral vascular disease of the upper limb; 2) patients or their families refuse to participate in the clinical trial. Withdrawal criteria: patients with anesthesia, surgical accidents and other adverse events during the experimental observation had to stop the trial. All the expenses for the treatment of the patients are paid by the Children's Heart Center of the second affiliated Hospital of Wenzhou Medical University, which does not increase the additional financial burden of the subjects. This study has been approved by the Ethics Committee of the Department of Cardiothoracic surgery of the second affiliated Hospital of Wenzhou Medical University and the Ethics Committee of Yuying Children's Hospital.
Self-control study on pre and post RIPC is performed in these patients, the cardiac tissues obtained before and after RIPC were randomized into control group and RIPC group respectively. After anesthesia and thoracotomy, a small amount of right atrial tissue is taken from above the purse-string suture area of the right atrium intubation. Placing the cuff on patients’ upper arm, inflate to 200mmHg to block the blood flow for 5 minutes then relax the cuff to restore perfusion for 5 minutes, repeated 4 times, and took a small amount of right atrial tissue again. After the specimen is collected, the operation begins. Echocardiography and electrocardiogram will be used to detect heart rhythm, cardiac correction and recovery of cardiac function during operation or at least 3 days after operation.
Grouping and Treatment of experimental animals
C57BL/6 mice, 6-8 weeks old, weighing 22-26g (provided by Vital River Laboratory Animal Technology Co., Ltd., Beijing, China), Mice are kept in a quiet room with 12/12h cycle light/dark at a constant temperature of 20~22 ℃ and provided with adequate water and food. All surgical procedures and postoperative care were carried out in accordance with the guidelines for the Care and use of Experimental Animals issued by the Chinese Academy of Health. All the operations involving mice were approved by the Animal Research Committee of Wenzhou Medical University(wydw2016-0334). Forty C57BL/6 mice are randomly grouped as follows: 1) Sham group(n=10): only undergo dissociation of the left femoral artery and thoracotomy.; 2) MIRI group(n=10): ligation and reperfusion of the left anterior descending branch are given on the basis of the separation of the left femoral artery and thoracotomy.; 3) RIPC+MIRI group(n=10): separating left femoral artery for RIPC, then ligate left anterior descending branch and reperfuse by thoracotomy; 4) RIPC group(n=10): separating left femoral artery for RIPC, then obtain the heart by thoracotomy.
RIPC in C57BL/6 mice
After endotracheal intubation and anesthetized with isoflurane, the limbs of mice are fixed with tape, a small incision is made along the left groin, the femoral artery is freed, and the left femoral artery is gently clamped with a microvascular clamp for 5 minutes, then relax the vascular clamp and restore perfusion for 5 minutes. Repeat 4 cycles.
MIRI model of mice
After isoflurane induced anesthesia, the mice are endotracheal intubated and adjusted to right lateral position. Thoracotomy is performed on the third or fourth intercostal on the left side to expose the pericardium. Open the pericardium with microscopic forceps and ligate the left anterior descending branch of coronary artery with 6-0 silk thread to induce myocardial ischemia, After 30 minutes of myocardial ischemia, loose ligation so that the heart can be reperfused. Meanwhile, indwell a small hose in chest to extract residual air in the chest, suture layer by layer rapidly to chest cavity. Then mice are resuscitated by 100% oxygen ventilation and transferred to a warm pad. 180 minutes later, the re-anesthetized mice are killed and the samples needed for the experiment were taken. The occlusion and reperfusion of the left anterior descending branch can be judged by the significant changes in myocardial color.
Total RNA Purification
Total RNA was extracted from collected myocardial tissue with the Trizol reagent (15596026; Ambion, Cambridge, UK). RNA content is quantified with 260/280 UV spectrophotometry. After being qualified, isolated high-purity total RNA is subjected to LncRNA microarray analysis or qRT-PCR testing.
LncRNA Microarray Analysis
Arraystarhuman and mouse LncRNA microarrays V3.0 provided by KangChen Bio-tech Co. Ltd. (Shanghai, China) are used to detect our RNA samples. RNA labeling and array hybridization
are conducted as previously described[9, 10]. Differential expression levels of LncRNAs and mRNAs between the RIPC group and the Sham group are compared. Among detected up-regulated RNAs, adjusted P value ＜0.05 and either whose FC value（fold change）＞2 & Chip intensity ＞500 or FC value ＞3 & Chip intensity ＞50 are considered as differentially expressed. As for down-regulated RNAs, adjusted P value ＜0.05 and either whose FC value＞2 & Chip intensity＞1000 or FC value＞3 & Chip intensity＞50 are defined differentially expressed.
GO and KEGG Pathway Analysis
KangChen Bio-tech Co. Ltd. (Shanghai, China) is commissioned to conduct GO and KEGG pathway analysis to systematically analyze differentially expressed genes and enrich important GO terms and KEGG pathways（P＜0.05）. The significance of the P value is evaluated by FDR (false discovery rate) and recommended FDR value is less than 0.05.
Western Blot Analysis in vivo
Extract total protein from mice’s myocardial tissue (5 mg) and determinate the protein concentration with Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). The following primary antibodies were used: 1:1000 diluted Rabbit anti-Bax (50599-2-Ig; Proteintech Group, Inc), Bcl2(26593-1-AP; Proteintech Group, Inc), Caspase3(66470-2-Ig; Proteintech Group, Inc), cleaved-Caspase3(WL01992; Wanlei Biotechnology Co., Ltd.), TNF-α (17590-1-AP; Proteintech Group, Inc), IL-6(WL02841; Wanlei Biotechnology Co., Ltd.)primary antibody. The next day, added 1:3000 diluted Goat Anti-Rabbit antibody (SA00001-2; Proteintech Group, Inc), use ECL for coloration, scan and analyze the results with the Gel imaging system. After quantification, ratio of each protein to GAPDH is to be used to compare and analyze.
Myocardial infarct size measurement
Assess myocardial infarct size through Evans Blue (E2129; Sigma, St. Louis, MO) / TTC (2,3,5-triphenyltetrazolium chloride, T8877; Sigma) double staining method. The viable tissue is stained red and white by TTC was defined as area at risk (AAR). Non-ischemic myocardial tissue is stained deep blue by Evans Blue. Infarct area (INF) looks pale after staining. The percent of infarcted area over area at risk (INF/AAR ratio, IAR%) is calculated.
Mice’s hearts are fixed in 4% paraformaldehyde. After 24h, embed fixed hearts with paraffin and slice the embedded hearts (5 μm thick). Sections are stained with HE according standard protocol and analyzed by light microscopy.
Plasma cTnI, TNF-α concentration
The serum levels of TNF-α, cTnI were measured using a mouse Enzyme-Linked ImmunoSorbent Assay kit ( Boyun Biotech Co., Ltd, Shanghai) in accordance with the manufacturer's instructions.
Extract total RNA from myocardial tissue using the Trizol reagent (15596026; Ambion, Cambridge, UK), quantatify RNA content by 260/280 UV spectrophotometry. The same volume of RNA solution was used to reverse transcriptase by the RT‐PCR kit (FSQ‐101; TOYOBO, Kita‐ku, Osaka, Japan). The expression of these LncRNAs are quantified by SYBR Green (170‐8882AP; Bio‐Rad, Hercules, CA) two‐step, real‐time RT‐PCR using CFX96 Touch Real‐Time PCR Detection System. The expression of LncRNA is normalized to GAPDH mRNA content and calculated using comparative methods.
Genes Homology Analysis & CeRNA Mechanism Analysis
Genes Homology Analysis and CeRNA Mechanism Analysis are performed by KangChen Bio-tech Co. Ltd. (Shanghai, China). Detected up-regulated homologous LncRNAs, whose chip intensity＞500, FC value ＞1.0, P＜0.05 and E-value ＜0.05 are selected. Detected down-regulated homologous LncRNAs, whose chip intensity＞50, FC value ＞1.0, P＜0.05 and E-value ＜0.05 are selected. Finally, the ceRNA mechanism analysis of stable differentially expressed homologous LncRNAs is performed.
All data in this article are expressed as mean±SEM. Comparisons between groups are assessed by T-test. All statistical analyses were performed using Graph Pad Prism Software (Version 8.0, La Jolla, CA). P < 0.05 was considered statistically significant.