Objective:To construct and identify the lentiviral vector of adenosine RNAi-adenosine A2a receptor (A2aR) in rats.Methods:Three pairs of short hairpin RNA(shRNA)-A2aR sequences (shRNA-A2aR 1, shRNA-A2aR 2, shRNA-A2aR 3) were designed, and three pairs of double-stranded shRNA oligos were respectively inserted into the shRNA virus vector to gain three kinds of shRNA lentiviral recombinant plasmid.The recombinant plasmid, packaging vector, and shuttle vector were co-transfected into 293T cells to obtain virus liquid.The experiment was performed in two parts.Part Ⅰ The rat primary cardiomyocytes were divided into 3 groups ( n= 6 each) by a random number table method: vehicle group (V group), shRNA-A2aR 1 group and shRNA-A2aR 3 group.Each group was transfected with virus solution of MOI 10 for 48 h. The expression of A2aR was detected by Western blot to select the most efficient lentivirus vector.Part Ⅱ The cardiomyocytes were randomly divided into 6 groups ( n=36 each): vehicle group (V group), MOI5 group, MOI10 group, MOI15 group and MOI20 group.Each group was transfected with the corresponding MOI virus liquid (the most effective lentivirus vector). At 24, 48, and 72 h of transfection, the cell viability and cell death were observed with a fluorescent microscope, and the A2aR expression was detected by Western blot to determine the interference efficiency. Results:Part Ⅰ Two types of shRNA-A2aR lentiviral vectors (shRNA-A2aR 1, 3) were successfully constructed, among which shRNA-A2aR 3 virus solution with a titer of 3.5×10 8 TU/ml had the best effect.Compared with group V and group shRNA-A2aR 1, the expression of A2aR in cardiomyocytes was significantly down-regulated ( P<0.01), and the interference efficiency of shRNA-A2aR 3 was 73% in shRNA-A2aR 3 group.Part Ⅱ shRNA-A2aR 3 was selected to screen out the transfection plan.The cell survival rate in each group was more than 85% at 24 h of transfection, the cell survival rate was more than 80% at 48 h of transfection in MOI5 and MOI10 groups; the cell survival rate in each group was less than 70% at 72 h of transfection.Under an inverted fluorescent microscope, a slightly lower fluorescence density was found in MOI5 group, the fluorescent density was higher and the cell condition was better at 48 h of transfection in MOI10 group and at 24 h of transfection in MOI20 group, and the cardiomyocyte viability was significantly decreased, and dead cells were increased at 72 h of transfection in each group.The results of Western blot showed that the interference efficiency at 48 h of transfection in MOI10 group, 48 h in MOI15 group, 24 and 48 h in MOI20 group was all > 70%. Conclusion:MOI of 10, transfection for 48 h or MOI of 20, transfection for 24 h is the optimal transfection protocol.

Objective:To evaluate the effect of activating adenosine A2B receptors on autophagy during myocardial ischemia-reperfusion (I/R) and the role of phosphatidylinositol 3-kinase/serine/threonine protein kinase (PI3K/Akt) signaling pathway in rats.Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, weighing 220-280 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (group Sham), myocardial I/R group (group I/R), adenosine A2B receptor agonist BAY 60-6583 group (group BAY) and BAY 60-6583+ PI3K inhibitor LY 294002 group (group BAY+ LY). Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion in group BAY.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion and LY 294002 10 mg/kg was intraperitoneally injected at 10 min before reperfusion in group BAY+ LY.Blood samples were obtained at the end of reperfusion for determination of concentrations of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct size (by Evan Blue and TTC double-staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 (LC3Ⅰ), LC3Ⅱ, Beclin-1 and phosphorylated Akt (p-Akt) (by Western blot). The ratio of LC3Ⅱ/LC3Ⅰ was calculated. Results:Compared with group Sham, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group I/R ( P<0.05). Compared with group I/R, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly decreased, the expression of p-Akt was up-regulated, the expression of Beclin-1 and LC3Ⅱ was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in the group BAY ( P<0.05), and no significant change was found in the parameters mentioned above in group BAY+ LY ( P>0.05). Compared with group BAY, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group BAY+ LY ( P<0.05). Conclusion:Activating adenosine A2B receptors can decrease autophagy of myocardial cells during myocardial I/R injury, and the mechanism may be related to activating PI3K/Akt signaling pathway in rats.

Objective:To evaluate the role of endoplasmic reticulum stress in myocardial ischemia-reperfusion (I/R) injury and the relationship with autophagy in rats.Methods:Thirty-six healthy adult male Sprague-Dawley rats, weighing 250-300 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (Sham group), myocardial I/R group (IR group), and endoplasmic reticulum stress inhibitor 4-PBA group (PBA group). Myocardial I/R was produced by occlusion of left anterior descending branch of coronary artery for 30 min followed by reperfusion for 120 min.Sham group only underwent thoracotomy without block of left anterior descending branch of coronary artery.Endoplasmic reticulum stress inhibitor 4-PBA 500 mg·kg -1·d -1 was given intragastrically for 3 consecutive days before the I/R model was developed in PBA group, while the equal volume of normal saline was given instead in Sham and IR groups.The blood samples from the iliac vein were collected at 120 min of reperfusion for determination of the plasma creatine kinase isoenzymes (CK-MB) and cardiac troponin I (cTnI) concentrations (by enzyme-linked immunosorbent assay). The rats were then sacrificed, and myocardial tissues were removed for detection of myocardial glucose-regulated protein 78 (GRP78) and microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and autophagy-related protein 5 (ATG5) expression (by Western blot). Result:Compared with Sham group, the concentrations of CK-MB and cTnI in plasma were significantly increased in IR and PBA groups, the expression of GRP78, ATG5 and LC3Ⅱ was up-regulated, and the pathological damage was aggravated in IR group ( P<0.05). Compared with IR group, the concentrations of CK-MB and cTnI in plasma were significantly decreased, the expression of GRP78, ATG5 and LC3Ⅱ was down-regulated ( P<0.05), and the pathological changes were significantly attenuated in PBA group. Conclusion:Endoplasmic reticulum stress is involved in the process of myocardial I/R injury, and the mechanism may be related to promotion of autophagy in rats.