The Effect And Mechanism Of Renalase On Sprague Dawley Rats With Myocardial Ischemia Reperfusion Injury

Objective:to study the effect of renalase on SD rats with myocardial ischemia-reperfusion (I/R) injury and explore the possible mechanism under three different conditions which include low level of renalase, normal level of renalase, and pretreatment with renalase recombinant protein.Methods:1. Cell experiments:Lentivirus vector mediated renalase gene silencing interference sequence (LV-Rnls-RNAi) transfer the H9C2cell, using QPCR and Western Blot to identify the inhibition ratio of renalase gene and protein, finding the best interference sequence.2. Renalase gene silencing of rats model:choosing15rats (male,10week,280-320g) and randomly dividing them into5groups (group Od, group4d, group7d, group10d, group14d). The Od group is blank control group; the other groups are injected the lentivirus (100ul,1E+8TU/ml) into the pericardial cavity of every rat. All rats are sacrificed in a set time. Using QPCR and Western Blot to identify the expression of renalase gene and protein and finding the best transfection time.3. myocardial I/R injury model:choosing36rats (male,10week,280-320g) and randomly dividing them into6groups:renalase gene interference (group RNAi), renalase gene interference negative control (group RNAi-NC), normal surgery (group Normal), Sham operation(group sham), pretreatment with renalase recombinant protein (group Protein), group Vehicle pretreatment (PBS), group RNAi and RNAi-NC were injected lentivirus into pericardial cavity10days ago before establishing I/R injury model; the method of establishing I/R injury model:blocking the left anterior descending coronary artery about45min and reperfusion24h; group Sham don’t block the bloodstream, Protein group and Vehicle group respectively subcutaneously were injected with renalase recombinant Protein (1.5mg/kg) and the same volume PBS10min before operation. Detecting the levels of renalase and NE by Elisa; measureing the myocardial necrosis and infarction area by double staining method; detecting myocardial cells apoptosis by TUNEL; using QPCR and Western Blot to detect myocardial tissue renalase expression.Results:1. The expression of renalase in group LV-Rnls-RNAi(19810-1), group LV-Rnls-RNAi (19811-1), and group LV-Rnls-RNAi (19813-1) decreased more obvious than group control, there is statistically significant difference(P<0.05), the inhibition ratio is63.47%,32.69%,93.66%respectively, and the inhibition ratio of group LV-Rnls-RNAi (19813-1) is83.1%to renalase protein production.2. There is no statistical difference in weight and blood pressure whether pre-operation or post-operation in each group (P<0.05). the renalase gene expression of group10d and14d decrease more obvious than group0d(P<0.05), but no statistical difference between them(P>0.05), and the renalase protein expression was inhibited by63.89%.3.(1) There is no statistical difference in weight and blood pressure whether pre-operation or post-operation of I/R in each group (P<0.05), and no statistical difference in infraction area after operation each group; group RNAi and group protein have significant difference versus group normal operation (P<0.05, n=3). The number of cells apoptosis of group RNAi increased more obvious than group control (P<0.05); and the group protein have inverse result.(2)The renalase gene expression of operative groups decreased versus group sham and have statistical difference (P<0.05); Group RNAi decreased more obvious than group RNAi-NC (P<0.05); the same result in renalase protein expression.(3) There in no statistical difference among each group about the level of pre-operation renalase (P<0.05); after operation, the renalase level of group protein increased more obvious than group vehicle (P=0.043); the NE level of group RNAi, group RNAi-NC, and group vehicle decreased more obvious after operation than respective the NE level of pro-operation, and have statistically significant difference (P<0.05).Conclusions:1. LV-Rnls-RNAi (19813-1) is the best interference sequence; That SD rats were injected Lentivirus into pericardial cavity can successfully establish renalase gene silencing model about10days after operation.2. The myocardial cells injured more serious after I/R when lack of renalase; renalase recombinant protein can protect against the cells injury of myocardial I/R.3. Renalase might protect against myocardial cells through anti-apoptosis. The renalase recombinant protein might become a drug that treat myocardial ischemia or myocardial I/R.