The Research On Antihypertensive Effect Of Metoprolol Influenced By Knockdown Of β1-AR In Myocardial Tissue Of Spontaneousely Hypertensive Rats Mediated By AAV9-shRNA-ADRB1-zsGreen

ObjectiveTo investigate the influence of knockdown of β1-AR in myocardium of SHRs on antihypertensive effect of metoprolol.Methods1. The experiment of packaging and identification of viral vectors: Confirmed by enzyme digestion and gene sequencing, wheather the preliminary plasmid100%identical to its accession number. The plasmid was then packaged to recombinant adeno-associated virus named AAV9-shRNA-ADRB1-ZsGreen and harvested. The purity, titer and in vitro activity of infection were detected.2. The experiment of SHR myocardial transfection:36SHRs were grouped into three randomly as follows:AAV9-shRNA-ADRB1-ZsGreen injection group(n=12); AAV9-shRNA-ADRB1-ZsGreen injection group (n=12); sham group(n=12); Virus vector used the pericardial cavity injection transfected SHR myocardial. Rats were killed and dissected the myocardial tissue after4weeks. The research on the expression of β1-AR in myocardial tissue among all rats groups. In transcription and translation levels, Real-time RT-PCR, Immunohistochemistry and Western blot were used to assay the expression ofβ1-AR.3. The experiment of metoprolol treatment:SHRs were meanwhile grouped into six randomly as follows:Group1, AAV9-shRNA-ADRB1-ZsGreen+metoprolol(n=6);Group2, AAV9-shRNA-ADRB1-ZsGreen+saline(n=6); Group3, AAV9-CMV-ADRB1-ZsGreen+metoprolol(n=6); Group4, AAV9-CMV-ADRB1-ZsGreen+saline(n=6); Group5, sham+metoprolol(n=6);Group6, sham+saline(n=6). Metoprolol and saline were given by gavage in4weeks. All of them were monitored their blood pressure weekly. Results1. Packaging and identification of virus vector:(1) Enzyme digestion and gene sequencing confirmed that the pAAV-ZsGreen-ADRB1-shRNA plasmid is correct;(2) Viral titer results showed that the purified AAV9-shRNA-ADRB1-ZsGreen titer was1.5x1012vg/mL;(3)293cells infected with a viral vector efficiency up to95%.2. The effect of SHR myocardial transfection:(1)Real-time RT-PCR results showed that AAV9-shRNA-ADRB1-ZsGreen injection could significantly decrease β1-AR mRNA expression compared with the negative control group (0.4142±0.3399vs.1.0933±0.5853, P<0.05);(2) Immunohistochemistry results showed that it could significantly decrease the (31-AR protein expression compared with the negative control and sham group (50.02±4.44vs.80.05±3.72vs.79.30±2.79, P<0.05);(3) Western blot results showed that it also could significantly decrease the β1-AR protein expression compared with the negative control group (0.5156±0.1199vs.0.8333±0.1728, P<0.05).3. The effect of knockdown of β1-AR in myocardial tissue of SHR on blood pressure and efficacy of metoprolol:After4weeks of the intervention,(1) systolic blood pressure in Group1, Group3, Group5was significantly lower than Group4and Group6(174.8±3.9mmHg vs.168.3±5.3mmHg vs.167.2±5.1mmHg vs.199.4±3.1mmHg vs.198.3±4.5mmHg, P<0.05);(2) Group2was significantly lower than Group6(178.1±6.3mmHg vs.198.3±4.5mmHg, P<0.05);(3) the SBP drop pressure amplitude of Group1were significantly lower than Group3and Group5(22.84±1.72vs.28.78±2.72vs.30.16±5.71, P<0.05);(4) Group1and Group2showed no significant difference in the SBP drop pressure amplitude (22.84±1.72mmHg vs.18.84±6.55mmHg, P>0.05)Conclusions1. The AAV9-shRNA-ADRB1-ZsGreen is successfully packaged.2. AAV9-shRNA-ADRB1-ZsGreen transfected SHR myocardial can significantly reduce the β1-AR expression in myocardial of SHRs.3. Knockdown of β1-AR in myocardial tissue of Spontaneousely Hypertensive Rats resulted in lower blood pressure and.insensitive to metoprolol.