SiRNA Silencing AT2 Receptor Inhibits no Generation in Cardiac Microvascular Endothelial Cells through Recombinant Human ACE2 Pathway

  • Yan Zhang

Abstract

To explore the NO formation after the impact of recombinant human Angiotensin Converting Enzyme 2 (rhACE2) via the AT2 pathway on the cardiac microvascular endothelial cells (CMVEC). Meanwhile, to investigate the mechanism of siRNA in this procedure. CMVEC were cultured in groups as follows: AngⅡ intervention group(for 24h); rhACE2 was added respectively after AngⅡ intervention; added AT2 receptor inhibitor (10umol/L) for 30min. And then, added rhACE2 (100 umol/L) 30min. Ransfect CMVEC by siRNA. The control group (normal CMVEC); the control group (normal CMVEC). Control group is negative siRNA (negative control, NCsiRNA). AT2 receptor protein expression was detected by western blot. We used NO content in cell culture supernatant was detected by Griess reagent measurement. Endothelial nitric oxide synthase (eNOS) mRNA in HUVEC expression was detected RT-PCR. We used NO fluorescent probe DAF-FM DA to detect intracellular NO formation and the activity of eNOS. Western blot detected the expression of phospho-eNOS. The content of NO in control group (11.513±0.392) was significantly higher than that in AngⅡ intervention group(3.495±0.362nmol/L) (P <0.05). The phosphor-eNOS expression levels and the NO contents of AngⅡ +rhACE2 treatment were significantly higher than those in AngⅡ group (P <0.05). But non-phospho-eNOS and eNOSmRNA protein expression were showed no significant difference between these (P > 0.05). And after AT2 pathway inhibitor (PD123319) intervented CMVEC, phosphor-eNOS expression were significantly lower than those in rhACE2 30min treated group (P <0.05). After siRNA transferened into CMVEC for 48h, Western blot test results showed that AT2 receptor’s protein expression decreased (P<0.05). AT2 siRNA transfected group’s NO level and eNOS activity was significantly reduced in negative control group and non-transfected control group. After rhACE2 acted on CMVEC, eNOS activity increased and NO production increased. AT2 receptor signaling pathway participated in this process.

Published
2019-07-15