Effects Of "Handle-region" Peptide And (Pro)renin Receptor-RNA Interference On Prorenin Activating PI3K\AKT Signal Pathway

Background:Atherosclerosis (AS) is the main disease that seriously affects the health of human being. The research of its mechanism has been going on for decades. It is confirmed that inflammatory reaction and endothelial dysfunction play a crucial role in formation and development of AS. Many risk factors can activate some signal pathway and inflammatory factors to cause endothelial dysfunction. AngiotensinⅡof renin-angiotensin system (RAS) plays an important role in AS. In the classical RAS, renin has been considered to have no biological activity except to enzymatically cleave angiotensinogen to angiotensinⅠ. Prorenin, as the precursor of renin, has always been assumed to have no function. But, the recognition about prorenin's function had emerged with the discovery of (pro) renin receptor [(P)RR]. Some research had showed that renin and prorenin exist biological function independent of angiotensinogen system by binding with (P)RR, including proliferation of mesangial cell, migration of smooth muscle cell, and so on. Very recently, our task group has been the pioneer in finding (pro)renin receptor (P)RR expression in human umbilical vein endothelial cell (HUVEC) by immunohistochemistry. Therefore, we hypothesized that prorenin and (P)RR might exist any similar pathophysiological functions in HUVEC as mesangial cell and smooth muscle cell."Handle-region" peptide(HRP) consists of 10 amino acid sequence of the prosegment of prorenin, which affects the binding of renin and prorenin with (P)RR. Therefore, HRP is considered the blocker of (P)RR. However, at present, the studies about the effect of HRP as (P)RR blocker is controversial. We wanted to observe whether HRP has effect on blocking (P)RR in HUVEC to protect endothelial function.Some studies showed that PI3K\AKT pathway can phosphorylate a variety of cytokines directly, such as NFκB, Bcl-2, TGF-β,and so on, which is related with vascular wall cell phenotype conversion, proliferation, migration and hypertrophy. So, we hypothesized that PI3K\AKT signal pathway may be a part of the mechanism of prorenin and (P)RR's founction.Objectives:To study whether prorenin could activate PI3K, AKT, NFκB, IL-6 in HUVECs through (P)RR independent of angiotensinⅡ(AngⅡ); and study whether HRP exists effect on (P)RR blocking.Methods:1. HUVECs were cultured in vitro.2. AT1/AT2 receptors of AngⅡwere blocked by Olmesartan(10-5M) and PD123319(10-4M) respectively to observe the prorenin(2×10-9M)'s effect on the expression of PI3K, p-AKT and NFκB-P65 in HUVECs, as well as IL-6 in cultural medium.3. (P)RR in HUVEC was treated by RNA interference with (F)RR siRNA, and HRP(1×10-6M,5×10-6M,10×10-6M) respectively, the changes of p-AKT, P65, IL-6 were observed.4. PI3K\AKT signal transduction pathway was blocked with wortm annin(0.5×10-6M), the changes of p-AKT, P65, IL-6 were observed.5. The efficiency of (P)RR interference is evaluated by RT-PCR.6. PI3K, p-AKT, p65 were evaluated by western blot.7. The concentration of IL-6 in cultural medium was evaluated by ELISA kitResults:1. Prorenin up-regulated the expression of PI3K, p-AKT, NFκB-P65, and IL-6.2. The activation of p-AKT, NFκB-P65 and IL-6 was inhibited by (P)RR-RNA interference.3. All of the three doses of HRP did not affect the activation of AKT, NFκB-P65 and IL-6 caused by renin/prorenin\(P)RR. 4. The activation of P65 and IL-6 was not inhibited by wortmannin.Conclusions:1. Prorenin can up-regulate the expression of PI3K, p-AKT, P65 and IL-6 independent of AngⅡgeneration.2. The activation of AKT, NFκB-P65, IL-6 by (pro)renin is through (P)RR.3. HRP does not have effect on (P)RR blocking.4. The expression of P65 and IL-6 may not be regulated through PI3K\AKT signal transduction pathway.