Roles Of PI3K/Akt/mTOR Signaling Pathway In Macrophage Autophagy And Atherosclerotic Plaque Vulnerability

Considerable evidence demonstrates that atherosclerosis (AS) is the development of a chronic inflammatory process, while the AS plaque acute inflammation could lead to plaque rupture and is the key factor of acute coronary syndrome (ACS). Inflammatory response, plaque vulnerability is the most important one of the internal factors. Macrophages in the plaque is the key factor of plaque inflammation reaction, and how to reduce the infiltration of macrophages in the plaque and to promote the stability of vulnerable plaques, has attracted great attention of domestic and foreign scholars. Previous studies have found that the changes of AS plaque macrophages are closely associated with autophagy, which is involved in the occurrence and development of AS. Autophagy is an important process to maintain cell homeostasis, which can be induced by hunger, pathogen invasion, cell differentiation, and so on. Autophagy possessed the capability of degrading the formation of long-lived protein, as well as cell organelles which were excess autologous or loss of the function growth process. PI3K/Akt/mTOR signaling pathway was detected that play an essential regulatory role in autophagy, and relative drugs which suppressed this signaling pathway for tumor treatment had achieved satisfactory results. Herein, this study was to select Akt inhibitor Casodex, mTOR inhibitor rapamycin, and mTOR-siRNA selectively inhibitor to suppress PI3K/Akt/mTOR signaling pathway, in order to promote the process of macrophage autophagy and to explore the potential mechanism and alternative strategy for inhibiting plaque inflammation and stablizing vulnerable plaques.Objective In present study, we aimed to investigate whether PI3K/Akt/mTOR is the signal transduction pathway of regulating the formation of atherosclerosis as well as macrophage autophagy in vulnerable plaques. Moreover, we attempted to explore that whether the selective inhibition of PI3K/Akt/mTOR pathway is able to reduce macrophages in vulnerable plaques by promoting the macrophage autophagy, in order to stabilize the atherosclerotic vulnerable plaque.Methods In vitro study, we used casodex (20μmol/l), rapamycin (10nmol/l) or mTOR-siRNA (30nmol/l) to treat mouse macrophage cell line RAW264.7, respectively. Inflammation related cytokines secreted by macrophages were measured by means of ELASA. Ultrastructural changes of macrophages were examined by transmission electron microscopy. mRNA or protein expression of protein kinase B (Akt), mammalian target of rapamycin (mTOR) and autophage related gene Beclin-1was assayed by real-time quantitative RT-PCR or Western blot, and expression of autophagy related indicator LC3-Ⅱ was detected by immunofluorescence or western blot. In vivo study, twenty-four New Zealand White rabbits underwent balloon-induced abdominal aortic wall injury and were fed on a diet of1%cholesterol for8weeks and then were randomly allocated to control group (n=8), casodex group (1.0mg/kg/d, n=8) and rapamycin group (0.5mg/kg/d, n=8). Four weeks after drug administrated, IVUS was carried out to observe the plaque imaging. Ultrastructural changes of macrophages, protein expression of macrophage, Akt, mTOR, LC3-Ⅱ were measured.Results In vitro study, more typical autophagosomes were detected in casodex, rapamycin or mTOR-siRN-treated cells. Expression levels of LC3-Ⅱ increased and Bekin-1and p-AKT, p-mTOR decreased significantly in the three treatment groups. Concentration of IL-10decreased while IFN-γ increased significantly in the treatment groups. In vivo study, IVUS found that EEMA, PA and PB decreased significantly in the casodex and rapamycin treated groups. Expression of LC3-Ⅱ increased significantly in the two treatment groups. Macrophage RAM-11and p-mTOR stainings were significantly reduced as compared with the control group.Conclusion In in vitro study, we successfully established the vitro research model of macrophage autophagy. Co-culture of macrophages and drugs were detected that related drugs and mTOR gene RNA interference could selectively inhibit PI3K/Akt/mTOR pathway via promoting macrophage autophagy. In in vivo study, we developed atherosclerotic vulnerable plaques in an animal model. Selectively inhibiting of PI3K/Akt/mTOR pathway showed that related drugs could induce macrophage autophagy, to reduce the infiltration of macrophages and to inhibite the inflammatory response in plaques, which is conducive to the stability of atherosclerotic vulnerable plaque.