Abstract 309: TLR4 Gene Silencing Attenuates Atherosclerosis Progression via Inhibition of PI3-kinase/Akt/NF-κB Signaling Pathway in Apolipoprotein E Knockout Mice
TLR4 activation induces the expression of IL-6 which may initiate or aggravate atherosclerotic lesions. The purpose of this study was to test the hypothesis that TLR4 gene silencing may attenuate atherosclerosis progression through inhibiting the expression of IL-6 induced by endotoxin.TLR4 knockdown ECV304 cell line (ECV304-TLR4) was constructed and cells were divided into four groups: control group (with fresh serum-free medium in ECV304 cells), LPS group (with LPS in ECV304 cells), LPS + LY294002 group (pretreatment with LY294002 and then adding LPS in ECV304 cells) and LPS + RNAi group (treatment with LPS in ECV304-TLR4). Cells were collected and the protein expression of P-AKT, AKT, P65, Iκ-α and IL-6 were assayed. Fifty-four apoE−/− mice were equally divided into two groups: group 1 received adenoviral RNAi vector containing TLR4 and group 2 received adenoviral RNAi vector containing scrambled siRNA as a negative control at the end of week 6. All mice were fed a high-fat diet and received celiac injections of LPS (1mg/kg, twice per week) for 10 weeks and were then sacrificed. Levels of serum lipids and IL-6 were measured. Plaque contents were evaluated by immunohistochemistry and the vulnerability index (VI) was calculated as:VI = (macrophage + lipid)/(SMC + collagen) contents. The results showed that TLR4 protein expression in vitro was significantly inhibited in ECV304-TLR4. Compared with control cells (0.67 ± 0.002), the NF-κB binding activity reached the maximum at 30min stimulation of LPS (1.89 ± 0.04),which was inhibited by RNAi (0.96 ± 0.04) (P < 0.05). LPS-induced protein expression of PI3K/Akt signaling pathway and expression of IL-6 were significantly suppressed in LPS + RNAi group (P < 0.05). In vivo study demonstrated that transfection of TLR4 RNAi adenoviral vector significantly increased collagen content and plaque cap thickness and decreased TLR4 and IL-6 expression and VI in plaques in group 1 compared with group 2 (P < 0.05). Plaque Area, lipid content and serum lipid levels between the two groups showed no significant difference. In conclusion, TLR4 gene silencing significantly reduces the expression of IL-6 and attenuates atherosclerosis progression probably through the inhibition of PI3-kinase/Akt/NF-κB signaling pathway.