Anti-inflammatory Effects Of A Novel Antiangiogenic Peptide And Its Mechanism

Purpose:H-RN, a novel antiangiogenic peptide derived from the kringle1domain ofhepatocyte growth factor (HGF), consists of the sequence RNPRGEEGGPW.Emerging evidence indicates that HGF and the kringle domain exhibitanti-inflammatory effects in inflammatory diseases. In the present study, we assessedthe anti-inflammatory effect of H-RN in cells and in models of experimental ocularinflammation, and demonstrated effects of H-RN on the PI3K/AKT/IKK pathway andNF-κB activation.Methods:1. Anti-inflammatory efficacy evaluation of H-RN in vitroLPS-stimulated RAW264.7murine macrophage cells wereused as one of the inflammatory models in vitro. The cell viabilities of RAW264.7cells were assessedusing the MTS cell proliferation assay kit. To evaluate its anti-inflammtory activity,cells were treated with H-RN or other drugs for30min before24h-combinedincubation with LPS. The protein expression of TNF-α and IL-6were assessed byELISA while mRNA levels were quantified by real-time PCR. The chemotaxis ofRAW264.7cells towards LPS were assessed with a transwell filter.HUVECs were incubated with H-RN or other drugs for18h, followed by6hstimulation with TNF-α. Quantification of monocyte-endothelial adhesion ispresented by the number of adhered U937cells per field adherent to the HUVECsunder a confocal laser scanning microscopy. Effects of H-RN on levels of ICAM-1,VCAM-1, E-selectin and P-selectin in TNF-α-stimulated HUVECs were determinedwith a human adhesion molecule multiplex kit.2. Anti-inflammatory efficacy evaluation of H-RN in vivoWistar rats were treated intravitreally with H-RN or other drugs in both eyesimmediately after LPS induction. Clinical manifestations were evaluated under abiomicroscope and scored24h after LPS injection. Effects of H-RN on proteinconcentration, infiltrating cell numbers, levels of TNF-α and IL-6in aqueous humor(AqH) of EIU rats were assessed. EIU rats’ eyeballs were fixed, sectioned and stainedwith H&E and the severity of inflammation was scored under a microscope.Lewis rats were immunized by injection of30μg R16peptide emulsified incomplete Freund’s adjuvant containing2.5mg/ml killed Mycobacterium tuberculosisinto the left hind footpads. On day6,9and12, Lewis rats were intravitreally treated with H-RN and other drugs in both eyes. Each day after immunization, clinical signswere monitored and scored. On day14, TNF-α, IL-6and IFN-γ levels in theAqH andICB-retina complex were assessed. On day16, histopathologic sections wereevaluated and scored blindly.3. Anti-inflammaoty mechanism reaserchThe localization of H-RN in RAW264.7cells was dertermined by monitoring thefluorescein isothiocyanate (FITC)-labled H-RN inside the cells. The intracellularlocations of NF-κB p65and pp65were assessed by immunofluorescence usingsecondary antibodies labeled with FITC or with rhodamine. Nuclear proteins wereanalyzed by western blot analysis using antibody against NF-κB p65or pp65. Theexpression levels of pp85, p85pAKTSer473, AKT, pIKKα/β, IKK or IκBα in whole cellextract were assessed by western blot.Results:1. Anti-inflammaoty effect of H-RN in vitroH-RN attenuated the LPS-induced mRNA and protein expression of tumornecrosis factor (TNF)-α and interleukin (IL)-6in RAW264.7cells and inhibited cellchemotactic migration towards LPS.H-RN also suppressed TNF-α-induced adhesion molecule expression in HUVECs,including ICAM-1, VCAM-1and E-selectin, which contributed to its suppressiveeffect on adherence of U937cells to endothelial cells.2. Anti-inflammaoty effect of H-RN in vivo H-RN concentration-dependently suppressed clinical manifestation, inhibitedocular inflammatory cytokine production and improved histopathologic scores in bothEIU and EAU rats.3. Anti-inflammaoty mechanismWestern blot and immunofluorescence staining analyses revealed that H-RNsignificantly suppressed LPS-induced phosphorylation of nuclear factor (NF)-κB-p65at Ser276. Based on examination of upstream pathways, we found that H-RNinhibited PI3K-p85and AKTSer473phosphorylation, which may result in theattenuation of LPS-induced IKK complex activation and IκB degradation.Conclusion：Intravitreal injection of H-RN, an antiangiogenic peptide derived from HGF K1,can inhibit inflammation in EIU and EAU clinically and histopathologically.Meanwhile, H-RN suppressed inflammatory cytokine production, as well as theadhesion and migration of inflammatory cells, possibly by interfering with thePI3K-AKT-IKK-IκBα-NF-κB signaling pathway.