Abstract 174: Development of a Novel Anti-inflammatory and Anti-osteoplastic Peptide Based on De-loop and Ef-loop in Rankl for Treatment of Ischemic Stroke

Abstract

Background & Purpose: We previously showed that stimulation of RANKL (receptor activator of nuclear factor-B ligand)/ RANK (receptor for RANKL) signal with recombinant RANKL worked as protective in ischemic brain through inhibiting TLR signaling pathways. However, augmentation of RANK signaling is also causative for increasing osteoclast differentiation and osteoporosis. To overcome the problem, we developed a novel RANKL-based peptide, which is anti-inflammatory and anti-osteoclastic. Methods: We made four peptides: MHP1 with complete DE loop and 66.7% of EF loop including complete βE and 72.3% of βD; MHP2 with complete DE loop, 22.2% of EF loop, and 10% of CD loop including complete βE and βD; MHP3 including complete CD loop and DE loop including complete βD; MHP4 including complete DE loop and βD. The action of the peptides were evaluated in MG6 cells, RAW 264.7 cells, primary osteoclast precursor cells, and 40 min-transient cerebral artery occlusion (tMCAo) model in C57BL6/J mice. Results: In LPS-stimulated MG6 and/or RAW 264.7 cells, MHP1 and MHP2 inhibited expression of IL-6 and TNFα, whereas MHP3 and 4 showed no effects. Knocking down of RANK revealed that the effects of MHP1 was dependent on RANK signaling, indicating that some components of DE loop, EF loop, βD, and βE in RANKL were required to show the anti-inflammatory effects in TLR4-induced inflammation. The anti-inflammatory effect was also effective in TLR2/6-induced inflammation but not RAGE-dependent inflammation, suggesting that effects of MHP1 might be specific TLRs signaling. In primary osteoclast precursor cells, MHP1 did not stimulate osteoclast differentiation and also inhibited RANKL-induced osteoclast differentiation. In the ischemic mice, the i.c.v. injection of MHP1 at 4 hrs after the ischemia showed less infarct volume and less activated microglia/macrophages in ischemic brain at 72 hrs after ischemia. Conclusions: Thus, MHP1 has a unique property, such as stimulation of RANK related anti-inflammatory signaling with inhibitory effects for RANK-induced osteoclast differentiation. This indicated that MHP1 could be one of novel candidates for preventing inflammation and osteoporosis in ischemic stroke.