LXW7

Microglia activation is closely linked to ischemia, various chronic neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis), and many other central nervous system diseases. Accumulating evidence suggests that depressing the microglial inflammatory response could be an effective treatment for inflammatory disorders. The integrin αvβ3 inhibitor LXW7 has a neuroprotective effect; however, its anti-inflammatory effects and underlying mechanism remain unclear. Thus, we examined whether LXW7 would inhibit inflammatory cytokines and mediators, and we evaluated the potential mechanisms of its neuroprotective effects. Nitrite analysis revealed LXW7 reduced the nitric oxide (NO) level. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) suggested that LXW7 suppressed the expression of proinflammatory genes for tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and anti-inflammatory gene interleukin 10 (IL-10) at the messenger ribonucleic acid level. Enzyme-linked immunosorbent assay results demonstrated that LXW7 treatment reduced the expression of prostaglandin E2 (PGE2), TNF-α, IL-1β and IL-10 at the protein level. Western blotting was conducted to confirm the upregulation of inflammatory factors, including iNOS and COX-2 at the protein level. LXW7 inhibited major genes in the Akt/NF-κB and c-Jun NH2-terminal kinase/ mitogen-activated protein kinases (JNK/MAPK) signaling pathways. Immunofluorescence revealed that LXW7 inhibited the nuclear translocation of nuclear factor kappa B (NF-κB). Thus, LXW7 effectively alleviated LPS-induced inflammatory damage and had neuroprotective effects. The anti-inflammatory effects of LXW7 may be associated with the inhibition of microglial activation via Akt/NF-κB and JNK/MAPK signaling pathways by blocking integrin αvβ3 receptor. The present study's findings suggest that LXW7 has a substantial therapeutic potential for treating inflammatory and neurodegenerative diseases.

CeO2 nanoparticles (nanoceria) have been used in many studies as a powerful free radical scavenger, and LXW7, a small-molecule peptide, can specifically target the integrin αvβ3, whose neuroprotective effects have also been demonstrated. The objective of this study is to observe the neuroprotective effect and potential mechanism of CeO2@PAA-LXW7, a new compound that couples CeO2@PAA (nanoceria modified with the functional group of polyacrylic acid) with LXW7 via a series of chemical reactions, in H2O2-induced NGF-differentiated PC12 cells. We examined the effects of LXW7, CeO2@PAA, and CeO2@PAA-LXW7 on the viability of primary hippocampal neurons and found that there was no significant difference under control conditions, but increased cellular viability was observed in the case of H2O2-induced injury. We used H2O2-induced NGF-differentiated PC12 cells as the classical injury model to investigate the neuroprotective effect of CeO2@PAA-LXW7. In this study, LXW7, CeO2@PAA, and CeO2@PAA-LXW7 inhibit H2O2-induced oxidative stress by reducing the production of reactive oxygen species (ROS) and regulating Bax/Bcl-2, cleaved caspase-3 and mitochondrial cytochrome C (cyto C) in the apoptotic signaling pathways. We found that the levels of phosphorylation of focal adhesion kinase (FAK) and of signal transducer and activator of transcription 3 (STAT3) increased significantly in H2O2-induced NGF-differentiated PC12 cells, whereas LXW7, CeO2@PAA, and CeO2@PAA-LXW7 suppressed the increase to different degrees. Among the abovementioned changes, the inhibitory effect of CeO2@PAA-LXW7 on H2O2-induced changes, including the increases in the levels of p-FAK and p-STAT3, is more obvious than that of LXW7 or CeO2@PAA alone. In summary, these results suggest that integrin signaling participates in the regulation of apoptosis via the regulation of ROS and of the apoptosis pathway in H2O2-induced NGF-differentiated PC12 cells. LXW7, CeO2@PAA, and CeO2@PAA-LXW7 can play neuroprotective roles by counteracting the oxidative stress and apoptosis induced by H2O2 in NGF-differentiated PC12 cells. CeO2@PAA-LXW7 exerting a more powerful synergistic effect via the conjunction of LXW7 and CeO2@PAA.

Microglia are the inherent immune effector cells in the central nervous system (CNS), are activated rapidly when the CNS is stimulated by ischaemia, infection, injury, etc. and participate in and aggravate the development of inflammatory reactions in the CNS. During the process of microglial activation, inflammatory factors such as TNF-α and IL-1β and an abundance of reactive oxygen species (ROS)/reactive nitrogen species (RNS), are released by damaged nerve cells. LXW7 is a small molecule peptide and specifically binds with integrin αvβ3. Cerium oxide nanoparticles (nanoceria) are strong free radical scavengers and are widely used in many studies. In this research, a model of inflammation was established using lipopolysaccharide (LPS) to induce BV2 microglia activation, and the effects of CeO2@PAA (synthetic nanoscale cerium oxide particles), LXW7 and CeO2@PAA-LXW7 were evaluated. We detected the expression level of inflammatory factors, the release of NO in BV2 cells and the generation of intracellular ROS. The expression levels of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) and their phosphorylated proteins were detected in BV2 microglia. We found that CeO2@PAA, LXW7 and CeO2@PAA-LXW7 all effectively inhibited the activation of BV2 microglia, reduced the production of cytokines and the release of NO and reduced the production of intracellular ROS. The three treatments all inhibited the phosphorylation of FAK and STAT3 in BV2 microglia. Regarding these effects, CeO2@PAA-LXW7 was more effective than the other two monotherapies. Our data indicate that CeO2@PAA, LXW7 and CeO2@PAA-LXW7 can exert a neuroprotective function by inhibiting the inflammatory response of LPS-induced BV2 microglia. LXW7 may inhibit the activation of FAK and STAT3 signals in combination with integrin αvβ3 to restrain neuroinflammation and the antioxidative stress effect of cerium oxide; hence, CeO2@PAA-LXW7 can exert a more robust anti-inflammatory and neuroprotective effect via synergistically suppressing the ability of LXW7 to influence the integrin pathway and the free radical-scavenging ability of CeO2@PAA.