Temporin-1CEa and its Analog LK2(6)A(L) Confer Neuroprotective Effects in Parkinson's Disease Model by Attenuating Neuroinflammation Via the NF-κB and MAPK Signaling Pathways.

Abstract

The formation of Parkinson's disease is affected by various factors, among which neuroinflammation mediated by microglial activation plays a crucial role in the advancement of neurodegenerative diseases. Antimicrobial peptides temporin-1CEa and its analog LK2(6)A(L) exhibit excellent anti-inflammatory activity. To understand the anti-neuroinflammatory mechanisms of antimicrobial peptides in an immortalized mouse microglial cell line (BV2 cells), and assess neuroprotective effects in a PC12 cell line (rat adrenal pheochromocytoma cell) and Caenorhabditis elegans. Lipopolysaccharide (LPS, 500 ng/mL) was used to induce neuroinflammation in microglial BV2 cells. The effects of antimicrobial peptides on inflammatory cytokines and anti-inflammatory pathways activated by microglia were evaluated using cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay (ELISA), real-time quantitative PCR(RT-qPCR), and western blotting (WB). An apoptosis model was established by treating PC12 cells with the supernatant of LPS-stimulated BV2 cells, and the influence of antimicrobial peptides on apoptosis was analyzed via flow cytometry (FCM), Western blotting, and caspase-3 and caspase-9 activity assays. In the transgenic nematode BZ555, an LPS (200 μg/mL)-induced model of cephalic dopaminergic neurons (CEPs) injury was developed to explore the protective effects of antimicrobial peptides on dopaminergic neuron damage, food-sensing behavior, body bending, neurotoxicity, and lifespan. Furthermore, NL5901 was employed to evaluate the capacity of antimicrobial peptides to clear the accumulation of alpha-synuclein (α-synuclein) and their impact on body bending, neurotoxicity, and lifespan. Temporin-1CEa and LK2(6)A(L) inhibited the release of pro-inflammatory mediators by downregulating the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. At 3.125 μM, both temporin-1CEa and LK2(6)A(L) inhibited the apoptosis of PC12 cells induced by activated BV2 cells. In vivo experiments in nematodes demonstrated that temporin-1CEa and LK2(6)A(L) alleviated the damage to dopaminergic neurons induced by LPS and mitigated the capability to mitigate the accumulation of α-synuclein. In this study, antimicrobial peptides were shown to control inflammatory factors by downregulating the NF-κB and MAPK signaling pathways, thereby providing valuable insights for the agents in the neuroinflammation model of Parkinson's disease. Additionally, an unexpected finding revealed that these peptides could effectively reduce the accumulation of α-synuclein, which is a critical pathogenic factor, as its aggregated forms significantly contribute to Parkinson's disease progression. Notably, temporin-1CEa and LK2(6)A(L) exerted neuroprotective effects on dopaminergic neurons by inhibiting neuroinflammation and clearing the accumulation of α-synuclein.