Background: Achyranthes bidentata polypeptide fraction k (ABPPk) has been shown to protect ischemic stroke and Parkinson's disease, and can inhibit neuroinflammation in lipopolysaccharide (LPS)-activated BV2 microglia. However, the effect of ABPPk responsible for alleviating microglial neurotoxicity remains unknown.
Methods: Primary microglia were cultured to investigate the effect of ABPPk on LPS-induced neuroinflammation. Microglia conditioned medium (MCM) was collected to stimulate primary cortical neurons and then the neuronal viability, lactate dehydrogenase (LDH) release, intracellular calcium influx, mitochondria membrane potential (MMP) were assessed, respectively. Postnatal day 5 Sprague-Dawley rat pups were intracerebral injected with LPS to establish an LPS-induced brain injury model. Double immunohistochemical staining for NeuN and Iba1 was performed to evaluate the effects of ABPPk on LPS-induced neuronal damage and microglial activation. TUNEL assay was conducted to detect cell apoptosis in LPS-injected brain. The effect of ABPPk on LPS-induced NADPH oxidase 2 (NOX2) expression and reactive oxygen species (ROS) production as well as the phosphorylation of protein kinase B (Akt) was detected. Moreover, LY294002 (a specific PI3K inhibitor) and SC79 (a specific Akt activator) were used to further reveal the underlying mechanism.
Results: ABPPk pretreatment inhibited LPS-induced NLRP3 and cleaved caspase 1 expressions as well as the mRNA levels of IL-1β and IL-18. Moreover, ABPPk inhibited glutamate release from LPS-activated microglia in a concentration-dependent manner. MCM stimulation resulted in characteristic neuronal toxicity including neuronal viability decrease, LDH release increase, calcium overload, and MMP drop. However, ABPPk pretreatment on microglia reduced the neurotoxicity of MCM. LPS intracerebral injection led to neuronal damage, microglial activation and cell apoptosis in the brain, while ABPPk preadministration significantly inhibited LPS-induced microglial activation and alleviated the brain injury. ABPPk pretreatment inhibited NOX2 expression and ROS production in LPS-activated primary microglia. Signaling pathway analysis showed that ABPPk promoted the phosphorylation of Akt in microglia and inhibited LPS-upregulated NOX2 expression, ROS production, and glutamate release, which can be eliminated by pharmacological inhibition of PI3K. Specific Akt activator could inhibit LPS-induced NOX2 expression, ROS production and glutamate release.
Conclusions: The present results suggested that ABPPk could alleviate neurotoxicity of LPS-activated microglia via PI3K/Akt dependent NOX2/ROS pathway.
Keywords: Achyranthes bidentata polypeptide; excitatory neurotoxicity; glutamate release; microglia.
2021 Annals of Translational Medicine. All rights reserved.