Chronotherapeutic neuroprotective effect of verapamil against lipopolysaccharide-induced neuroinflammation in mice through modulation of calcium-dependent genes

Abstract

Background: Neuroinflammation is a major mechanism in neurodegenerative diseases such as Alzheimer's disease (AD), which is a major healthcare problem. Notwithstanding of ample researches figured out possible molecular mechanisms underlying the pathophysiology of AD, there is no definitive therapeutics that aid in neuroprotection. Therefore, searching for new agents and potential targets is a critical demand. We aimed to investigate the neuroprotective effect of verapamil (VRP) against lipopolysaccharide (LPS)-induced neuroinflammation in mice and whether the time of VRP administration could affect its efficacy.

Methods: Forty male albino mice were used and were divided into normal control, LPS only, morning VRP, and evening VRP. Y-maze and pole climbing test were performed as behavioral tests. Hematoxylin and eosin together with Bielschowsky silver staining were done to visualize neuroinflammation and phosphorylated tau protein (pTAU); respectively. Additionally, the state of mitochondria, the levels of microglia-activation markers, inflammatory cytokines, intracellular Ca²⁺, pTAU, and Ca²⁺-dependent genes involving Ca²⁺/ calmodulin dependent kinase II (CAMKII) isoforms, protein kinase A (PKA), cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF), with the level of VRP in the brain tissue were measured.

Results: LPS successfully induced neuroinflammation and hyperphosphorylation of tau protein, which was indicated by elevated levels of microglia markers, inflammatory cytokines, and intracellular Ca²⁺ with compromised mitochondria and downregulated CAMKII isoforms, PKA, CREB and BDNF. Pretreatment with VRP showed significant enhancement in the architecture of the brain and in the behavioral tests as indicated by the measured parameters. Moreover, morning VRP exhibited better neuroprotective profile compared to the evening therapy.

Conclusions: VRP highlighted a multilevel of neuroprotection through anti-inflammatory activity, Ca²⁺ blockage, and regulation of Ca²⁺-dependent genes. Furthermore, chronotherapy of VRP administration should be consider to achieve best therapeutic efficacy.

Keywords: Alzheimer’s disease; CAMKII; Calcium; Chronotherapy; Lipopolysaccharide; Verapamil.