Allicin alleviates traumatic brain injury-induced neuroinflammation by enhancing PKC-δ-mediated mitophagy

Abstract

Background

Traumatic brain injury (TBI) leads to neuroinflammation, which is a key contributor to the negative prognosis in TBI patients. Recent evidence indicates that allicin can prevent neuronal injury after TBI. However, whether allicin alleviates neuroinflammation by promoting mitophagy is unclear.

Purpose

We investigated the suppressive effects of allicin on neuroinflammation and clarified the role of mitophagy in the underlying mechanism.

Study design/methods

The controlled cortical impact (CCI) was employed to effectively mimic TBI in a living system. Cellular mechanical damage was modeled in vitro using a Bv2 cell stretch model. Neuroinflammation was assessed by evaluating levels of TNF-α, IL-1β, IL-6, ROS, IL-4 and IL-10, along with the expression of NLRP3 and TLR4 proteins. RNA-sequence and KEGG analyses revealed allicin-regulated molecular processes in the Bv2 cell stretch model. Immunofluorescence staining was performed to label both the autophagy marker protein LC3 and the outer mitochondrial membrane (OMM) marker COX IV. Lipid MS and lipidomic analyses were used to determine the CL levels in the OMM and IMM. The characteristic bilayer structure of mitochondria was observed using transmission electron microscopy (TEM). PKC-δ expression and phosphorylated phospholipid scramblase-3 (PLS3) levels were detected via western blotting. Stretched Bv2 cells and primary neurons were cocultured to assess the anti-neuroinflammatory effects of allicin. Neuro-rehabilitation was assessed using behavioral experiments such as the rotarod and morris water maze (MWM) tests.

Results

Allicin treatment reduced TNF-α, IL-1β, IL-6, ROS levels, and the expression of NLRP3 and TLR4 proteins in mice with CCI, while IL-4 and IL-10 levels remained unchanged. Additionally, allicin reduced tissue lesions and cell death after CCI. The transcriptomic analysis revealed that mitophagy was important in allicin-related molecular pathways. The translocation of CL from IMM to OMM was facilitated by allicin, as demonstrated by flow cytometry and lipidomic analyses. Importantly, allicin increased PKC-δ expression and PLS3 phosphorylation in the CL-related mitophagy process in both the CCI and Bv2 cell stretch models. These findings suggest that allicin reduces mitophagy-related neuroinflammation and further prevents neuronal injury in vitro. Rottlerin, a selective PKC-δ inhibitor, effectively diminished allicin's capacity to reduce neuroinflammation, correlating with worsened motor function and cognitive abilities. Thus, CCI-induced behavioral deficits were also ameliorated by the administration of allicin via a PKC-δ-related mitophagy.

Conclusions

This study uncovers a novel mechanism where allicin enhances PKC-δ expression and PLS3 phosphorylation, facilitating CL translocation to the OMM and activating mitophagy, thereby reducing TBI-induced neuroinflammation.