Neuroprotective Effect of β-Caryophyllene on Cerebral Ischemia-Reperfusion Injury via Regulation of Necroptotic Neuronal Death and Inflammation: In Vivo and in Vitro.

Mei Yang,Jie Lou,Lu Xu,Xiaocui Tian,Qian Zhang,Minghang Li,Ruidi An,Yongjiu Lv,Zhi Dong

doi:10.3389/fnins.2017.00583

Abstract

Necrotic cell death is a hallmark feature of ischemic stroke and it may facilitate inflammation by releasing intracellular components after cell-membrane rupture. Previous studies reported that β-caryophyllene (BCP) mitigates cerebral ischemia-reperfusion (I/R) injury, but the underlying mechanism remains unclear. We explored whether BCP exerts a neuroprotective effect in cerebral I/R injury through inhibiting necroptotic cell death and inflammation. Primary neurons with and without BCP (0.2, 1, 5, 25 μM) treatment were exposed to oxygen-glucose deprivation and re-oxygenation (OGD/R). Neuron damage, neuronal death type and mixed lineage kinase domain-like (MLKL) protein expression were assessed 48 h after OGD/R. Furthermore, mice underwent I/R procedures with or without BCP (8, 24, 72 mg/kg, ip.). Neurologic dysfunction, cerebral infarct volumes, cell death, cytokine levels, necroptosis core molecules, and HMGB1-TLR4 signaling were determined at 48 h after I/R. BCP (5 μM) significantly reduced necroptotic neurons and MLKL protein expression following OGD/R. BCP (24, 72 mg/kg, ip.) reduced infarct volumes, neuronal necrosis, receptor-interaction protein kinase-1 (RIPK1), receptor-interaction protein kinase-3 (RIPK3) expression, and MLKL phosphorylation after I/R injury. BCP also decreased high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) levels. Thus, BCP alleviates ischemic brain damage potentially by inhibiting necroptotic neuronal death and inflammatory response. This study suggests a novel application for BCP as a neuroprotective agent.

Highlights

Stroke is a common cerebrovascular disease associated with high disability and mortality worldwide, and ∼85% of strokes are caused by cerebral ischemia (Flynn et al, 2008)
To investigate whether ischemic neuronal death occurs via necroptosis after cerebral ischemia in vitro, oxygen-glucose deprivation and re-oxygenation (OGD/R)-induced neuronal death was measured by different cell death assays
Nec-1, which inhibits necroptosis, reduced increased necrotic cells and Lactate dehydrogenase (LDH) leakage rate after OGD/R injury from 80.84 ± 2.00 and 51.69 ± 1.62% to 39.24 ± 0.96 and 22.76 ± 2.75%, indicating that necroptotic neuronal death occurs in primary neurons following ischemia in vitro

Summary

Introduction

Stroke is a common cerebrovascular disease associated with high disability and mortality worldwide, and ∼85% of strokes are caused by cerebral ischemia (Flynn et al, 2008). Tissue plasminogen activator (tPA) is an effective therapeutic agent that has been approved by the FDA for ischemic stroke. Necrotic cell death is a hallmark feature of ischemic stroke, yet lacks therapeutics to target necrosis because it has long been characterized as an unregulated and irreversible cell death. Necrostain-1 (Nec-1), a specific small-molecule inhibitor of RIPK1, can inhibit necroptosis through blocking the formation of necrosome and phosphor-MLKL signal (Wang H. et al, 2014). Nec-1 has been used to ameliorate tissue injury in murine models of ischemiareperfusion (I/R) in the brain (Degterev et al, 2005; Xu et al, 2010; Yin et al, 2015), which revealed that necroptosis is involved in brain injury caused by I/R and may serve as a putative therapeutic target to treat cerebral I/R injury

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