Enmein Decreases Synaptic Glutamate Release and Protects against Kainic Acid-Induced Brain Injury in Rats

Abstract

This study investigated the effects of enmein, an active constituent of Isodon japonicus Hara, on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) and evaluated its neuroprotective potential in a rat model of kainic acid (KA)-induced glutamate excitotoxicity. Enmein inhibited depolarization-induced glutamate release, FM1-43 release, and Ca2+ elevation in cortical nerve terminals but had no effect on the membrane potential. Removing extracellular Ca2+ and blocking vesicular glutamate transporters, N- and P/Q-type Ca2+ channels, or protein kinase C (PKC) prevented the inhibition of glutamate release by enmein. Enmein also decreased the phosphorylation of PKC, PKC-α, and myristoylated alanine-rich C kinase substrates in synaptosomes. In the KA rat model, intraperitoneal administration of enmein 30 min before intraperitoneal injection of KA reduced neuronal cell death, glial cell activation, and glutamate elevation in the hippocampus. Furthermore, in the hippocampi of KA rats, enmein increased the expression of synaptic markers (synaptophysin and postsynaptic density protein 95) and excitatory amino acid transporters 2 and 3, which are responsible for glutamate clearance, whereas enmein decreased the expression of glial fibrillary acidic protein (GFAP) and CD11b. These results indicate that enmein not only inhibited glutamate release from cortical synaptosomes by suppressing Ca2+ influx and PKC but also increased KA-induced hippocampal neuronal death by suppressing gliosis and decreasing glutamate levels by increasing glutamate uptake.

Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation

“…Since excess glutamate is observed in the hippocampi of KA-treated rats and is associated with neuronal death, 28,31 we assessed the glutamate concentration in the hippocampi three days after KA injection by HPLC (Fig. 4A).…”

“…The slides were rehydrated and stained with cresyl violet solution (#ab246816; Abcam, Cambridge, United Kingdom) to assess the neuronal morphology in the hippocampus. NeuN staining was performed as described previously 27,28 to visualize neurons. Briefly, after fixating and blocking, the slides were incubated overnight with NeuN antibody (1 : 500, #MAB377; Merck Millipore, Billerica, MA, USA) in blocking solution.…”

“…Proteins were prepared for blocking with the relevant primary antibody as previously reported by our laboratory. 28,29 Primary antibodies included β-actin (1 : 5000; #3700; Cell Signaling, Beverly, MA, USA), NeuN (1 : 3000; #MAB377; Merck Millipore, Billerica, MA, USA), GFAP (1 : 5000; #3670; Cell Signaling, Beverly, MA, USA), EAAT1 (1 : 5000; #ab416; Abcam, Cambridge, UK), EAAT2 (1 : 50 000; #ab41621; Abcam, Cambridge, UK), EAAT3 (1 : 10 000; #14 501; Cell Signaling, Beverly, MA, USA), GS (1 : 50 000; #ab176562; Abcam, Cambridge, UK), glutaminase (1 : 10 000; #701965; Invitrogen, Waltham, MA, USA), GluA1 (1 : 10 000; #MA5-27694; Invitrogen, Waltham, MA, USA), GluA2 (1 : 10 000; #MA5-17 084; Invitrogen, Waltham, MA, USA), GluN2A (1 : 3000; #4205; Cell Signaling, Beverly, MA, USA), GluN2B (1 : 3000; #4207; Cell Signaling, Beverly, MA, USA), IL-1β (1 : 2000; #ab283818; Abcam, Cambridge, UK), IL-6 (1 : 800; #ab9324; Abcam, Cambridge, UK), TNF-α (1 : 300; #ab6671; Abcam, Cambridge, UK), IL-10 (1 : 400; #sc-8438; Santa Cruz Biotechnology, Dallas, TX, USA), HMGB1 (1 : 10 000; #ab79823; Abcam, Cambridge, UK), IL-1R1 (1 : 500; #ab106278; Abcam, Cambridge, UK), or TLR4 (1 : 5000; #ab13556; Abcam, Cambridge, UK) antibodies. Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 5000; #GTX213110-01, #GTX213111-01; Genetex, Zeeland, MI, USA) and visualized using an enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, UK).…”

“…The slides were rehydrated and stained with cresyl violet solution (#ab246816; Abcam, Cambridge, United Kingdom) to assess the neuronal morphology in the hippocampus. NeuN staining was performed as described previously 27,28 Staining for glial fibrillary acidic protein (GFAP) was performed as previously described by Lu et al 28 In brief, the 5 sections taken from each brain were treated with primary antibodies (GFAP, 1 : 1000; #3670; Cell Signaling, Beverly, MA, USA) at 4 °C overnight. The sections were then incubated with goat biotinylated anti-mouse secondary antibodies (1 : 200; Vector Laboratories, Burlingame, CA, USA) for 2 h and subsequently incubated with ExtrAvidin peroxidase (1 : 1000; Sigma-Aldrich, St Louis, MO) for 1 h at room temperature.…”

“…Proteins were prepared for blocking with the relevant primary antibody as previously reported by our laboratory. 28,29…”