Journal List > Korean J Physiol Pharmacol > v.14(5) > 1025690

Byun, Han, Hong, Hwang, Kwon, Lee, Kim, Kim, and Chun: Bark Constituents from Mushroom-detoxified Rhus verniciflua Suppress Kainic Acid-induced Neuronal Cell Death in Mouse Hippocampus

Abstract

Urushinol, a plant allergen, has significantly restricted the medical application of Rhus verniciflua, although it has been reported to possess a wide variety of biological activities such as anti-inflammatory, antioxidant, and anti-cancer actions. To reduce the urushinol content while maintaining the beneficial biological activities, mushroom-mediated fermentation of Rhus verniciflua was carried out and this method resulted in significantly attenuated allergenicity [1]. In the present study, to examine the neuroprotective properties of mushroom-fermented stem bark of Rhus verniciflua, two constituents were isolated from mushroom-fermented bark and their neuroprotective properties were examined in a mouse model of kainic acid (KA)-induced excitotoxicity. KA resulted in significant apoptotic neuronal cell death in the CA3 region of mouse hippocampus. However, seven daily administrations of RVH-1 or RVH-2 prior to KA injection significantly attenuated KA-induced pyramidal neuronal cell death in the CA3 region. Furthermore, pretreatment with RVH-1 and RVH-2 also suppressed KA-induced microglial activation in the mouse hippocampus. The present study demonstrates that RVH-1 and RVH-2 isolated from Rhus verniciflua and detoxified using mushroom species possess neuroprotective properties against KA-induced excitotoxicity. This leads to the possibility that detoxified Rhus verniciflua can be a valuable asset in herbal medicine.

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Fig. 1.
Chemical structure of RVH-1 (stigma-4-ene-3-one) and RVH-2 (stigma-4-ene-3,6-dione).
kjpp-14-279f1.tif
Fig. 2.
Representative neuroprotective effects of RVH-1 and RVH-2 on KA-induced neuronal cell death in CA3 region of mouse hippocampus. Hippocampal cell death was examined with cresyl violet staining. Intracerebroventricular (icv) injection of KA showed marked loss of neurons in the CA3 region of hippocampus (KA). However, RVH-1 or RVH-2 treatment prior to KA injection showed attenuation of neuronal cell loss compared to KA alone. RVH-2 appeared to be more protective than RVH-1, albeit not significantly so. Neuronal cell death was not observed in vehicle (Cont)-, RVH-1-only-, or RVH-2-only-treated mice. More than three mice were used for each group. Scale bar: 100 μm.
kjpp-14-279f2.tif
Fig. 3.
Representative (A) and quantitative (B) analysis of neuronal cell death with Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) assay. A considerable number of TUNEL-positive neurons appeared with KA treatment within the CA3 region after 24 hr. Pre-administration of RVH-1 or RVH-2 significantly reduced the number of TUNEL-positive cells compared to the KA-only group. RVH-2 pretreatment showed fewer TUNEL-positive cells than RVH-1. A single treatment of RVH-1 or RVH-2 had no noticeable effects on cell survival. More than three mice were used for each group. Quantitative data represent three independent experiments and are expressed as mean±SEM. ∗∗p<0.01 indicates statistically significant difference from the KA-only treated group. Scale bar: 100 μm.
kjpp-14-279f3.tif
Fig. 4.
Representative images of neuronal protection by RVH-1 and RVH-2 against KA-induced neuronal cell death in the CA3 region of hippocampus. In order to further elucidate the neuroprotective properties of RVH-1 and RVH-2, NeuN immunoreactivity, which specifically stains pyramidal neurons in the hippocampus, was examined. Pretreatment of RVH-1 or RVH-2 attenuated KA-induced death of pyramidal neurons in the CA3 region of hippocampus. RVH-2 showed more neuronal protection against KA-induced excitotoxicity compared to RVH-1. RVH-1 and RVH-2 showed no noticeable change in neuronal viability. More than three mice were used for each group. Scale bar: 100 μm.
kjpp-14-279f4.tif
Fig. 5.
Suppressive effects of RVH-1 and RVH-2 on KA-induced microglial activation. Given the previous report that KA-mediated neuronal death accompanies microglial activation, expression of OX-6, a microglial activation marker, was examined with immunohistochemical staining at 24 hr after KA or vehicle treatments. KA resulted in increased microglial activation (KA). However, KA-induced microglial activation was attenuated with RVH-1 or RVH-2 pretreatment. RVH-2 appears to be more suppressive than RVH-1. RVH-1 or RVH-2 showed negligible effects on microglial activation. Representative images were obtained from three independent experiments. More than three mice were used for each group. Scale bar: 100 μm.
kjpp-14-279f5.tif
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