Bark Constituents from Mushroom-detoxified Rhus verniciflua Suppress Kainic Acid-induced Neuronal Cell Death in Mouse Hippocampus

Jong-Seon Byun; Yoon Hee Han; Sung-Jun Hong; Sung-Mi Hwang; Yong-Soo Kwon; Hee Jae Lee; Sung-Soo Kim; Myong-Jo Kim; Wanjoo Chun

doi:10.4196/kjpp.2010.14.5.279

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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

Original Article

Korean J Physiol Pharmacol 2010;14(5):279-283.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.5.279

Bark Constituents from Mushroom-detoxified Rhus verniciflua Suppress Kainic Acid-induced Neuronal Cell Death in Mouse Hippocampus

Jong-Seon Byun¹, Yoon Hee Han^1,⁴, Sung-Jun Hong¹, Sung-Mi Hwang¹, Yong-Soo Kwon², Hee Jae Lee¹, Sung-Soo Kim¹, Myong-Jo Kim^3,^†, Wanjoo Chun^1,^∗

¹Department of Pharmacology, College of Medicine, Kangwon National University, Chuncheon 200-701, Korea

²College of Pharmacy, Kangwon National University, Chuncheon 200-701, Korea

³Division of Bio-resources Technology, Kangwon National University, Chuncheon 200-701, Korea

⁴Department of Radiology, Ilsan Paik Hospital, Inje University, School of Medicine, Goyang 411-706, Korea

^∗Corresponding to: Wanjoo Chun, Department of Pharmacology, College of Medicine, Kangwon National University, Hyoja-2-dong, Chuncheon 200-701, Korea. (Tel) 82-33-250-8853, (Fax) 82-33-250-8809, (E-mail) wchun@kangwon.ac.kr

^†Co-corresponding author: Myong-Jo Kim, Division of Bio-resources Technology, Kangwon National University, Hyoja-2-dong, Chuncheon 200-701, Korea. (Tel) 82-33-250-6413, (Fax) 82-33-250-6413, (E-mail) kimmjo@kangwon.ac.kr

Received 19 July 2010 Revised 25 August 2010 Accepted 1 September 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Kainic acid, Neuroprotection, Stigma-4-en-3-one, Stigma-4-en-3,6-dione

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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).

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.

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.

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.

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.

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