Neuroprotective effect of modified Wu-Zi-Yan-Zong granule, a traditional Chinese herbal medicine, on CoCl2-induced PC12 cells

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Abstract

Aim of the study

To investigate the neuroprotective effect of aqueous extract of modified Wu-Zi-Yan-Zong granule (MWG), a traditional Chinese herbal medicine, against CoCl2-induced neurotoxicity in PC12 cells.

Materials and methods

Cell viability assay, apoptosis rate assay, ROS detection and mitochondrial membrane potential (MMP) assay were performed. In addition, cytochrome c, caspase-3, PARP and MAPKs were also detected by Western blotting.

Results

MWG extract increased viability and suppresses early and middle/late stage apoptosis in a dose-dependent manner in CoCl2-induced PC12 cells. Moreover, MWG extract decreased the level of intracellular reactive oxygen species (ROS), increased MMP, regulated Bcl-2 family protein expression (Bcl-2 and Bcl-XL) and inhibited the release of cytochrome c from the mitochondria. In addition, MWG extract attenuated activation of caspase-3 and poly ADP-ribose polymerase (PARP) and inhibited the phosphorylation of ERK, c-Jun NH2-terminal kinase (JNK) and p38 MAPKs.

Conclusions

MWG extract exhibited significant neuroprotective effect on PC12 cells, and this effect may be associated with the suppression of ROS generation and inhibition of mitochondria-mediated caspase and MAPK signaling pathways.

Introduction

Senile dementia (SD) is one of the most common nervous system diseases in aged humans around the world. Recent researches reveal that hypoxic/ischemic neurological damage induced by aging is an important risk factor and actively involved in the pathogenesis of SD (Rao and Howard, 1998, Martin et al., 2008). Along with aging, chronic cerebral hypoxia or ischemia usually induces progressive neuronal cells death and further leads to amnesia and memory deterioration. However, little progress has been made over the past decades in developing novel and effective agents for senile dementia and age-related cognitive impairment.

The use of herbal medicine for the treatment of neuronal disorders, especially for senile dementia, has a long history in China. In clinical practice of traditional Chinese medicine, herbal formulas are widely used for anti-dementia and proved to own fewer side effects than western medicine. Modified Wu-Zi-Yan-Zong granule (MWG) is a traditional Chinese medicine and has clinically proven a good therapeutic effect on its anti-dementia properties. MWG, consisting of six medicinal plants (Cuscuta chinensis Lam., Lycium barbarum L., Rubus chingii Hu., Schizandra chinensis (Turcz.) Baill., Plantago asiatica L. and Epimedium brevicornu Maxim.), derived from traditional Chinese kidney-reinforcing medicine Wu-Zi-Yan-Zong Formula which was firstly documented in ancient Chinese medical works “She Sheng Zhong Miao Fang” (Volume 11) by Shi-Che Zhang in 1550 AD, and also recorded in Chinese Pharmacopoeia (The Pharmacopoeia Commission of PRC, 2005). In traditional Chinese medicine (TCM) theory, it is hold that decrease of memory ability is closely related to senility and kidney deficiency. Therefore the anti-dementia effect of MWG originates directly from its original reinforcing kidney effect. Previous clinical double-blind, randomized controlled trial and magnetic resonance imaging studies indicate that MWG improves both the memory quotient and hippocampal volume of patients with mild cognitive impairment (Fu et al., 2006, Fu et al., 2007). However, the underlying mechanism of MWG on its neuroprotective effect has not been totally explored until now.

In the present study, we examined the neuroprotective effect of MWG water extract against CoCl2-induced neurotoxicity in PC12 cells, which is widely used in vitro model simulating neuronal damage and apoptosis under hypoxia/ischemia insults (Hou et al., 2003, Jung and Kim, 2004, Jung et al., 2008), so as to deeply understand the potential mechanism behind the neuroprotective and anti-dementia activity of MWG.

Section snippets

Plant material and preparation of the extract

In this study, all traditional Chinese herbs were purchased from Tong Ren Tang Chinese Medicine Co., Ltd. (Beijing, China) and carefully authenticated by Dr. Peng-Fei Tu (School of Pharmaceutical Science, Peking University, Beijing, China) according to the Chinese pharmacopoeia (The Pharmacopoeia Commission of PRC, 2005). For MWG water extract preparation, Cuscuta chinensis Lam. (200 g), Lycium barbarum L. (200 g), Rubus chingii Hu. (100 g), Schizandra chinensis (Turcz.) Baill. (25 g), Plantago

Protective effect of MWG extract on neurotoxicity of PC12 cells

In a preliminary experiment, CCK-8 assay data showed that MWG extract up to 400 μg/mL did not show any significant toxicity for 48 h (Fig. 1A). Moreover, approximately 62.5 ± 12.4% of PC12 cells survived a treatment with CoCl2 (250 μM) for 24 h, and cell viability increased to 96.29 ± 1.7% (at 400 μg/mL) when the cells were pretreated with MWG extract for 1 h (Fig. 1B). Therefore, the results demonstrate that MWG extract protects PC12 cells from CoCl2-induced cell death in a dose-dependent manner.

Protective effect of MWG extract on apoptosis of PC12 cells

The

Discussion

Previous clinical practice has shown that the MWG possesses good anti-dementia effects on elderly patients (Fu et al., 2006, Fu et al., 2007). Here, our results indicate that MWG possesses neuroprotective and anti-apoptotic properties under the hypoxic/ischemic conditions induced by CoCl2. MWG extract could scavenge ROS in a dose-dependent manner, which was interpreted as a result of its anti-lipid peroxidation properties in a previous clinical trial (Wang et al., 2004). Moreover, our data

Acknowledgements

This work was supported by the Korea Institute of Science and Technology institutional program (Grant Nos. 2Z03270 and 2Z03380, Republic of Korea), National Natural Science Funds of China (Grant Nos. 30973813 and 30672760, China) and Research Fund for the Doctoral Program of Ministry of Education (Grant No. 20070001707, China).

References (21)

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