Joongpoongtang 05 (JP05) confers neuroprotection via anti-apoptotic activities in Neuro-2a cells during oxygen–glucose deprivation and reperfusion Ramalingam Mahesh a , Hyo Won Jung a , Chang-Ho Han b , Cheong-Weon Cho c , Yong-Ki Park a,⇑ a Oriental Medicine R&D Center, Dongguk University, Gyeongju 780-714, Republic of Korea b Department of Internal Medicine, Dongguk University Ilsan Oriental Hospital, Dongguk University, Gyeonggi-Do 410-773, Republic of Korea c College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea article info Article history: Received 9 July 2010 Accepted 26 October 2010 Available online 30 October 2010 Keywords: Neuro-2a Oxygen–glucose deprivation Anti-apoptosis HO-1 NF-jB Akt abstract To evaluate the anti-apoptotic effects of Joongpoongtang 05 (JP05), a mixture of plant extracts, on a Neuro-2a (N2a) cell model of oxygen and glucose deprivation (OGD)/reperfusion (OGDR), a neuroblas- toma cell injury model was induced by OGDR. This model allowed us to investigate cerebral ischemic changes and the protective effects of JP05. JP05 treatment significantly enhanced cell viability and reduced the levels of lactate dehydrogenase, nitric oxide, reactive oxygen species and the oxidants/anti- oxidants balance in neuronal cells as compared to the untreated OGDR group. Here, JP05 reduced OGDR- induced expressions of heme oxygenase-1 and nitric oxide synthase, which may contribute to the neuroprotection. JP05 also partially reversed the effects of OGDR on NF-jB and activated Akt production. Our findings suggest that JP05 confers neuroprotective effects via anti-apoptotic property against OGDR-induced free radical injury in N2a cells. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Oxidative stress has been linked to the modification of diverse cellular macromolecules such as nucleic acids, lipids, and proteins. These modifications lead to altered function, mutagenesis, or cell death. Oxygen is an essential molecule for metabolism and energy generation, but the high rate of oxygen consumption of the brain is also the primary source for the generation of reactive oxygen spe- cies (ROS) (Ames et al., 1993; Winrow et al., 1993) such as super- oxide (O Å 2 ), hydrogen peroxide (H 2 O 2 ), and peroxynitrite (ONOO  ). Excess ROS are harmful because of their potent ability to interact with and modify a wide range of cellular damages in the pathogen- esis of neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and stroke (Choi et al., 2002). Stroke, one of the most frequent causes of disability and death worldwide, is a neurological condition that develops when a por- tion of the brain is deprived of oxygen and glucose. The damage caused to neurons during ischemia is due to a reduction in the oxy- gen and glucose supply, i.e., oxygen and glucose deprivation (OGD). The OGD insult, followed by reoxygenation (OGDR), is thought to mimic the pathological conditions of ischemia (Mattson et al., 2000). Although ischemia cannot be reproduced in a neuronal cul- ture, OGD mimics ischemic conditions and provides a convenient model to test pharmacological compounds for neuroprotective po- tency (Goldberg and Choi, 1993; Ruscher et al., 2007). Upon path- ological stress, inhibiting death cascades could provide neuroprotection. Promoting the endogenous survival/repair mech- anism may bear equal, if not greater, importance in achieving neu- roprotection (Park et al., 2004). In recent years, several studies have highlighted the ability of herbal medicines to promote a vari- ety of pharmacological and biological activities. This work describes, Joongpoongtang 05 (JP05), a mixture of the 12 herbs: Astragali Radix, Salviae Miltiorrhizae, Angelicae Gigantis Radix, Paeoniae Radix Rubra, Achyranthis Radix, Cnidii Rhizoma, Lum- bricus, Percicae Semen, Carthami Flos, Cinnamomi Ramulus, Polygalae Radix and Acori Graminei Rhizoma. These herbs have long been used as a prescription for stroke, senile and vascular dementia, ischemic brain, and heart damages and have also been reported to have neu- roprotective effects (Irie and Keung, 2003; Chang et al., 2005; Kang et al., 2006; Kim et al., 2006; Jeong et al., 2008). Recently, the vaso- protective effects of JP05, acting through the Akt-dependent eNOS and ERK pathways in brain endothelial cells, were reported by our laboratory (Son et al., 2010). However, the mechanisms and effec- tiveness of JP05 for treatment of neurodegenerative diseases are poorly understood. In this study, we investigated whether JP05 protects N2a neuroblastoma cells from OGDR, an in vitro model of ischemic stroke. Edaravone (3-methyl-1-phenly-2-pyrazolin-5-one) is a free- radical scavenger which was used as positive control in this study, has been evaluated as a neuroprotective compound in several models of cerebral diseases. The protective effects of edaravone 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.10.016 ⇑ Corresponding author. Tel./fax: +82 547702647. E-mail address: yongki@dongguk.ac.kr (Y.-K. Park). Toxicology in Vitro 25 (2011) 177–184 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit