In this study, we investigated the effect of Rhei Rhizoma (RR; 大黃) water extract on gene expression in a hypoxia model of cultured rat hippocampal neurons. RR water extract (2.5 μg/ml) was added to the culture media on day 10 in vitro (DIV10), and a hypoxic shock (2% O2/5% CO2 , 37 o C, 3 h) was given on DIV13. After maintaining the cultures in normoxia for 24 hr, total RNA was isolated and used for microarray analysis. The MA-plot indicated that most genes were up-or downregulated within 2-fold. There were more downregulated genes (725 ea) than upregulated ones (472 ea) when larger than Global M value 0.2 (i.e., >15% increase) or smaller than Global M value -0.2 (i.e., >15% decrease) were considered. Antiapoptosis genes such as Tegt (2.4-fold), Nfkb1 (2.4-fold) Veg (1.8-fold), Ngfr (1.6-fold) were upregulated, while pro-apoptosis genes such as Bad (-64%), Cstb (-66%) were downregulated. Genes for combating environmental stress (stress response genes) such as Defb3 (2.7-fold), Cygb (2.2-fold), Ahsg (2.18-fold), Alox5 (2-fold) were upregulated. Genes for cell proliferation (cell cycle-related genes) such as Erbb2 (1.84-fold), Mapk12 gene (1.8-fold) was upregulated. Therefore, RR water extracts upregulate many pro-survival genes while downregulating many pro-death genes. It is interpreted that these genes, in combination with other regulated genes, can promote neuronal survival in a stress such as hypoxia. [40,46]. Effects of RR on lowering serum cholesterol are attributed to rhein, which are transformed from anthranoids sennoside A and B by bacterial enzymes in large intestine [55]. Rhaponticin in the rhizome of RR has extensive anti-allergic and anti-thrombotic properties [46]. RR extracts decreased serum creatinine levels in diabetic patients with neuropathy and retinopathy [18].Studies on the nervous system are relatively scarce. It has been reported that RR improves memory ability. By comparing the effects of the Compound Tong Jiang Oral Liquid with Da Huang added (TJ) and Qi Yin Oral Liquid (QY) without Da Huang on senile persons' memory ability, Tian et al. [55] discovered that TJ improves senile persons' memory ability, in addition to shortened interval and duration of defecation. However, effects of RR on the nervous system are not studied at the cellular level. Previously, our laboratory reported that RR suppresses production of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (MMP) in a hypoxia model of rat hippocampal neurons in culture [34]. Here, we report results of a microarray analysis on the expression of genes in hypoxia.
Materials and MethodsPreparation of water-extracts of RR Rhei Rhizoma (RR; 大黃) was selected according to the Korean Pharmacopoeia and obtained from Dongguk University Oriental Hospital (Gyeongju, Korea). Distilled water was added to the RR powder and it was agitated for 4 hr at room temperature (RT) followed by overnight at 4 o C. After centrifugation (15,000 rpm, 15 min, RT), the supernatant was filter-sterilized (pore size 0.2 μm) and stored at -20 ...