Nov 2, 2015

1. Protection of neuron cells from apoptosis by Korean red ginseng via ERβ/PI3K signalling Dong-Kwon Rhee School of Pharmacy SungKyunKwan University Nov 2, 2015

2. Introduction ► What is Korean ginseng? ▪ Deciduous perennial plant belonging to Araliaceae family. ▪ Root has been used for medicinal purpose ▪ Scientific name: Panax ginseng C. A, Meyer ▪ Entomology of Panax Pan (Greek): all＋ axos (Greek):cure⇒“cure-all” ▪ Other species belonging to Panaxgenus: American、Japanese、Chinese、Himalayan ▪ WHO approved Korean ginseng for medicinal use but not American and Chinese ginseng.

3. Korean ginseng

4. KoreanRed Ginseng (KRG)

5. Introduction to Stress Selye H. (1936) J Neuropsychiatry Clin Neurosci. 10:230 : Non-specific response of body Stress can be defined as physical and psychological modifications that disrupt the homeostasis and the balance of organisms. Stress is known as one of the most important reasons of several diseases (Johnson et al., 1992)

6. Anti-stress effects of ginseng -Ginseng and ginsenoside studies have been focused on biochemical parameters such as plasma glucose, cholesterol, serum corticosterone, proinflammatory cytokines (IL-1β, IL-6, and TNF-α) , malondialdehyde levels, free radical scavenging activity. Also glutathione level and superoxide dismutase activity in substantia nigra were determined in mice brain. -Moreover, ginseng was found to increase expression of TLR4 and the release of cytokines. -However, effect of ginseng on gene expression levels in stressed brain remains unknown.

7. Oxidative nature of brain • Oxidative stress such as free radicals has been implicated as a major pathological mechanism of the brain disorders such as Alzheimer's disease, brain trauma, and Parkinson's disease (Basso et al., 2004; McGrath et al., 2001). Since brain contains relatively large amount of iron and lipid content of myelin sheaths as well as high rate of oxidative metabolism and limited antioxidant capacity than the other organs, it is highly vulnerable to oxidative damage (Choi, 1993; Floyd, 1999; Herbert et al., 1994). In the brain, stress stimulates secretion of glucocorticoids, which augment the extracellular accumulation of glutamate in the hippocampus. Since glutamate can induce neuronal excitotoxicity and leads to TNFα release, this sequence results in translocation of NF-κB to neuronal nuclei and activation of the induction of iNOS and COX2, which are the two major enzymes responsible for the neurological damage. Subsequent generation of free radicals impairs DNA, oxidizes protein and lipid, and finally results in neural degeneration and cell death (Madrigal et al., 2006).

8. Structure of ginsenoside: similar to estrogen -> Estrogen receptor (ER) in brain? ERα and β Ginseng has been used as an alternative medicine for estrogen without any side effects

9. Decrease of cell death by KRG in vivo TUNEL assay Western blot Control A + IMO Immobilization stress B Con 100mg/kg KRG PBS 50mg Bcl-2 PBS + IMO β-actin ** KRG 100 mg/Kg + IMO * ** X 40 C Bcl-2 2.0 *** D J Ethnopharmacol. 148(2):474-85. *** 400 1.5 300 1.0 Bcl-2/GAPDH Apoptotic cells/cm2 of view fields 200 (A) Mice were administered 100 mg/kg of KRG for 1 week twice a day and the brains of mice were subject to TUNEL assay. Representative data from 3 mice samples are shown. Apoptotic cells were counted in randomly chosen x40 histological fields. (B) Mice were administered 50 and 100 mg/kg of RG for 1 week twice a day and then IMO stressed  for 45 min. The protein was isolated from the brains of mice and then subjected to Western blot. Five mice per group  were used for these experiments. 0.5 100 0 Con 50mg 100 mg/kg KRG PBS 0 Con PBS KRG + IMO + IMO

10. Anti-inflammatory effect of KRG A *** B 140 * * 120 TNF-α ELISA, OD 650 nm 100 80 TNF-α, Relative to control 60 D 40 * * 0.20 20 0 0.5 Con 1 2 0.15 Con LPS PBS + IMO, hr nmol MDA/mg protein 50 mg/Kg 100 mg/Kg 0.10 + IMO 0.05 C 0.00 *** *** Con 25 PBS 100 mg/Kg KRG 10000 + IMO 1000 RQ of COX-2 J Ethnopharmacol. 148(2):474-85. 100 10 1 0.1 Con PBS KRG +IMO (A) After IMO, mice brain homogenates were used for TNF-α ELISA. (B) RG was administered for 1 week followed by IMO stress for 30 min. Mice brain homogenates were used for TNF-α ELISA assay. (C) RG 100 mg/Kg was administered for 1 week and followed by IMO stress for 45 min. qPCR result. (D) After administration of the specified RG dose per mouse for 1 week, mice were IMO stressed for 45 min, and the brain MDA level was determined.

11. Activation of ERβ by RG inhibits apoptosis in stressed brain cells SK-N-SH neuron cell Mice brain KRG Extract ER-β PADI4 J Ethnopharmacol. 2013; 148(2):474-85. Oxidative Stress APOPTOSIS

12. KRG inhibits apoptosis in an oxidatively- stressed brain via TACE Stress KRG PADI4 TACE TNF-α NF-κB Translocation ROS, inflammation Apoptosis J Ginseng Res. 2013 Jul;37(3): 315-23.

13. Aim: How KRG modulates gene expression in cell survival in brain cells stressed by oxidative ?

14. Inhibition of ER-β-mediated inhibition of apoptotic signaling during oxidative stress in vitro H2O2 (0.5 mM) B A PBS KRG PBS Treatment time 2h 2h 2h 2h KRG Ctrl Ctrl 120 * ER-β *** Bcl-2 90 *** p-p53 60 Cell viability (%) Caspase-3 30 β-actin 0 Ctrl siCO siER-β Ctrl Ctrl siER-β siCO siCO siER-β H2O2 (0.5 mM) C PBS ** KRG ** 1.5 1.5 *** *** 1.0 1.0 Blc-2/β-actin 0.5 0.5 ER-β/β-actin 0 0 siER-β siER-β Ctrl siCo Ctrl siCo * *** ** antiapoptotic protein Bcl2 ** 1.5 1.5 *** proapoptotic factors p-p53 & caspase-3 1.0 1.0 p-p53/β-actin 0.5 0.5 Caspase-3/β-actin SK-N-SH cells were transfected for 24 h with 40 nM siER-β. Following transfection, cells were incubated with KRG (1 mg/mL) for 48 h and then with 0.5 mM H2O2 for 2 h. (A) Cell viabilities were determined by MTT assays. (B) Cell lysates were used to estimate protein expression levels by western blot analysis. (C) Quantification of results from (B). 0 0 siER-β Ctrl siCo siER-β Ctrl siCo J Ginseng Res. 2015 Jan;39(1):69-75.

15. H2O2 (0.5 mM) A B PBS KRG Treatment time 120 PBS 2h 2h Ctrl 2h Ctrl 2h KRG ER-β 90 ** *** Bcl-2 *** 60 p-p53 Cell viability (%) 30 Caspase-3 β-actin 0 Ctrl DMSO PHTPP Ctrl Ctrl PHTTP DMSO DMSO PHTTP H2O2 (0.5 mM) C PBS 1.5 ** * 1.5 KRG ER-β inhibitor PHTPP inhibits apoptosis in oxidatively stressed brain cells. *** * 1.0 ** 1.0 0.5 Blc-2/β-actin 0.5 ER-β/β-actin 0 0 Ctrl Ctrl DMSO DMSO PHTPP PHTPP ** 1.5 *** ** 1.5 *** *** * 1.0 1.0 Caspase3/β-actin pp53/β-actin 0.5 0.5 0 SK-N-SH cells were incubated sequentially with KRG (1 mg/mL) for 48 h, 5 µM PHTPP for 5 h, and 0.5 mM H2O2 for 2 h. (A) Cell viabilities were determined by MTT assays. (B) Cell lysates were used to estimate protein expression levels by western blot analysis. (C) Quantification of at least three independent experimental results from (B). 0 Ctrl Ctrl DMSO DMSO PHTPP PHTPP J Ginseng Res. 2015 Jan;39(1):69-75.

16. Functional ER-β inhibition suppresses AKT/PI3K signaling in oxidatively stressed brain cells. A B PBS KRG PBS H2O2 (0.5 mM) 0.5h 0.5h Ctrl 0.5h Ctrl 0.5h * KRG 1.5 1.5 PI3K * * * 1.0 1.0 * p-Akt p-Akt/β-actin PI3K/β-actin 0.5 0.5 Akt 0 0 β-actin Ctrl Ctrl siCO siCO siER-β siER-β Ctrl Ctrl siCO siCO siER-β siER-β PBS C KRG D 0.5h 0.5h H2O2 (0.5 mM) Ctrl 0.5h Ctrl 0.5h PBS KRG * 1.5 1.5 *** PI3K 1.0 ** 1.0 p-Akt PI3K/β-actin * p-Akt/β-actin 0.5 0.5 Akt 0 0 β-actin Ctrl Ctrl DMSO PHTTP DMSO PHTTP Ctrl Ctrl (A) SK-N-SH cells were transfected with 40 nM siER-β for 24 h and then incubated with 1 mg/mL of KRG for 48 h. (C) SK-N-SH cells were incubated with 1 mg/mL of KRG for 48 h and then exposed to the ERβ inhibitor PHTPP (5 µM) for 5 h. Following the indicated treatments, cells from (A) and (C) were incubated with H2O2 for 30 min, and western blots were performed to estimate gene expression levels in cell lysates. (B, D) Quantification of results from (A, C). DMSO DMSO PHTTP PHTTP J Ginseng Res. 2015 Jan;39(1):69-75.

17. Inhibition of PI3K/AKT signaling counteracts apoptosis in oxidatively stressed brain cells. H2O2 (0.5 mM) A B PBS-treatment PBS KRG KRG-treatment Treatment time ** * * Ctrl 0.5h 0.5h Ctrl 0.5h 0.5h 1.5 1.5 ** 1.0 p-Akt 1.0 Bcl-2/actin p-Akt/actin 0.5 0.5 Akt 0 0 Bcl-2 *** ** 1.5 1.5 * p-p53 1.0 1.0 p-p53/actin Caspase-3 Caspase-3/actin 0.5 0.5 β-actin 0 0 Ctrl Ctrl Ctrl Ctrl DMSO DMSO DMSO DMSO LY294002 LY294002 LY294002 LY294002 H2O2 (0.5 mM) C D (A, C) SK-N-SH cells were incubated with 1 mg/mL of KRG for 48 h. Subsequently, cells were exposed for 6 h to 80 µM of PI3K inhibitor LY294002 (A) or 50 µM of AKT inhibitor VIII (C). Cells were then treated with H2O2 for 30 min. Cell lysates were subject to western blot analysis to estimate protein levels of interest following treatment. (B, D) Quantification of experimental results from (A, C). PBS KRG *** *** Treatment time ** 1.5 1.5 0.5h 0.5h 0.5h 0.5h Ctrl Ctrl * * 1.0 1.0 Bcl-2 Bcl-2/actin Caspase-3/actin 0.5 0.5 p-p53 0 0 Ctrl Ctrl Caspase-3 DMSO DMSO AKT inhibitor AKT inhibitor β-actin Ctrl Ctrl DMSO DMSO AKT inhibitor AKT inhibitor J Ginseng Res. 2015 Jan;39(1):69-75.

18. Upregulation of ER-β by KRG inhibits apoptosis in oxidatively stressed brain cells. Conclusion Oxidative Stress KRG Oxidative stress suppresses ER-β, which is required for the activation of PI3K/AKT signals. Activation of PI3K/AKT signals induces anti-apoptotic factors or represses pro-apoptotic factors. KRG induces ER-β, leading to the upregulation of PI3K/AKT signals, thereby inhibiting apoptosis in the brain. ER-β PI3K Akt Caspase-3 Bcl-2 pp53 J Ginseng Res. 2015 Jan;39(1):69-75.

19. Acknowledgement Dept. of Mol. Science & Tech., Ajou University, School of Pharmacy, Sungkyunkwan University Cuong Thach Nguyen Eun-Hye Kim Truc Thanh Luong In-Hye Kim Mi-Jeong Lee Jung-Ah Ha Suhkneung Pyo Soo-Cheol Lee Sangdun Choi

20. http://www.ginsengsociety.org/abstract/2016_spring/

21. Thanks for your attention http://www.ginsengsociety.org/abstract/2016_spring/