Effects of Ginsenoside Rb3 on NMDA current and KA current in rat cultured hippocampal neurons SGK BDNF GCR

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1 DOI: /j.cnki.zyyl Effects of Ginsenoside Rb3 on NMDA current and KA current in rat cultured hippocampal neurons Shen Hongmei Jiang Zhenglin Department of Physilogy Institute of Nautical Medicine Nantong University Nantong Objective To observe the influence of ginsenoside Rb3 on the N-methyl-D-aspartic acid receptors and kainate acid receptors in rat's cultured hippocampal neurons. Methods Hippocampal neurons from embryo of rats were cultured in vitro and then using the patch-clamp technique we examed the effect of ginsenoside Rb3 on the peak amplitude steady state amplitude the percentage of steady state amplitude obove the peak amplitude the time constate of NMDA current but also observed the influence of ginsenoside Rb3 on KA current. Results ginsenoside Rb3 inhibited the steady state applitude of NMDA current the percentage of steady state amplitude above the peak amplitude the time constant of NMDA current. However ginsenoside Rb3 did not effect the amplitude of KA current. Conclusions Ginsenoside Rb3 can induce the inactivation of NMDA receptors selectively. Key words ginsenoside Rb3 N-methyl-D-aspartic acid kainate acid * SGK \BDNF \GCR ** M M CORT + 3nM RU M CORT g /L M CORT + 40uM 24h SYTO13 - PI MTT Western Blot GCR BDNF SGK GCR BDNF SGK RU g /L 40μM GCR BDNF SGK GCR BDNF SGK SGK BDNF GCR - - HPA 1 GC Megazyme 0. 1g /L GCR 40μM Corticosteron US Biological 10-4 M RU38486 GC Sigma 3nM h SD 1. 3 SYTO13 Invitrogen PI Amresco SP 1 MSG 2 DMEM Hyclone Gibcol Neurobasal B27 Gibcol DMSO Amresco MTT Sigma SGK CST GCR Merck IgG- 3 4 HRP IgG-HRP NSE BDNF SANTA CRUZ 1. 4 B5060EK CO 2 Heraeus Bio-hazard TH-3560 * 2010JW07 J50301 **

2 17 XDS-1B Olympus 5 A B CORT JA1003 ELX M C RU38486 BIO-TEC FR M CORT + 3nM RU38486 D 10-4 M CORT g /L E 10-4 M CORT + 40μM h SD 5 24h SYTO13 - PI MTT West- 75% ern blot SYTO13 - PI DMEM 96 n = 6 96 mm % Trypsin & 0. 02% EDTA 6ml 2ul SYTO uM 15ml 30min 15min 2ul PI 10min 5 ~ 6ml 15min HEPES buffer 20% BSA DMEM 3min 2 5 ~ 6ml MTT 96 3min 50ml 150ul n = 6 MTT 5-6ml 3 ~ 5mg /ml 20ul 4h 150ulDMSO 4 50ml 10min 570nm OD 0. 5 ~ / Western Blot ml ul 24 1ml 6 2ml GCR BDNF SGK DBI CO 2 24h neurobasal + BCA B n = 4 Nissl NSE 20μl μg 10 Nissl 6 n = 6 4% SDS-PAGE 5% 1 Nissl 37 4 PBST min 2 95% 5 70% PBST ECL NSE 6 2 n = 6NSE SP h 1b 10d 1a 2d 1c 7d NSE

3 18 NSE 2b 2a nissl 2. 3 SYTO13-PI 3a 3b SYTO13 PI 2 SGK BDNF GCR /β-actin x 珋 ± s 4 n SGK BDNF GCR ± ± ± RU ± ± ± RU ± * ± * ± * 2. 4 MTT 1 MTT OD570nm x 珋 ± s n OD 570nm ± ± RU nM ± g /L ± μM ± P < * P < ± * ± * ± * ± * ± * ± * * P < GCR BDNF SGK GCR BDNF SGK 3 GCR RU GC GCR GC-GCR GCR GCR 10-4 M 6 7 GCR RU38486 GCR HPA 8 9 BDNF 10 BDNF 2. 5 Western blot SGK BDNF GCR TrkB B 11 BDNF-TrkB ras Ras GC-GCR BDNF 12 ~ 14 / SGK 2002 Kuen SGK

4 19 SGK 149 ~ SGK 6 Burnstein KL Jewell CM Cidlowski JA. Human glucocorti-coid receptor cdna contains sequences sufficient for receptor down regulation. J Biol SGK 16 Chem ~ Song LN. Effects of retinoic acid and its dexamethasone on proliferation differentiation and glucocorticoid receptor expression in cultured human osteosarcoma cells. Oncol Res ~ 114 SGK BDNF 8 Boyer P. Do anxiety and depression have a common pathophysiological SGK BDNF mechanism Acta Psychiatr Scand ~ 29 Western 9 MA Lynch. Age related impairment in long-term potentiation in hippocampus a role for the cytokine interleukin 1β. Prog Neurobiol GCR SGK BDNF GCR RU38486 GCR 571 ~ 589 Maiyar 10 Murer MG Yan Q Raisman VR. Brain-derived neurotrophic factor in GCR SGK the control human brain and in Alzheimer's disease and Parkinson's disease. SGK Petti Pang Bai Lu. Regulation of late phase LTP and long-term memory in normal and aging hippocampus role of secreted proteins TPA and BD- NF. Ageing research reviews ~ 430 GCR BDNF SGK 12 Kp lan DR Hemp stead BL Martin Z et al. The trk protoon -cogene GCR product A signal transduction receptor for nerve growth factor. Science SGK BDNF ~ Sugimoto T Ikuroda H Horii Y et al. Signal transduction pathways through TRK-A and TRK-B receptors in human neuroblastoma cells. J Cancer Res ~ Feng Hao Lu Limin Huang Ying et al. Blockade of NMDA receptor enhances corticosterone induced downregulation of brain derived neurotrophic 1 Cui Y Yan Z Hou S et al. Effect of ShuDi-huang on the transmitter and factor gene expression in the rat hippocampus through camp response element receptor of amino acid in brain and learning and memory of dementia model. China J Chin Mater Med ~ 866 binding protein pathway. Acta Physiologica Sinica ~ Tsai KJ Chen SK Ma YL et al. sgk a primary glucocor- ticoid-induced c-fos NGF ~ gene facilitates memory consolidation of spatial learning in rats. PNAS ~ Ding Xuepeng Chen Xuequn Du Jizeng. The roles of SGK and GR in ~ 46 the spatial learning and memory enhancement. Bulletin of science and tech- 4 Kubo M Asano T Matsuda H et al. Studies on Rehmanniae RadixⅢ. The relation between changes of constituents and improvable effects on hemorheology with the processing of roots Rehmanniae glutinosa. Yakugaku Zasshi ~ SUN Gang WAN Shun-lun QIU Jian et al. New aspects of the mechanisms of glucocorticoid actions on the brain. chin J Neurosci Prog Neuro- bio ~ 124 nology ~ Maiyar AC Phu PT Huang AJ et al. Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum / glucocorticoid-inducible protein kinase sgk gene promoter by the p53 tumor suppressor protein. Mol docrinol ~ 329 Gene expression of SGK BDNF GCR following mannotr-iose of SHUDI treatment in hippocampal neurons with high dose corticosterone injury Zhang Lina Jin Guoqin Department of Biochemistry College of Basic Medicine of Shanghai University of TCM Shanghai Objective To observe the effect of mannotriose in SHUDI on gene expression of the learning and memory related signaling molecules. in injured hippocampal neurons by high dose of corticosterone. Methods Hippocampal neurons prime cultured were prepared from newborn 24h rats after 7 d incubation the cultured neurons were treated with drug and divided into 5 groups. 1 blank group 2 model group containing10-4 M CORT 3 RU38486 group containing 10-4 M CORT + 3nM RU mannotriose group containing10-4 M CORT g /L mannotriose 5 positive control group containing10-4 M CORT + 40μM aricept. 48 hours later SYTO13-PI double-fluorescence staining MTT assay and Western Blot was carried out. Results Compared with the control group both the cell survival rate by SYTO13-PI double-fluorescence staining and cell viability by MTT decreased markedly in model group P < GCR BDNF and SGK protein expression significantly de-

5 20 creased. Mannotriose as Aricept and RU38486 as increased the above indices to varying degrees. Conclusion 10-4 M CORT had injured the hippocampus neurons. Mannotriose of SHUDI as a positive control aricept as could significantly prevent the hippocampus neuron from injury probably retarding learning and memory functional deterioration induced by high dose of CORT. The regulatory mechanism maybe is that through signal transdu- ction pathway on learning and memory the mannotriose change the learning and memory-related signal transduction molecules gene expression such as GCR BDNF and SGK. Key words mannotriose hippocampal neuron signal transduction SGK BDNF GCR * 4' ** '- - 4'-methyl ether-scutellarein 4-MS JAR RT-PCR Survivin Caspase 9 Caspase 3 p38 MAPK Western blot Survivin Caspase 9 Caspase 3 p38 MAPK Bax cytochrome C RT-PCR 20 40μg /ml 4-MS 24h JAR Survivin mrna Caspase 9 Caspase 3 p38 MAPK mrna Western blot JAR Survivin Bax cytochrome C p38 Caspase 3 Caspase 9 4-MS Bax cytochrome C Caspase 9 Caspase 3 p38 MAPK Survivin JAR 4'- - 4-MS JAR p38 MAPK Verbena officinalis C 4 '- - 4 '-methery cutellarein 4- MS JAR JAR Ca Ca MS 4-MS JAR 1 NEOMARKERS Survivin p-p38 BioLegend Bax cytochrome C Pro-caspase 9 sigma PVDF Bio-Rad ECL-Plus AMERSHAM ECL POERCE 1. 4 HERA cell 150 CO 2 HERAEUS OLYMPUS JEM1010 FACSCalibur BD ScanArray JAR 4000 PACKARD Cel Doc Bio-Rad PCR JAR Bioer Rotor-Gene 3000 Realtime PCR Corbett Research 1. 5 JAR % CO % 2 ~ MS JAR % 75 ml 24 h 40μg /L 1. 2 JAR 48h 72 h 2 PBS Gibcol 20% 2000 rpm 10 min 2. 5% 618 LKB-V SIGMA Trizol PCR cdna DNA Marker DL2000 TAKARA PCR RT-PCR Caspase 3 Caspase 9 Survivin p38 mr- Caspase 3 Caspase 9 NA RNA JAR 24h μg /ml 48h 4 RNA ROCHE D260 D280 * BK **