ACTA UN IVER SITATIS TR ADITIO N IS M EDICALIS SIN EN SIS PHAR M ACO LO GIAEQ UE SHAN GHAI Vol. 29 N o. 3 M ay 2015

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1 70 ACTA UN IVER SITATIS TR ADITIO N IS M EDICALIS SIN EN SIS PHAR M ACO LO GIAEQ UE SHAN GHAI Vol. 29 N o. 3 M ay S -PPD protopanaxadiol PPD 20 S -PPD 20 S -PPD MTT DAPI Calpain Calpain 3 3- PCR XBP-1 CHOP mrna Western blot Caspase-3 Caspase-8 bax bcl-2 ATF-6 IRE1 PERK ATF-4 CHOP 20 S -PPD 10 μmol /L 6 h 5 μmol 24 h 20 S -PPD 10 μmol /L 6 h DAPI 3 3- Calpain Western blot Caspase-3 Caspase-8 bax bcl-2 ATF-6 IRE1 PCR XBP-1s mrna PERK ATF-4 CHOP mrna 20 S -PPD IRE1 PERK bcl-2 R A DOI / j x protopanaxadiol PPD S -PPD Rbl Rb2 Rb2 Rc Rd Rg3 Rh2 20 HPLC 98% DMSO S R PPD 10 mmol /L μm PPD - 20 DMEM S -PPD HUVECs 4 ScienCell Research Laboratories 20 S -PPD 10% DMEM 37 5% CO DMEM Thermo 1 Fisher MTT JW02 Genmed Trizol SuperScriptTM II RNase H- datp dgtp dctp dttp Invitrogen SYBR Premix Ex Taq GC Perfect Real Time zhangtdmj@ hotmail. com DAPI Sigma-Aldrich Calpain Activity Assay Kit Abnova TaKaRa GAPDH Epitomics-an

2 Abcam Caspase-3 Caspase-8 bax bcl-2 Cell Signaling Technology ATF-4 CHOP Proteintech IRE1p ATF-6 RFU Prosci PERK Santa Cruz Biotechnology 2 ml 80% GAPDH F 5 -TCTGATTTGGTCGTAT TGGG-3 GAPDH R 5 -CGCTCCTGGAAGATGGT GAT bp XBP-1s F 5 -CCGACGGGACCC CTAAAG-3 XBP-1s R 5 -ACTGGGCCTGCACCT GCT bp CHOP F 5 -GGAGCTGGAAGCCT GGTATGAGG-3 CHOP R 5 -TCCCTGGTCAGGC GCTCGATTTCC bp Hettich PCR Eppendorf RNA 2. 0 μg RNA Synergy H1 BioTek PCR PCR Olympus min s s 40 Zeiss StrataGene Mx3000P PCR Agilent GAPDH Bio-Rad Western blot min / 96 5% 1 h 20 S -PPD PBST HRP DMSO 6 1 h PBST 4 ECL 6 h 24 h 5 mg /ml MTT 4 h 1. 3 SPSS DMSO 150 μl 10 min 490 nm OD P < % = OD - OD 2 / OD - OD S -PPD HUVECs 2 ml 80% MTT 20 S -PPD HUVECs 10 μmol /L 20 S -PPD DMSO PBS 3 2 ng /ml DAPI 1 ml 15 min PBS 6 h HUVECs 6 h 5 μmol /L 20 S -PPD Calpain DAPI 10 μmol /L 20 S -PPD 6 h 25 cm 2 80% 10 μmol /L 20 S - PPD DMSO 2 ~ 12 h Calpain μg h 400 nm 505 nm 10 μmol /L 20 S - PPD DMSO 6 h min s s s s r /min 40 μg x ± s 10 μmol /L 20 S -PPD HUVECs 24 h S -PPD HUVECs Western blot 10 μmol /L 20 S -PPD 6 h Caspase-3 17 kd

3 72 ACTA UN IVER SITATIS TR ADITIO N IS M EDICALIS SIN EN SIS PHAR M ACO LO GIAEQ UE SHAN GHAI Vol. 29 N o. 3 M ay **P < S -PPD HUVECs 10 μmol /L 20 S -PPD Western blot Caspase-8 bax bcl-2 20 S -PPD Calpain S -PPD HUVECs μmol /L 20 S -PPD 6 h 20 S -PPD HUVECs S -PPD HUVECs DAPI S -PPD HUVECs Caspase S -PPD HUVECs Calpain S -PPD HUVECs Western blot 10 μmol /L 20 S -PPD 6 h ATF-6 IRE1 PERK ATF-4 CHOP 10 μmol /L 20 S -PPD 6 h PCR XBP-1s CHOP mrna S -PPD HUVECs Western blot

4 S -PPD HUVECs 20 S -PPD HUVECs XBP-1s CHOP mrna n = 3 x ± s 20 S -PPD XBP-1s 20 S -PPD HUVECs 200 CHOP 0 h ± ± h ± ** ± ** 12 h ± **## ± **## 0 h **P < h ##P < PPD PPD PPD PPD 20 S -PPD HUVECs 5 μmol /L 20 S -PPD 20 S -PPD 20 S -PPD HUVECs PPD HUVECs enos NO 9 10 μmol /L 20 S -PPD HUVECs6 h DAPI Caspase-3 17 kd 20 S -PPD HUVECs Caspase-3 Caspase-8 Caspase-9 Calpain Caspase S -PPD Caspase-8 20 S -PPD HUVECs S -PPD

5 74 ACTA UN IVER SITATIS TR ADITIO N IS M EDICALIS SIN EN SIS PHAR M ACO LO GIAEQ UE SHAN GHAI Vol. 29 N o. 3 M ay S -PPD 6 h 20 S -PPD HUVECs IRE1 PERK CHOP bcl-2 20 S -PPD Ca 2 + unfolded protein response UPR PERK IRE-1 ATF-6 12 PERK 12 eif2α ATF-4 CHOP 13 IRE-1 X 1 X box-binding protein-1 XBP-1 mrna 26 XBP-1 mrna XBP-1 UPR 14 ATF μmol /L 20 S -PPD 6 h ATF-6 ATF-6 20 S -PPD UPR 10 μmol /L 20 S -PPD 6 h IRE1 XBP- 1s mrna PERK ATF- 4 CHOP mrna IRE1 PERK 20 S - PPD HUVECs UPR and total assignment of 1 H NMR spectra J Am Gourley M Williamson JS. Calpain Caspase S -PPD Calpain Caspase S -PPD HUVECs bcl CHOP CHOP bcl μmol /L 20 S - PPD 6 h CHOP mrna bcl-2 Cell Biol bax 20 S -PPD 1 Dong H Bai LP Wong VK et al. The in vitro structure-related anti-cancer activity of ginsenosides and their derivatives J. Molecules Wang W Rayburn ER Zhao Y et al. Novel ginsenosides 25-OH- PPD and 25- OCH3- PPD as experimental therapy for pancreatic cancer anticancer activity and mechanisms of action J. Cancer Lett Li G Wang Z Sun Y et al. Ginsenoside 20 S -protopanaxadiol inhibits the proliferation and invasion of human fibrosarcoma HT1080 cells J. Basic Clin Pharmacol Toxicol Bagri A Kouros-Mehr H Leong KG et al. Use of anti-vegf adjuvant therapy in cancer challenges and rationale J. Trends Mol Med J Usami Y Liu YN Lin AS et al. Antitumor agents S protopanaxadiol and 20 S -protopanaxatriol as antiangiogenic agents J Nat Prod 7 Folkman J. Fighting cancer by attacking its blood supply J. Sci Angiogenesis new targets for the development of anticancer chemotherapies J. Curr Pharm Des Leung KW Leung FP Mak NK et al. Protopanaxadiol and protopanaxatriol bind to glucocorticoid and oestrogen receptors in endothelial cells J. Br J Pharmacol Chai WS Zhu XM Li SH et al. Role of Bcl-2 family members in caspase-3 /9-dependent apoptosis during Pseudomonas aeruginosa infection in U937 cells J. Apoptosis Feng B Yao PM Li Y et al. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages J. Nat Kim I Xu W Reed JC. Cell death and endoplasmic reticulum

6 stress disease relevance and therapeutic opportunities J. Nat Rev Drug Discov Harding HP Novoa I Zhang Y et al. Regulated translation initiation controls stress- induced gene expression in mammalian cells J. Mol Cell Yoshida H Matsui T Yamamoto A et al. XBP1 mrna is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor J. Cell Ye J Rawson RB Komuro R et al. Mol Cell. ER stress induces cleavage of membrane- bound ATF6 by the same proteases that process SREBPs J. Mol Cell Xu C Bailly-Maitre B Reed JC. Endoplasmic reticulum stress cell life and death decisions J. J Clin Invest Hetz CA. ER stress signaling and the BCL-2 family of proteins from adaptation to irreversible cellular damage J. Antioxid Redox Signal Szegezdi E Macdonald DC Ni Chonghaile T et al. Bcl-2 family on guard at the ER J. Am J Physiol Cell Physiol McCullough KD Martindale JL Klotz LO et al. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state J. Mol Cell Biol S -protopanaxadiol Activates ER Stress to Induce Cell Apoptosis in Human Umbilical Vein Endothelial Cells ZHANG Xue-li 1 HAN Zhi-fen 2 XIA Hua-ying 1 JIANG Jia-ye 2 ZHANG Tong 2 1. College of Basic Medicine of Shanghai University of Traditional Chinese Medicine 2. Experiment Center for Teaching and Learning of Shanghai University of Traditional Chinese Medicine ABSTRACT Objective To observe the effects of 20 S -PPD on apoptosis and study the mechanism of 20 S -PPD inducing cell apoptosis in human umbilical vein endothelial cells HUVECs. Methods HUVECs were treated with 20 S - PPD and cell proliferation was detected by MTT assay. Cytoplasmic calpain activities were detected through Calpain Activity Assay Kit. Morphologies of endoplasmic reticulum were observed by ER fluorescent probe 3 3-dihexyloxacarbocyanine iodide staining. Splicing XBP-1 and CHOP mrna levels were detected by Real-time quantitative PCR analysis. Caspase-3 Caspase- 8 bax bcl-2 ATF-6 phosphorylated IRE1 phosphorylated PERK and CHOP protein levels were detected by Western blot analysis. Results The cell growth of HUVECs was inhibited after treatment with 20 S -PPD above 10 μmol /L for 6 h or inhibited after treatment with 20 S -PPD above 5 μmol /L for 24h. After 10 μmol /L 20 S -PPD treatments obvious condensed chromatins and fragmented nuclear bodies were observed in apoptotic cells by DAPI staining. The networks of ER appeared to be fragmented and aggregated to small patches distributed randomly around nucleus by 3 3-dihexyloxacarbocyanine iodide staining while cytoplasmic calpain activities were not changed significantly. Cleaved Caspase-3 was detected in 10 μmol /L 20 S -PPD treated HUVECs Caspase-8 bax were not changed significantly while bcl-2 protein levels decreased significantly after 10 μmol /L 20 S -PPD treatments. ATF6 was not changed significantly phosphorylated IRE1 and splicing XBP-1 mrna increased significantly phosphorylated PERK and ATF-4 increased significantly. CHOP mrna and protein levels increased significantly after 10 μmol /L 20 S -PPD treatments. Conclusion 20 S -PPD induces apoptosis in HUVECs by activating IRE1 and PERK signal pathway through ER stress to down-regulate bcl-2. KEYWORDS protopanaxadiol ER stress human umbilical vein endothelial cells apoptosis