Abstract
Cellular adaptation to hypoxia involves regulation of specific genes such as vascular endothelial growth factor (VEGF), erythropoietin (EPO) and hypoxia inducible factor (HIF)-1. In this study, we have evaluated the protective effect of picroliv (a purified iridoid glycoside fraction from roots of Picrorhiza kurrooa with hepatoprotective, anti-inflammatory and antioxidant properties) against hypoxic injury by examining lactate dehydrogenase (LDH) release in Hep 3B and Glioma cells. The expression of hypoxia regulated genes, VEGF and HIF-1 was studied in human umbilical vein endothelial cells (HUVEC), Hep 3B and Glioma cells. Picroliv reduced the cellular damage caused by hypoxia as revealed by a significant reduction in LDH release compared to untreated control. The expression of VEGF and HIF-1 subunits (HIF-1α and HIF-1β) was enhanced by treatment with picroliv during normoxia and hypoxia in HUVEC and Hep 3B cells and on reoxygenation the expression of these genes was significantly reduced as revealed by mRNA analysis using RT-PCR. Simultaneous treatment with picroliv during hypoxia inhibited VEGF and HIF-1 expression in Glioma cells whereas the expression was not reduced by picroliv treatment during reoxygenation as evidenced by both RT-PCR and Northern hybridization. VEGF expression as revealed by immunofluorescence studies correlates well with the regulations observed in the MRNA expression. We have also examined the kinase activity of tyrosine phosphorylated proteins and protein kinase C (PKC) in Glioma cells treated with picroliv during hypoxia/reoxygenation. A selective inhibition of protein tyrosine kinase activity leading to tyrosine dephosphorylation of several proteins including 80 kd protein, and a reduction in PKC was seen in cells treated with picroliv and hypoxia. These findings suggest that picroliv may act as a protective agent against hypoxia/reoxygenation induced injuries, and the underlying mechanism may involve a novel signal transduction pathway.
Similar content being viewed by others
References
Ladoux A, Frelin C: Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochem Biophys Res Commun 195: 1005–1010, 1993
Shweiki D, Itin A, Soffer D, Keshet E: Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359: 843–845, 1992
Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT: Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 246: 1309–1312, 1989
Fandrey J, Bunn HF: In vivo and in vitro regulation of erythropoietin mRNA: Measurement by competitive polymerase chain reaction. Blood 81: 617–623, 1993
Hernandez RE, Ladoux A, Frelin C: Differential regulation of cardiac heme oxygenase-1 and vascular endothelial growth factor mRNA expressions by hemin, heavy metals, heat shock and anoxia. FEBS Lett 382: 229–233, 1996
Semenza GL, Roth PH, Fang HM, Wang GL: Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem 269: 23757–23763, 1994
Marti HH, Jung HH, Pfeilschrifter J, Bauer C: Hypoxia and cobalt stimulate lactate dehydrogenase (LDH) activity in vascular smooth muscle cells. Pflügers Arch 429: 216–222, 1994
Firth JD, Ebert BL, Pugh CW, Ratcliffe PJ: Oxygen regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: Similarities with the erythropoietin 3′ enhancer. Proc Natl Acad Sci USA 91: 6496–6500, 1994
Folkman J, Shing Y: Angiogenesis. J Biol Chem 276: 10931–10934, 1992
Klagsbrun M, Soker S: VEGF/VPF: The angiogenesis factor found? Curr Biol 3: 699–702, 1993
Goldman CK, Kim J, Wong W, King V, Brock T, Gillespie GY: Epidermal growth factor production by human malignant glioma cells: A model of glioblastoma multiform pathophysiology. Mol Biol Cell 4: 121–133, 1993
Goldberg MA, Schneider TJ: Similarities between the oxygen-sensing mechanisms regulating the expression of vascular endothelial growth factor and erythropoietin. J Biol Chem 269: 4335–4359, 1994
Garrido C, Saule S, Gospodarowicz D: Transcriptional regulation of vascular endothelial growth factor gene expression in ovarian bovine granulosa cells. Growth factors 8: 109–117, 1993
Harada S, Nagy JA, Sullivan KA, Thomas KA, Endo N, Rodan GA, Rodan SB: Induction of vascular endothelial growth factor expression by prostaglandin E1 and E2 in osteoblasts. J Clin Invest 93: 2490–2493, 1994
Cullinan-Bove K, Koos RD: Vascular endothelial growth factor/ vascular permeability factor expression in the rat uterus: Rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology 133: 829–837, 1993
Wang GL, Jiang B-H, Rue EA, Semenza GL: Hypoxia inducible factor 1 is a basic-helixloop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Nat Acad Sci USA 92: 5510–5514, 1995
Semenza GL, Wang GL: A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 12: 5447–5454, 1992
Guillemin K, Karsnow MA: The hypoxic response: Huffing and HIFing. Cell 89: 9–12, 1997
Forsythe JA, Jiang B-H, Iyer NV, Agani F, Leung SW, Koos RD, Semenza GL: Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16: 4604–4613, 1996
Frede S, Fandrey J, Jelkmann W: Role of protein kinase C in hepatic erythropoietin synthesis. Exp Haematol 23: 96–98, 1995
Wang GL, Jiang B-H, Semenza GL: Effect of protein kinase and phosphotase inhibitors on expression of hypoxia inducible factor-1. Biochem Biophys Res Commun 216: 669–675, 1995
Chander R, Kapoor NK, Dhawan BN: Picroliv, picroside-1 and kutkoside from Picrorhiza kurrooa are scavengers of superoxide anions. Biochem Pharmacol 44: 180–183, 1992
Rastogi R, Saksena S, Garg NK, Kapoor NK, Agarwal DP, Dhawan BN: Picroliv protects against alcohol-induced chronic hepatotoxicity in rats. Planta Med 62: 283–285, 1996
Dwivedi Y, Rastogi R, Garg NK, Dhawan BN: Perfusion with picroliv reverses biochemical changes induced in livers of rats toxicated with galactosamine or thioacetamide. Planta Med 59: 418–420, 1993
Dwivedi Y, Rastogi R, Garg NK, Dhawan BN: Picroliv and its components kutkoside and picroside 1 protect against galactosamine-induced damage in rats. Pharmacol Toxicol 71: 383–387, 1992
Dhawan BN: Picroliv – A new hepatoprotective agent from an Indian medicinal plant, Picrorhiza kurrooa. Med Chem Res 5: 595–605, 1995
Chander R, Kapoor NK, Dhawan BN: Picroliv affects gamma-glutamyl cycle in liver and brain of Mastomys natalensis infected with Plasmodium berghei. Ind J Exp Biol 32: 324–327, 1994
Chander R, Kapoor NK, Dhawan BN: Effect of picroliv on glutathione metabolism in liver and brain of Mastomys natalensis infected with Plasmodium berghi. Ind J Exp Biol 30: 711–714, 1992
Tandon S, Rastogi R, Shukla R, Kapoor NK, Srimal RC, Dhawan BN: Protective activity of picroliv from Picrorhiza kurrooa against myocardial ischemia induced by coronary ligation or isoproterenol. Med Sci Res 23: 515–518, 1995
Singh AK, Sidhu GS, Deepa T, Maheshwari RK: Curcumin inhibits the proliferation and cell cycle progression of human umbilical vein endothelial cells. Cancer Lett 107: 109–115, 1996
Maheshwari RK, Srikantan V, Bhartiya D, Kleinman HK, Grant DS: Differential effects of interferon gamma and alpha on in vitro model of angiogenesis. J Cell Physiol 146: 164–169, 1991
Visen PKS, Saraswat B, Patnaik GK, Agarwal DP, Dhawan BN: Protective activity of picroliv isolated from Picrorhiza kurrooa against ethanol toxicity in isolated rat hepatocytes. Ind J Pharm 28: 98–101, 1996
Dwivedi Y, Rastogi R, Sharma SK, Mehrotra R, Garg NK, Dhawan BN: Picroliv protects against monocrotaline-induced hepatic damage in rats. Pharmacol Res 23: 399–407, 1991
Fellman V, Raivio KO: Reperfusion injury as the mechanism of brain damage after perinatal asphyxia. Pediatric Res 41: 599–606, 1997
Horakova L, Stolc S, Chromikova Z, Pekarova A, Derkova L: Mechanisms of hippocampal reoxygenation injury. Treatment with antioxidants. Neuropharmocology 36: 177–184, 1997
Kuroki M, Voest EE, Amano S, Beerepoot LV, Takashima S, Tolentino M, Kim RY, Rohan RM, Colby KA, Yeo K-T, Adamis AP: Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. J Clin Invest 98: 1667–1675, 1996
Gulati S, Singh AK, Irazu C, Orak J, Rajagopalan PR, Fitts CT, Singh I: Ischemia reperfusion injury: Biochemical alterations in peroxisomes of rat kidney. Arch Biochem Biophys 295: 90–100, 1992
Metzger J, Lauterburg BH: Post ischemic ATP levels predict hepatic function 24 h following ischemia in the rat. Experientia 44: 455–457, 1988
Watson BD, Buston R, Goldberg WJ, Santiso M, Ginsberg MD: Lipid peroxidation in vivo induced by reversible global ischemia in rat brain. J Neurochem 42: 268–274, 1984
White BC, Aust SD, Ardors KE, Aronson LD: Brain injury by ischemic anoxia hypothesis extension-a tale of two ions. Ann Emer Med 13: 862–867, 1984
Ferrara N, Davis-Smyth T: The biology of vascular endothelial growth factor. Endocrine Rev 18: 4–25, 1997
Beerepoot LV, Shima DT, Kuroki M, Yeo K-T, Vost EE: Up-regulation of vascular endothelial growth factor production by iron chelators. Cancer Res 56: 3747–3751, 1996
Bunn HF, Poyton RO: Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev 76: 839–885, 1996
Rozengurt E, Rodriguez-Pena A, Coombs M, Sinnett-Smith J: Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells; role of Ca2+-sensitive phopholipid dependent protein kinase. Proc Nat Acad Sci USA 81: 5748–5752, 1984
Hayashi A, Koroma BM, Imai K, Dejuan E: Increase of protein tyrosine phosphorylation in rat retina after ischemia-reperfusion injury. Invest Opth Vis Sci 37: 2146–2s156, 1996.
Braunton JL, Wong V, Wang W, Salter MW, Roder J, Liu M, Wang YT: Reduction of tyrosine kinase activity and protein tyrosine dephosphorylation by anoxic stimulation in vitro. Neuroscience 82: 161–170, 1998
Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield K, Razvi S, Walsh K, Symes JF: Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischemic limb. Lancet 348: 370–374, 1996
Harada K, Friedman M, Lopez JJ, Wang SY, Li J, Prasad PV, Pearlman JD, Edelman ER, Sellke FW, Simons M: Vascular endothelial growth factor in chronic myocardial ischemia. Am J Physiol 270: H1791–H1802, 1996
Pearlman JD, Hibberd MG, Chuang ML, Harada K, Lopez JJ, Gladstone SR, Friedman M, Sellke FW, Simons M: Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis. Nature Med 1: 1085–1089, 1995
Author information
Authors and Affiliations
Additional information
Affiliation
Rights and permissions
About this article
Cite this article
Gaddipati, J.P., Madhavan, S., Sidhu, G.S. et al. Picroliv - a natural product protects cells and regulates the gene expression during hypoxia/reoxygenation. Mol Cell Biochem 194, 271–281 (1999). https://doi.org/10.1023/A:1006982028460
Issue Date:
DOI: https://doi.org/10.1023/A:1006982028460