姜黄素对辐射诱导的胰腺外分泌细胞损伤的保护作用

摘要/Abstract

摘要： 将大鼠胰腺外分泌细胞(AR42J)分为实验组与对照组,两组细胞根据姜黄素处理水平又分为A/B/C/D/E 5个姜黄素处理组,分别经0、2、4、8、16 μM姜黄素处理,实验组予以一次性大剂量X射线照射(6 Gy),模拟急性辐射损伤事件。照后收集细胞蛋白行免疫印迹试验(Wensten-blot)检测凋亡相关蛋白PARP、BCL-2,应激及能量代谢相关因子HIF-1ɑ、LDHA、PDH表达情况,MTT法检测细胞增殖活力,荧光酶标仪检测细胞ATP及ROS生成情况。清洁级大鼠24只随机分为对照组、单纯加药组、单纯照射组、照射+加药组,予以12 Gy X射线照射或假照射,射后24小时处死并解剖取出胰腺组织,Wensten-blot检查能量代谢相关蛋白表达情况,以探讨姜黄素在胰腺外分泌细胞辐射损伤中的作用及其机制。结果表明,与对照组相比,单纯照射组细胞在受照24 h后,凋亡蛋白PARP表达升高,BCL-2蛋白下调,线粒体膜电位下降表明线粒体受损。辐照促使缺氧诱导因子HIF-1α表达上调,介导糖酵解的关键因子LDHA表达增强,PDH表达下调。细胞增殖活力下降,ROS生成增多,沉默HIF-1表达在一定程度上抑制了辐照诱导的这种能量代谢转变。经姜黄素预处理,纠正了HIF-1α及LDHA的表达上调,部分恢复PDH表达,有效清除氧自由基,并且对细胞的增殖能力及ATP生成具有正向作用。动物实验显示辐照诱导HIF-1α表达加强,调控能量代谢相关蛋白表达发生改变,LDHA表达增强,PDH表达下调;经姜黄素预处理,纠正了HIF-1α上调所介导的能量代谢相关蛋白的表达变化,趋势与细胞实验相符。以上结果说明,胰腺外分泌细胞在电离辐射损伤下,通过上调HIF-1α表达,促使能量代谢发生转变,糖酵解途径加强,而经线粒体的有氧氧化受到抑制。姜黄素通过下调HIF-1α,纠正了辐射诱导的细胞能量代谢紊乱,并有效清除氧自由基,保护线粒体,从而发挥其对胰腺外分泌细胞的辐射损伤的保护作用。

关键词: 姜黄素, 辐射防护, HIF-1α, 能量代谢, 线粒体

Abstract: In order to elucidate the stress response and energy metabolism of pancreatic exocrine cells induced by ionizing radiation, and to explore the role of curcumin in pancreatic exocrine cell radiation injury and its mechanism,the experiment of Rat pancreatic exocrine cells (AR42J) were established. The rats were divided into experimental group and control group. The two groups were divided into five subgroups of A/B/C/D/E, which were treated with 0, 2, 4, 8 and 16 μM curcumin, repectively. The experimental group was given a one-time high-dose X-ray irradiation (6 Gy), simulating acute radiation damage events. The expression of PARP, BCL-2, stress and energy metabolism related factors HIF-1ɑ, LDHA and PDH were detected by Wensten-blot. The cell viability was detected by MTT assay, Fluorescence detection of cell ATP and ROS production. The experimental results were further validated by in vivo experiments. 24 clean-grade rats were randomly divided into control group, simple drug-added group, simple irradiation group, irradiation + dosing group. Rats in each group were exposed to 12 Gy X-ray or simulated irradiation. The rats were killed 24 hours after irradiation, and the pancreatic tissue was dissected. The expression of energy metabolism related proteins was examined by Wensten-blot method. The results demonstrate: 1) Compared with the control group, the expression of PARP was increased and the BCL-2 protein was slightly down-regulated after 24 hours of irradiation. Meanwhile, the decrease of mitochondrial membrane potential (ΔΨm) was observed which indicated mitochondria damage. 2) The expression of HIF-1α was significantly increased by irradiation, and LDHA, the key factor of glycolysis was significantly increased and PDH expression was down-regulated. 3) MTT assay showed a decrease in cell proliferation and the production of ROS was increased by microplate reader. 4) Silence HIF-1α by RNA technology had eliminated irradiation-induced energy metabolism transformation partly. In the experimental group, the increased expression of HIF-1α and LDHA was reversed by curcumin treatment, PDH was partially restored, the free radical scavenging was effectively removed, and the proliferation of cells and the ATP production were increased. 5) The animal experiments have got the same results. The expression of HIF-1α is up-regulated by ionizing radiation stress in pancreatic exocrine cells, which leads to the change of energy metabolism as the enhancement of glycolysis pathway and the inhibition of aerobic oxidation of mitochondria. Curcumin reduces the energy metabolism disorder induced by radiation by down-regulating HIF-1α, and effectively scavenges oxygen free radicals and protects mitochondria, thus exerting its protective effect on pancreatic exocrine cells.

Key words: curcumin, radiation protection, HIF-1α, energy metabolism, mitochondria

中图分类号:

R146

刘显荣, 何丽艳, 卢先州. 姜黄素对辐射诱导的胰腺外分泌细胞损伤的保护作用[J]. 辐射防护, 2019, 39(2): 157-167.

LIU Xianrong, HE Liyan, LU Xianzhou. Protective effect of curcumin on radiation-inducedpancreatic exocrine cell damage[J]. RADIATION PROTECTION, 2019, 39(2): 157-167.

参考文献

[1] Ifigeneia M, Zacharenia N, Maria S, et al. Complex DNA damage: A route to radiation-induced genomic instability and carcinogenesis[J]. Cancers, 2017, 9(12):91-111.
[2] Goel A, Aggarwal B B. Curcumin, the golden spice from Indian saffron, is a chemosensitizer and radiosensitizer for tumors and chemoprotector and radioprotector for normal organs [J]. Nutr Cancer, 2010, 62(7): 919-930.
[3] Vashisht M, Rani P, Dahiya S, et al. Curcumin primed exosomes reverses LPS-induced pro-inflammatory gene expression in buffalo granulosa cells[J]. Journal of Cellular Biochemistry, 2017, 119(2) 1 488-1 500.
[4] Fadus M C, Lau C, Bikhchandani J, et al. Curcumin: An age-old anti-inflammatory and anti-neoplastic agent[J]. Journal of Traditional & Complementary Medicine, 2017, 7(3):339-346.
[5] YANG L, ZHENG Z, QIAN C, et al. Curcumin-functionalized silk biomaterials for anti-aging utility[J]. J Colloid Interface Sci, 2017, 496: 66-77.
[6] LI J, ZHOU Y, ZHANG W, et al. Relief of oxidative stress and cardiomyocyte apoptosis by using curcumin nanoparticles[J]. Colloids & Surfaces B Biointerfaces, 2017, 153:174-182.
[7] LIU Y, CHENG F, LUO Y, et al. PEGylated curcumin derivative attenuates hepatic steatosis via CREB/PPAR-γ/CD36 pathway[J]. BioMed Research International, 2017, 2017(1):1-11.
[8] Soltani B, Ghaemi N, Sadeghizadeh M, et al. Curcumin confers protection to irradiated THP-1 cells while its nanoformulation sensitizes these cells via apoptosis induction[J]. Cell Biology and Toxicology, 2016, 32(6):543-561.
[9] Mehta H J, Patel V, Sadikot R T. Curcumin and lung cancer—a review[J]. Target Oncol, 2014, 9(4): 295-310.
[10] Oliver F J, Rubia G de la, Rolli V, et al. Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant[J]. J Biol Chem, 1998, 273(50): 33 533-33 539.
[11] Gaziev A I. Pathways for maintenance of mitochondrial DNA integrity and mitochondrial functions in cells exposed to ionizing radiation[J]. Radiats Biol Radioecol, 2013, 53(2): 117-136.
[12] Prithivirajsingh S, Story M D, Bergh, et al. Accumulation of the common mitochondrial DNA deletion induced by ionizing radiation[J]. FEBS Lett, 2004, 571(1-3): 227-232.
[13] Iijima T, Mishima T, Tohyama M, et al. Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron[J]. Neurochemistry International, 2003, 43(3):260-269.
[14] Rabbani Z N, Mi J, Zhang Y, et al. Hypoxia inducible aactor 1α signaling in fractionated radiation-induced lung injury: Role of oxidative stress and tissue hypoxia[J]. Radiation Research, 2010, 173(2):165-174.
[15] Higgins D F, Kimura K, Iwano M, et al. Hypoxia-inducible factor signaling in the development of tissue fibrosis[J]. Cell Cycle, 2008, 7(9):1 128-1 132.
[16] Kim W Y, Oh S H, Woo J K, et al. Targeting heat shock Protein 90 overrides the resistance of lung cancer cells by blocking radiation-induced stabilization of hypoxia-inducible factor-1α[J]. Cancer Research, 2009, 69(4):1 624-1 632.
[17] Semenza G L, Jiang B H, Leung S W, et al. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase a gene promoters contain essential binding sites for hypoxia-inducible factor 1[J]. Journal of Biological Chemistry, 1997, 271(51):32 529-32 537.
[18] Lunt S Y, Vander Heiden MG. Aerobic glycolysis: meeting the metabolic requirements of cell proliferation[J]. Annu Rev Cell Dev Biol, 2011, 27: 441-464.
[19] CHAN S Y, ZHANG Y Y, Hemann C, et al. MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2[J]. Cell Metabolism, 2009, 10(4):273-284.
[20] Corn, Paul G. Hypoxic regulation of miR-210: Shrinking targets expand HIF-1’s Influence[J]. Cancer Biology & Therapy, 2008, 7(2):265-267.
[21] Soltani B, Ghaemi N, Sadeghizadeh M, et al. Redox maintenance and concerted modulation of gene expression and signaling pathways by a nanoformulation of curcumin protects peripheral blood mononuclear cells against gamma radiation[J]. Chemico-Biological Interactions, 2016, 257:81-93.
[22] Thresiamma K C, George J, Kuttan R. Protective effect of curcumin, ellagic acid and bixin on radiation induced toxicity[J]. Indian J Exp Biol, 1996, 34(9): 845-847.
[23] Ak T, Gulcin I. Antioxidant and radical scavenging properties of curcumin[J]. Chem Biol Interact, 2008, 174(1): 27-37.
[24] 杨爱洁, 何信佳, 安永恒,等. 不同分割照射对胰腺生物学效应的影响[J].中华放射医学与防护杂志, 2011, 31(6):653-656
[25] ICRP. Recommendations of International Commission on Radiological Protection[M]. Oxford: Elsevier Science Publication, 2008: 103.
[26] Ahmadu-Suka F, Gillette E L, Withrow S J, et al. Exocrine pancreatic function following intraoperative irradiation of the canine pancreas[J]. Cancer, 1988, 62(6):1 091-1 095.
[27] Yamaguchi K, Nakamura K, Kimura M, et al. Intraoperative radiation enhances decline of pancreatic exocrine function after pancreatic head resection[J]. Digestive Diseases and Sciences, 2000, 45(6):1 084-1 090.
[28] Hoekstra H J, Restrepo C, Kinsella T J, et al. Histopathological effects of intraoperative radiotherapy on pancreas and adjacent tissues: a postmortem analysis[J]. Journal of Surgical Oncology, 2010, 37(2):104-108.
[29] Yamamori T, Yasui H, Yamazumi M, et al. Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint[J]. Free Radic Biol Med, 2012, 53(2): 260-270.
[30] Kam W W,Banati R B. Effects of ionizing radiation on mitochondria[J]. Free Radic Biol Med, 2013, 65: 607-619.
[31] Leach J K, Tuyle G V, Lin P S, et al. Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen[J]. Cancer Research, 2001, 61(10):3 894-3 901.
[32] Yu-Jue W, Wen L, Chi C, et al. Irradiation induced injury reduces energy metabolism in small intestine of tibet minipigs[J]. PLoS ONE, 2013, 8(3):e58970.
[33] Lall R, Ganapathy S, Yang M, et al. Low-dose radiation exposure induces a HIF-1-mediated adaptive and protective metabolic response[J]. Cell Death and Differentiation, 2014, 21(5):836-844.
[34] Kozyreva E V, Eliseenko N N. Ability of energy metabolism of cells to activate repair of its genetic material after radiation damage[J]. Radiats Biol Radioecol, 2002, 42(6): 632-635.
[35] Hashimoto T, Hussien R, Oommen S, et al. Lactate sensitive transcription factor network in L6 cells: activation of MCT1 and mitochondrial biogenesis.[J]. Faseb Journal, 2007, 21(10):2 602-2 612.
[36] Priyanka A, Anusree S S, Nisha V M, et al. Curcumin improves hypoxia induced dysfunctions in 3T3-L1 adipocytes by protecting mitochondria and down regulating inflammation[J]. BioFactors, 2014, 40(5):513-523.
[37] Kaelin W G. ROS: really involved in oxygen sensing[J]. Cell Metabolism, 2005, 1(6):357-358.
[38] Klimova T, Chandel N S. Mitochondrial complex III regulates hypoxic activation of HIF[J]. Cell Death Differ, 2008, 15(4): 660-666.
[39] Meiser J, Krämer L, Sapcariu S C, et al. Pro-inflammatory macrophages sustain pyruvate oxidation through pyruvate dehydrogenase for the synthesis of itaconate and to enable cytokine expression[J]. J Biol Chem, 2016, 291(8): 3 932-3 946.
[40] Requejo-Aguilar R, Lopez-Fabuel I, Fernandez E, et al. PINK1 deficiency sustains cell proliferation by reprogramming glucose metabolism through HIF1[J]. Nature Communications, 2014, 5 (1):1-9.
[41] Eigner D, Scholz D. Ferula asa-foetida and curcuma longa in traditional medical treatment and diet in Nepal[J]. J Ethnopharmacol, 1999, 67(1): 1-6.
[42] Basnet P, Skalko-Basnet N. Curcumin: an anti-inflammatory molecule from a curry spice on the path to cancer treatment[J]. Molecules, 2011, 16(6): 4 567-4 598.
[43] Sahu R P, Batra S, Srivastava S K. Activation of ATM/Chk1 by curcumin causes cell cycle arrest and apoptosis in human pancreatic cancer cells[J]. Br J Cancer, 2009, 100(9): 1 425-1 433.
[44] Cheng A L, Hsu C H, Lin J K, et al. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions[J]. Anticancer research, 2000, 21(4B):2 895-2 900.
[45] Bisht S, Feldmann G, Soni S, et al. Polymeric nanoparticle-encapsulated curcumin (“nanocurcumin”): a novel strategy for human cancer therapy[J]. Journal of Nanobiotechnology, 2007, 5(1):3-3.
[46] Ranjan A P, Mukerjee A, Helson L, et al. Efficacy of liposomal curcumin in a human pancreatic tumor xenograft model: Inhibition of tumor growth and angiogenesis[J]. Anticancer Res, 2013, 33(9):3 603-3 609.
[47] Hegge A B, Vukicevic M, Bruzell E, et al. Solid dispersions for preparation of phototoxic supersaturated solutions for antimicrobial photodynamic therapy (aPDT): Studies on curcumin and curcuminoides L[J]. Eur J Pharm Biopharm, 2013, 83(1): 95-105.