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Journal of ZheJiang University(Medical Science)  2016, Vol. 45 Issue (5): 493-500    DOI: 10.3785/j.issn.1008-9292.2016.09.07
    
Tripotolide ameliorates inflammation and apoptosis induced by focal cerebral ischemia/reperfusion in rats
BAI Shi1,2, SUN Yayi2, WU Lijuan1, WU Zhongmin1, FANG Marong2
1. School of Medicine, Taizhou University, Taizhou 318000, China;
2. Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou 310058, China
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Abstract  

Objective: To investigate the effects of triptolide on inflammation and apoptosis induced by focal cerebral ischemia/reperfusion in rats. Methods: The rat model of focal cerebral ischemia/reperfusion injury was established according to Longa's method. A total of 80 SD rats were randomly divided into 5 groups:normal control, sham group, DMSO group, middle cerebral artery occlusion (MCAO) group, and MCAO with tripolide treatment group. TTC staining was used to examine the site and volume of cerebral infarction, and Longa score was employed for neurological disorders measurement. Number of astrocytes was measured by fluorescence staining, and neuronal apoptosis was determined by TUNEL staining. The expressions of inducible nitric oxide synthase(iNOS), cyclooxygenase 2(COX-2) and NF-κB proteins were detected by immunohistochemistry, and the expression of iNOS, COX-2 mRNA was detected by real-time PCR. Results: Compared with DMSO group and MCAO group, brain edema was improved (80.03±0.46)% (P<0.05), infarct volume was reduced (8.3±1.4)% (P<0.01), Longa score was decreased (1.38±0.20, P<0.05) in triptolide treatment group. Meanwhile triptolide also dramatically reduced the number of GFAP-positive astrocytes (P<0.05), alleviated protein expression of COX-2 (91.67±1.31), iNOS (95.24±5.07) and NF-κB (75.03±2.06) triggered by MCAO (all P<0.05), and induced a down-regulation of cell apoptosis as showed by TUNEL assay (64.15±3.52, P<0.05). Conclusion: Triptolide can reduce the cerebral infarction volume, attenuate brain edema and ameliorate the neurological deficits induced by cerebral ischemia-reperfusion injury rats, indicating that it might be used as a potential anti-inflammatory agent.



Key wordsTriptolide/therapeutic use      Brain ischemia/drug therapy      Reperfusion injury/drug therapy      Neuritis/drug therapy      NF-kappa B/metabolism      Cyclooxygenase 2/metabolism      Nitric oxide synthase/metabolism      Disease models, animal     
Received: 07 April 2016      Published: 25 September 2016
CLC:  R743  
  R96  
Cite this article:

BAI Shi, SUN Yayi, WU Lijuan, WU Zhongmin, FANG Marong. Tripotolide ameliorates inflammation and apoptosis induced by focal cerebral ischemia/reperfusion in rats. Journal of ZheJiang University(Medical Science), 2016, 45(5): 493-500.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2016.09.07     OR     http://www.zjujournals.com/med/Y2016/V45/I5/493


雷公藤甲素缓解局灶性脑组织缺血再灌注损伤诱发的炎症及其相关凋亡的研究

目的:探讨雷公藤甲素对局灶性脑组织缺血再灌注损伤大鼠脑组织的抗炎、抗凋亡等神经保护机制。方法:参照Longa的方法建立大鼠局灶性脑组织缺血再灌注损伤模型,80只SD大鼠随机分为健康对照组、手术对照组、溶剂对照组、模型对照组、雷公藤甲素组。TTC染色确定脑梗死的部位和体积,Longa法对神经功能障碍程度进行评分,荧光染色观察星形胶质细胞数量,TUNEL染色检测神经元凋亡情况,免疫组织化学检测诱导型一氧化氮合酶(iNOS)、环氧化酶-2(COX-2)、NF-κB的蛋白表达情况,实时定量PCR测定各组大鼠iNOS和COX-2 mRNA表达量的变化。结果:相较于溶剂对照组和模型对照组,雷公藤甲素组大鼠大脑中动脉分布区域脑组织水肿改善明显(80.03±0.46)%(P<0.05),诱发梗死体积缩小(8.3±1.4)%(P<0.01),神经功能障碍程度较轻,Longa评分为1.38±0.20(P<0.05);炎症相关因素如GFAP阳性星形胶质细胞的数量减少(P<0.05),COX-2、iNOS和NF-κB蛋白表达下降,分别为91.67±1.31、95.24±5.07和75.03±12.06(均P<0.05),且损伤区凋亡作用减轻,TUNEL阳性细胞数为64.15±3.52(P<0.05)。结论:在大鼠局灶性脑组织缺血再灌注模型中,雷公藤甲素能够对抗炎症及其相关凋亡,起到减轻脑水肿及梗死范围,缓解神经功能性障碍的保护作用。


关键词: 雷公藤内酯/治疗应用,  脑组织缺血/药物疗法,  再灌注损伤/药物疗法,  神经炎/药物疗法,  NF-&kappa,  B/代谢,  环氧化酶2/代谢,  一氧化氮合酶/代谢,  疾病模型,  动物 
[[1]]   BAI S, HU Z, YANG Y, et al.Anti-inflammatory and neuroprotective effects of triptolide via the NF-κB signaling pathway in a rat MCAO model[J].Anat Rec (Hoboken), 2016, 299(2):256-266.
[[2]]   XU Z, XU R X, LIU B S, et al.Time window characteristics of cultured rat hippocampal rat hippocampal neurons subjected to ischemia and reperfusion[J].Chin J Traumatol, 2005, 8(3):179-182.
[[3]]   CHAN P H.Reactive oxygen radicals in signaling and damage in the ischemie brain[J].J Cereb Blood Flow Metab, 2001, 21(1):2-14.
[[4]]   ZHAO G, VASZAR L T, QIU D, et al.Anti-inflammatory effects of triptolide in human bronchial epithelial cells[J].Am J Physiol Lung Cell Mol Physiol, 2000, 279(5):L958-L966.
[[5]]   WANG X, LIANG X B, Li F Q, et al.Therapeutic strategies for Parkinson's disease:the ancient meets the future-traditional Chinese herbal medicine, electroacupuncture, gene therapy and stem cells[J].Neurochem Res, 2008, 33(10):1956-1963.
[[6]]   GENG Y, FANG M, WANG J, et al.Triptolide down-regulates COX-2 expression and PGE2 release by suppressing the activity of NF-κB and MAP kinases in lipopolysaccharide-treated PC12 cells[J].Phytother Res, 2012, 26(3):337-343.
[[7]]   XUE B, JIAO J, ZHANG L, et al.Triptolide upregulates NGF synthesis in rat astrocyte cultures[J].Neurochem Res, 2007, 32(7):1113-1119.
[[8]]   SU Z, YUAN Y, CAO L, et al.Triptolide promotes spinal cord repair by inhibiting astrogliosis and inflammation[J].Glia, 2010, 58(8):901-915.
[[9]]   LIU Y, CHEN H L, YANG G.Extract of Tripterygium wilfordii Hook F protect dopaminergic neurons against lipopolysaccharide-induced inflammatory damage[J].Am J Chin Med, 2010, 38(4):801-814.
[[10]]   GUAN T, LIU Q, QIAN Y, et al.Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke[J].Eur J Pharmacol, 2013, 714(1-3):303-311.
[[11]]   陈卫伟, 杨留才, 潘施文, 等.线栓法制备SD大鼠局灶性脑缺血再灌注损伤模型[J].中国组织工程研究与临床康复, 2011, 15(50):9377-9380.CHEN Weiwei, YANG Liucai, PAN Shiwen, et al.Preparation of Sprague-Dawley rat models of focal cerebral ischemia-reperfusion injury by suture method[J].Chinese Journal of Tissue Engineering Research, 2011, 15(50):9377-9380.(in Chinese)
[[12]]   LONGA E Z, WEINSTEIN P R, CARLSON S, et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke, 1989, 20(1):84-91.
[[13]]   MDZINARISHVILI A, KIEWERT C, KUMAR V, et al.Bilobalide prevents ischemia-induced edema formation in vitro and in vivo[J].Neuroscience, 2007, 144(1):217-222.
[[14]]   YANG F, LIU Y, TU J, et al.Activated astrocytes enhance the dopaminergic differentiation of stem cells and promote brain repair through bFGF[J/OL].Nat Commun, 2014, 5:5627.
[[15]]   李琳, 张志强.脑缺血再灌注损伤中细胞凋亡的研究进展[J].中华物理医学与康复杂志, 2005, 27(1):60-62.LI Lin, ZHANG Zhiqiang.Research progress of apoptosis in ischemia-reperfusion injury[J].Chinese Journal of Physical Medicine and Rehabiltation, 2005, 27(1):60-62.
[[16]]   BRESGEN N, KARLHUBER G, KRIZBAI I, et al.Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations, micronuclei, and apoptosis[J].J Neurosci Res, 2003, 72:327-333.
[[17]]   DOHMEN C, KUMURA E, ROSNER G, et al.Extracellular correlates of glutamate toxicity in short-term cerebral ischemia and reperfusion:a direct in vivo comparison between white and gray matter[J].Brain Res, 2005, 1037(1-2):43-51.
[[18]]   WEI J, QUAST M.Effect of nitric oxide synthesis inhibitor on a hyperglycemia rat model of reversible focal ischemia:detection of excitatory amino acids release and hydroxyl radical formation[J].Brain Res, 1998, 791(1-2):146-156.
[[19]]   CEULEMANS AO, ZGAVC T, KOOIJMAN R, et al.The dual role of the neuroinflammatory response after ischemic stroke:modulatory effects of hypothermia[J].J Neuroinflammation, 2010, 7:74.
[[20]]   SHICHITA T, SAKAGUCHI R, SUZUKI M, et al.Post-ischemic inflammation in the brain[J].Front Immunol, 2012, 3(3):132.
[[21]]   DENES A, THORNTON P, ROTHWELL N J, et al.Inflammation and brain injury:acute cerebral ischaemia, peripheral and central inflammation[J].Brain Behav Immun, 2010, 24(5):708-723.
[[22]]   YE Z, WANG N, XIA P, et al.Delayed administration of parecoxib, a specific COX-2 inhibitor, attenuated postischemic neuronal apoptosis by phosphorylation Akt and GSK-3β[J].Neurochem Res, 2012, 37(2):321-329.
[[23]]   CLEMENS J A, STEPHENSON D T, YIN T, et al.Drug-induced neuroprotection from global ischemia is associated with prevention of persistent but not transient activation of nuclear factor-kappaB in rats[J].Stroke, 1998, 29(3):677-682.
[[24]]   YASUDA Y, SHIMODA T, UNO K, et al.Temporal and sequential changes of glial cells and cytokine expression during neuronal degeneration after transient global ischemia in rats[J].J Neuroinflammation, 2011, 8:70.
[[25]]   QIU D, ZHAO G, AOKI Y, et al.Immunosuppressant PG490(Triptolide) inhibits T-cell interleukin-2 expression at the level of purine-box/nuclear factor of activated T-cells and NF-kappaB transcriptional activation[J].J Biol Chem, 1999, 274(19):13443-13450.
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