Skip to main content

Advertisement

SpringerLink
Log in

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway

  • Original Research
  • Published:
Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Abstract

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood–brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

References

  • Abbott NJ, Rönnbäck L, Hansson E (2006) Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci 7:41–53

    Article  CAS  PubMed  Google Scholar 

  • Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ (2010) Structure and function of the blood-brain barrier. Neurobiol Dis 37:13–25

    Article  CAS  PubMed  Google Scholar 

  • Abeysinghe HC, Phillips EL, Chin-Cheng H, Beart PM, Roulston CL (2016) Modulating Astrocyte Transition after Stroke to Promote Brain Rescue and Functional Recovery: emerging Targets Include Rho Kinase. Int J Mol Sci 17:288

    Article  PubMed  PubMed Central  Google Scholar 

  • Al Ahmad A, Gassmann M, Ogunshola OO (2012) Involvement of oxidative stress in hypoxia-induced blood-brain barrier breakdown. Microvasc Res 84:222–225

    Article  CAS  PubMed  Google Scholar 

  • Allen C, Srivastava K, Bayraktutan U (2010) Small GTPase RhoA and its effector rho kinase mediate oxygen glucose deprivation-evoked in vitro cerebral barrier dysfunction. Stroke 41:2056–2063

    Article  CAS  PubMed  Google Scholar 

  • Aslam M, Schluter KD, Rohrbach S, Rafiq A, Nazli S, Piper HM, Noll T, Schulz R, Gündüz D (2013) Hypoxia-reoxygenation-induced endothelial barrier failure role of RhoA, Rac1 and myosin light chain kinase. J Physiol 591:461–473

    Article  CAS  PubMed  Google Scholar 

  • Cardoso FL, Brites D, Brito MA (2010) Looking at the blood-brain barrier: molecular anatomy and possible investigation approaches. Brain Res Rev 64:328–363

    Article  CAS  PubMed  Google Scholar 

  • Chandra J, Samali A, Orrenius S (2000) Triggering and modulation of apoptosis by oxidative stress. Free Rad Biolo Med 29:323–333

    Article  CAS  Google Scholar 

  • Chang CY, Kao TK, Chen WY, Ou YC, Li JR, Liao SL, Raung SL, Chen CJ (2015) Tetramethylpyrazine inhibits neutrophil activation following permanent cerebral ischemia in rats. Biochem Biophys Res Commun 463:421–427

    Article  CAS  PubMed  Google Scholar 

  • Cohen-Kashi Malina K, Cooper I, Teichberg VI (2009) Closing the gap between the in vivo and in vitro blood-brain barrier tightness. Brain Res 1284:12–21

    Article  CAS  PubMed  Google Scholar 

  • Doyle KP, Simon RP, Stenzel-Poore MP (2008) Mechanisms of ischemic brain damage. Neuropharmacology 55:310–318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Durán WN, Breslin JW, Sánchez FA (2010) The NO cascade, eNOS location, and microvascular permeability. Cardiovasc Res 87:254–261

    Article  PubMed  PubMed Central  Google Scholar 

  • Fujii M, Duris K, Altay O, Soejima Y, Sherchan P, Zhang JH (2012) Inhibition of Rho kinase by hydroxyfasudil attenuates brain edema after subarachnoid hemorrhage in rats. Neurochem Int 60:327–333

    Article  CAS  PubMed  Google Scholar 

  • Fukata Y, Amano M, Kaubuchi K (2001) Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci 22:32–39

    Article  CAS  PubMed  Google Scholar 

  • Gao HC, Zhao H, Zhang WQ, Li YQ, Ren LQ (2013) The role of the Rho/Rock signaling pathway in the pathogenesis of acute ischemic myocardial fibrosis in rat models. Exp Ther Med 5:1123–1128

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gibson CL, Srivastava K, Sprigg N, Bath PM, Bayraktutan U (2014) Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions. J Neurochem 129:816–826

    Article  CAS  PubMed  Google Scholar 

  • Gu Y, Dee CM, Shen J (2011) Interaction of free radicals, matrix metalloproteinases and caveolin-1 impacts blood-brain barrier permeability. Front Biosci (Schol Ed) 3:1216–1231

    Article  Google Scholar 

  • Gwag BJ, Lobner D, Koh JY, Wie MB, Choi DW (1995) Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro. Neuroscience 68:615–619

    Article  CAS  PubMed  Google Scholar 

  • Han J, Shuvaev VV, Muzykantov VR (2011) Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor. J Pharmacol Exp Ther 338:82–91

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harvey KA, Welch Z, Sliva D, Siddiqui RA (2010) Role of Rho kinase in sphingosine 1-phosphate-mediated endothelial and smooth muscle cell migration and differentiation. Mol Cell Biochem 342:7–19

    Article  CAS  PubMed  Google Scholar 

  • Hawkins BT, Davis TP (2005) The blood brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173–185

    Article  CAS  PubMed  Google Scholar 

  • Hippenstiel S, Schmeck B, N’Guessan PD, Seybold J, Krüll M, Preissner K, Eichel-Streiber CV, Suttorp N (2002) Rho protein inactivation induced apoptosis of cultured human endothelial cells. Am J Physiol Lung Cell Mol Physiol 283:L830–L838

    Article  CAS  PubMed  Google Scholar 

  • Inan S, Büyükafşar K (2008) Antiepileptic effects of two Rho-kinase inhibitors, Y-27632 and fasudil, in mice. Br J Pharmacol 155:44–51

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jung HJ, Jeon YH, Bokara KK, Koo BN, Lee WT, Park KA, Lee JE (2013) Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways. Life Sci 92:42–50

    Article  CAS  PubMed  Google Scholar 

  • Kao TK, Ou YC, Kuo JS, Chen WY, Liao SL, Wu CW, Chen CJ, Ling NN, Zhang YH, Peng WH (2006) Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats. Neurochem Int 48:166–176

    Article  CAS  PubMed  Google Scholar 

  • Kao TK, Chang CY, Ou YC, Chen WY, Kuan YH, Pan HC, Liao SL, Li GZ, Chen CJ (2013) Tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats. Exp Neurol 247:188–201

    Article  CAS  PubMed  Google Scholar 

  • Kaur C, Ling EA (2008) Blood brain barrier in hypoxic-ischemic conditions. Curr Neurovasc Res 5:71–81

    Article  CAS  PubMed  Google Scholar 

  • Kim GW, Sugawara T, Chan PH (2000) Involvement of oxidative stress and caspase-3 in cortical infarction after photothrombotic ischemia in mice. J Cereb Blood Flow Metab 20:1690–1701

    Article  CAS  PubMed  Google Scholar 

  • Koumura A, Hamanaka J, Kawasaki K, Tsuruma K, Shimazawa M, Hozumi I, Inuzuka T, Hara H (2011) Fasudil and ozagrel in combination show neuroprotective effects on cerebral infarction after murine middle cerebral artery occlusion. J Pharmacol Exp Ther 338:337–344

    Article  CAS  PubMed  Google Scholar 

  • Lai JM, Hsieh CL, Chang ZF (2003) Caspase activation during phorbol ester-induced apoptosis requires ROCK-dependent myosin-mediated contraction. J Cell Sci 116:3491–3501

    Article  CAS  PubMed  Google Scholar 

  • Liu C, Wu J, Zou MH (2012) Activation of AMP-activated protein kinase alleviates high glucose-induced dysfunction of brain microvascular endothelial cell tight junction dynamics. Free Radic Biol Med 53:1213–1221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lochhead JJ, McCaffrey G, Quigley CE, Finch J, DeMarco KM, Nametz N, Davis TP (2010) Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation. J Cereb Blood Flow Metab 30:1625–1636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lu CS, Yin MH, Huang YH, Pao C, Sung CF (1978) Radix salviae miltiorrhizae and Rhizoma ligustici wallichii in coronary heart disease. Chin Med J 4:43–46

    Google Scholar 

  • Olmez I, Ozyurt H (2012) Reactive oxygen species and ischemic cerebrovascular disease. Neurochem Int 60:208–212

    Article  CAS  PubMed  Google Scholar 

  • Pun PB, Lu J, Moochhala S (2009) Involvement of ROS in BBB dysfunction. Free Radic Res 43:348–364

    Article  CAS  PubMed  Google Scholar 

  • Raad M, El Tal T, Gul R, Mondello S, Zhang Z, Boustany RM, Guingab J, Wang KK, Kobeissy F (2012) Neuroproteomics approach and neurosystems biology analysis: ROCK inhibitors as promising therapeutic targets in neurodegeneration and neurotrauma. Electrophoresis 33:3659–3668

    Article  CAS  PubMed  Google Scholar 

  • Raju J, Patlolla JM, Swamy MV, Rao CV (2004) Diosgenin, a steroid saponin of Trigonella foenum graecum (Fenugreek), inhibits azoxymethane-induced aberrant crypt foci formation in F344 rats and induces apoptosis in HT-29 human colon cancer cells. Cancer Epidemiol Biomarkers Prev 13:1392–1398

    CAS  PubMed  Google Scholar 

  • Raz L, Zhang QG, Zhou CF, Han D, Gulati P, Yang LC, Yang F, Wang RM, Brann DW (2010) Role of Rac1 GTPase in NADPH oxidase activation and cognitive impairment following cerebral ischemia in the rat. PLoS One 5:e12606

    Article  PubMed  PubMed Central  Google Scholar 

  • Rikitake Y, Kim HH, Huang Z, Seto M, Yano K, Asano T, Moskowitz MA, Liao JK (2005) Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke 36:2251–2257

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rizzo MT, Leaver HA (2010) Brain endothelial cell death: modes, signaling pathways, and relevance to neural development, homeostasis, and disease. Mol Neurobiol 42:52–63

    Article  CAS  PubMed  Google Scholar 

  • Royall JA, Ischiropoulos H (1993) Evaluation of 2′,7′-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302:348–355

    Article  CAS  PubMed  Google Scholar 

  • Sawada N, Liao JK (2009) Targeting eNOS and beyond: emerging heterogeneity of the role of endothelial Rho proteins in stroke protection. Expert Rev Neurother 9:1171–1186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sawada N, Kim HH, Moskowitz MA, Liao JK (2009) Rac1 is a critical mediator of endothelium-derived neurotrophic activity. Sci Signal 2:ra10

    Article  PubMed  PubMed Central  Google Scholar 

  • Shuvaev VV, Muzykantov VR (2011) Targeted modulation of reactive oxygen species in the vascular endothelium. J Control Release 153:56–63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song J, Kang SM, Lee WT, Park KA, Lee KM, Lee JE (2014) The Beneficial Effect of Melatonin in Brain Endothelial Cells against Oxygen-Glucose Deprivation Followed by Reperfusion-Induced Injury. Oxid Med Cell Longev 2014:639531

    PubMed  PubMed Central  Google Scholar 

  • Takemoto M, Sun J, Hiroki J, Shimokawa H, Liao JK (2002) Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation 106:57–62

    Article  CAS  PubMed  Google Scholar 

  • Tan F, Fu W, Cheng N, Meng DI, Gu Y (2015) Ligustrazine reduces blood-brain barrier permeability in a rat model of focal cerebral ischemia and reperfusion. Exp Ther Med 9:1757–1762

    PubMed  PubMed Central  Google Scholar 

  • Tcherkezian J, Lamarche-vane N (2007) Current knowledge of the large RhoGAP family of proteins. Biol Cell 99:67–86

    Article  CAS  PubMed  Google Scholar 

  • Tsai TH, Liang C (2001) Pharmacokinetics of tetramethylpyrazine in rat blood and brain using microdialysis. Int J Pharm 216:61–66

    Article  CAS  PubMed  Google Scholar 

  • van Nieuw Amerongen GP, Beckers CM, Achekar ID, Zeeman S, Musters RJ, van Hinsbergh VW (2007) Involvement of Rho Kinase in Endothelial Barrier Maintenance. Arterioscler Thromb Vasc Biol 27:2332–2339

    Article  PubMed  Google Scholar 

  • Villar-Cheda B, Dominguez-Meijide A, Joglar B, Rodriguez-Perez AI, Guerra MJ, Labandeira-Garcia JL (2012) Involvement of microglial RhoA/Rho-kinase pathway activation in the dopaminergic neuron death. Role of angiotensin via angiotensin type 1 receptors. Neurobiol Dis 47:268–279

    Article  CAS  PubMed  Google Scholar 

  • Wang N, Liu QY, Peng DY, Dai M, Tang JY, Li JL (2003) Experimental studies on protective effects and mechanisms of Tongqiao Huoxue Decoction in mice undergone repetitious cerebral ischemia reperfusion. Chin J Exp Tradit Med Form 10:22–24

    CAS  Google Scholar 

  • Wang N, Liu QY, Peng DY, Dai M, Zhang XB, Hu HB (2004) Studies on protective effects and mechanisms of Tongqiaohuoxue Decoction in rats of cerebral ischemia. Chin J Inform Tradit Chin Med 11:407–409

    Google Scholar 

  • Wang N, Liu QY, Peng DY, Dai M, Zhu Q (2005) Effects of Tongqiaohuoxue decoction on contents of CGRP, ET, IL-1β, TNF-α in the brains of rats of cerebral ischemia. Chin Tradit Pat Med 27:1295–1297

    Google Scholar 

  • Wang J, Hou J, Zhang P, Li D, Zhang C, Liu J (2012) Geniposide reduces inflammatory responses of oxygen-glucose deprived rat microglial cells via inhibition of the TLR4 signaling pathway. Neurochem Res 37:2235–2248

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Wang N, Cai B, Wang GY, Li J, Piao XX (2015) In vitro model of the blood-brain barrier established by co-culture of primary cerebral microvascular endothelial and astrocyte cells. Neural Regen Res 10:2011–2017

    Article  PubMed  PubMed Central  Google Scholar 

  • Xiao X, Liu Y, Qi C, Qiu F, Chen X, Zhang J, Yang P (2010) Neuroprotection and enhanced neurogenesis by tetramethylpyrazine in adult rat brain after focal ischemia. Neurol Res 32:547–555

    Article  CAS  PubMed  Google Scholar 

  • Yagita Y, Kitagawa K, Sasaki T, Terasaki Y, Todo K, Omura-Matsuoka E, Kaibuchi K, Hori M (2007) Rho-kinase activation in endothelial cells contributes to expansion of infarction after focal cerebral ischemia. J Neurosci Res 85:2460–2469

    Article  CAS  PubMed  Google Scholar 

  • Yan YY, Wang XM, Jiang Y, Chen H, He JT, Mang J, Shao YK, Xu ZX (2015) The role of Rho/Rho-kinase pathway and the neuroprotective effects of fasudil in chronic cerebral ischemia. Neural Regen Res 10:1441–1449

    Article  PubMed  PubMed Central  Google Scholar 

  • Yeh LH, Park YJ, Hansalia RJ, Ahmed IS, Deshpande SS, Goldschmidt-Clermont PJ, Irani K, Alevriadou BR (1999) Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS. Am J Physiol 276:C838–C847

    CAS  PubMed  Google Scholar 

  • Zhang C, Teng F, Tu J, Zhang D (2014a) Ultrasound-Enhanced Protective Effect of Tetramethylpyrazine against Cerebral Ischemia Reperfusion Injury. PLoS One 9:e113673

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang M, Gao F, Teng F, Zhang C (2014b) Tetramethylpyrazine promotes the proliferation and migration of brain endothelial cells. Mol Med Rep 10:29–32

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou C, Zhao XM, Li XF, Wang C, Zhang XT, Liu XZ, Ding XF, Xiang SL, Zhang J (2013) Curcumin inhibits AP-2γ-induced apoptosis in the human malignant testicular germ cells in vitro. Acta Pharmacol Sin 34:1192–1200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The study was supported by the National Natural Science Foundation of China (Nos. 30973979, 81374005) and “Twelfth Five Year” National Science and Technology Support Program of China (No. 2012BAI26B03).

Author information

Author notes

    Authors and Affiliations

    1. Key Laboratory of Xin’an Medicine, Ministry of Education, Hefei, 230012, China

      Guang Yang, Chen Qian, Ning Wang, Chenyu Lin, Yan Wang, Guangyun Wang & Xinxin Piao

    2. Anhui University of Chinese Medicine, Qian Jiang Road 1, Hefei, 230012, China

      Guang Yang, Chen Qian, Ning Wang, Chenyu Lin, Yan Wang, Guangyun Wang & Xinxin Piao

    3. Institute for Pharmacodynamics and Safety Evaluation of Chinese Medicine, Anhui Academy of Chinese Medicine, Hefei, 230012, China

      Guang Yang, Chen Qian, Ning Wang, Chenyu Lin, Yan Wang, Guangyun Wang & Xinxin Piao

    Authors
    1. Guang Yang
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Chen Qian
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Ning Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Chenyu Lin
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Yan Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Guangyun Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Xinxin Piao
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Ning Wang.

    Ethics declarations

    Conflict of Interest

    None.

    Ethical statement

    On behalf of, and having obtained permission from all the authors, I declare that: (a) the material has not been published in whole or in part elsewhere; (b) the paper is not currently being considered for publication elsewhere; (c) all authors have been personally and actively involved in substantive work leading to the report, and will hold themselves jointly and individually responsible for its content; (d) whole research process was approved by the institutional Animal Care and Use Committee of the Anhui University of Chinese medicine and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. I testify to the accuracy of the above on behalf of all the authors.

    Additional information

    Guang Yang and Chen Qian have contributed equally to this work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    Supplementary material 1 (DOCX 15 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Yang, G., Qian, C., Wang, N. et al. Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway. Cell Mol Neurobiol 37, 619–633 (2017). https://doi.org/10.1007/s10571-016-0398-4

    Download citation

    • Received:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10571-016-0398-4

    Keywords

    Search

    Navigation