Ligustrazine suppresses renal NMDAR1 and caspase-3 expressions in a mouse model of sepsis-associated acute kidney injury

  • Jing Ying
  • Jin WuEmail author
  • Yiwei Zhang
  • Yangyang Han
  • Xinger Qian
  • Qiuhong Yang
  • Yongjie Chen
  • Yijun Chen
  • Hao Zhu


Sepsis-associated acute kidney injury (AKI) is a life threatening condition with high morbidity and mortality. The pathogenesis of AKI is associated with apoptosis. In this study, we investigated the effects of ligustrazine (LGZ) on experimental sepsis-associated AKI in mice. Sepsis-associated AKI was induced in a mice model using cecal ligation and puncture (CLP) method. Mice were administered LGZ (10, 30, and 60 mg/kg) via tail vein injection 0.5 h before CLP surgery. Mice survival was evaluated. Renal water content was detected. Urine samples were collected for ELISA of Kim1. Kidneys were collected for nucleic acid analysis and histological examination. Pathological assessment was used to determine the effect of LGZ on sepsis-associated AKI. Caspase-3 expression in kidney was assessed by immunohistochemistry. Renal NMDAR1 level was also determined. Treatment of LGZ improved mice survival rate; the effect was significant when administered at a high LGZ dose (60 mg/kg). Renal water content of mice undergoing CLP was significantly reduced by LGZ treatment. Both middle-dose and high-dose LGZ treatments reduced urine Kim1 level in sepsis-associated AKI mice. The severity of AKI in septic mice was reduced by middle-dose and high-dose LGZ administration. Immunohistochemical analysis revealed decreased caspase-3 and NMDAR1 levels in the kidney following middle-dose and high-dose LGZ treatments. RT-PCR assay showed a significant reduction in NMDAR1 mRNA expression in the kidney of middle-dose and high-dose LGZ-treated mice. LGZ exhibited protective effects against sepsis-associated AKI in mice, possibly via downregulation of renal NMDAR1 expression and its anti-apoptotic action by inhibiting caspase-3.


Ligustrazine Acute kidney injury Sepsis Apoptosis Caspase-3 NMDAR1 



This study was funded by Ningbo Natural Science Foundation (Grant Number 2017A610192).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethic approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. (Institutional Animal Care and Use Committee of Hangzhou Hibio Technology Co. Ltd., IACUC Protocol Number: HBFM3.68-2015).

Informed consent

Not applicable.


  1. 1.
    Barbar SD, Clere-Jehl R, Bourredjem A, Hernu R, Montini F, Bruyere R, Lebert C, Bohe J, Badie J, Eraldi JP, Rigaud JP, Levy B, Siami S, Louis G, Bouadma L, Constantin JM, Mercier E, Klouche K, du Cheyron D, Piton G, Annane D, Jaber S, van der Linden T, Blasco G, Mira JP, Schwebel C, Chimot L, Guiot P, Nay MA, Meziani F, Helms J, Roger C, Louart B, Trusson R, Dargent A, Binquet C, Quenot JP (2018) Timing of renal-replacement therapy in patients with acute kidney injury and sepsis. N Engl J Med 379(15):1431–1442. CrossRefPubMedGoogle Scholar
  2. 2.
    Bagshaw SM, Lapinsky S, Dial S, Arabi Y, Dodek P, Wood G, Ellis P, Guzman J, Marshall J, Parrillo JE, Skrobik Y, Kumar A (2009) Acute kidney injury in septic shock: clinical outcomes and impact of duration of hypotension prior to initiation of antimicrobial therapy. Intensive Care Med 35(5):871–881. CrossRefPubMedGoogle Scholar
  3. 3.
    Bates DW, Su L, Yu DT, Chertow GM, Seger DL, Gomes DR, Dasbach EJ, Platt R (2001) Mortality and costs of acute renal failure associated with amphotericin B therapy. Clin Infect Dis 32(5):686–693. CrossRefPubMedGoogle Scholar
  4. 4.
    Levy EM, Viscoli CM, Horwitz RI (1996) The effect of acute renal failure on mortality. A cohort analysis. JAMA 275(19):1489–1494CrossRefGoogle Scholar
  5. 5.
    Poston JT, Koyner JL (2019) Sepsis associated acute kidney injury. BMJ 364:k4891. CrossRefPubMedGoogle Scholar
  6. 6.
    Havasi A, Borkan SC (2011) Apoptosis and acute kidney injury. Kidney Int 80(1):29–40. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kockara A, Kayatas M (2013) Renal cell apoptosis and new treatment options in sepsis-induced acute kidney injury. Ren Fail 35(2):291–294. CrossRefPubMedGoogle Scholar
  8. 8.
    Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thornberry NA, Wong WW, Yuan J (1996) Human ICE/CED-3 protease nomenclature. Cell 87(2):171CrossRefGoogle Scholar
  9. 9.
    Earnshaw WC, Martins LM, Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68:383–424. CrossRefPubMedGoogle Scholar
  10. 10.
    Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281(5381):1312–1316CrossRefGoogle Scholar
  11. 11.
    Kunduzova OR, Escourrou G, Seguelas MH, Delagrange P, De La Farge F, Cambon C, Parini A (2003) Prevention of apoptotic and necrotic cell death, caspase-3 activation, and renal dysfunction by melatonin after ischemia/reperfusion. FASEB J 17(8):872–874. CrossRefPubMedGoogle Scholar
  12. 12.
    Yang B, Lan S, Dieude M, Sabo-Vatasescu JP, Karakeussian-Rimbaud A, Turgeon J, Qi S, Gunaratnam L, Patey N, Hebert MJ (2018) Caspase-3 is a pivotal regulator of microvascular rarefaction and renal fibrosis after ischemia–reperfusion injury. J Am Soc Nephrol 29(7):1900–1916. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Lee SY, Lee YS, Choi HM, Ko YS, Lee HY, Jo SK, Cho WY, Kim HK (2012) Distinct pathophysiologic mechanisms of septic acute kidney injury: role of immune suppression and renal tubular cell apoptosis in murine model of septic acute kidney injury. Crit Care Med 40(11):2997–3006. CrossRefPubMedGoogle Scholar
  14. 14.
    Arora S, Kaur T, Kaur A, Singh AP (2014) Glycine aggravates ischemia reperfusion-induced acute kidney injury through N-methyl-d-aspartate receptor activation in rats. Mol Cell Biochem 393(1–2):123–131. CrossRefPubMedGoogle Scholar
  15. 15.
    Kaur A, Kaur T, Singh B, Pathak D, Singh Buttar H, Pal Singh A (2016) Curcumin alleviates ischemia reperfusion-induced acute kidney injury through NMDA receptor antagonism in rats. Ren Fail 38(9):1462–1467. CrossRefPubMedGoogle Scholar
  16. 16.
    Pundir M, Arora S, Kaur T, Singh R, Singh AP (2013) Effect of modulating the allosteric sites of N-methyl-d-aspartate receptors in ischemia-reperfusion induced acute kidney injury. J Surg Res 183(2):668–677. CrossRefPubMedGoogle Scholar
  17. 17.
    Simon RP, Swan JH, Griffiths T, Meldrum BS (1984) Blockade of N-methyl-d-aspartate receptors may protect against ischemic damage in the brain. Science 226(4676):850–852CrossRefGoogle Scholar
  18. 18.
    Tu W, Xu X, Peng L, Zhong X, Zhang W, Soundarapandian MM, Balel C, Wang M, Jia N, Zhang W, Lew F, Chan SL, Chen Y, Lu Y (2010) DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke. Cell 140(2):222–234. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y, Shibasaki F, McKeon F, Bobo T, Franke TF, Reed JC (1999) Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284(5412):339–343CrossRefGoogle Scholar
  20. 20.
    Husi H, Sanchez-Nino MD, Delles C, Mullen W, Vlahou A, Ortiz A, Mischak H (2013) A combinatorial approach of proteomics and systems biology in unravelling the mechanisms of acute kidney injury (AKI): involvement of NMDA receptor GRIN1 in murine AKI. BMC Syst Biol 7:110. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wang B, Ni Q, Wang X, Lin L (2012) Meta-analysis of the clinical effect of ligustrazine on diabetic nephropathy. Am J Chin Med 40(1):25–37. CrossRefPubMedGoogle Scholar
  22. 22.
    Gao BT (1989) The effects of ligustrazine, aspirin and beta-histine on platelet aggregation in patients with acute ischemic stroke. Zhonghua shen jing jing shen ke za zhi = Chin J Neurol Psychiatry 22(3):148–151Google Scholar
  23. 23.
    Yang XG, Jiang C (2010) Ligustrazine as a salvage agent for patients with relapsed or refractory non-Hodgkin’s lymphoma. Chin Med J 123(22):3206–3211PubMedGoogle Scholar
  24. 24.
    Feng L, Xiong Y, Cheng F, Zhang L, Li S, Li Y (2004) Effect of ligustrazine on ischemia-reperfusion injury in murine kidney. Transpl Proc 36(7):1949–1951. CrossRefGoogle Scholar
  25. 25.
    Shao Z, Wang L, Liu S, Wang X (2017) Tetramethylpyrazine protects neurons from oxygen-glucose deprivation-induced death. Med Sci Monit 23:5277–5282. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Yu B, Ruan M, Liang T, Huang SW, Liu SJ, Cheng HB, Shen XC (2017) Tetramethylpyrazine phosphate and borneol combination therapy synergistically attenuated ischemia–reperfusion injury of the hypothalamus and striatum via regulation of apoptosis and autophagy in a rat model. Am J Transl Res 9(11):4807–4820PubMedPubMedCentralGoogle Scholar
  27. 27.
    Chen JL, Zhou T, Chen WX, Zhu JS, Chen NW, Zhang MJ, Wu YL (2003) Effect of tetramethylpyrazine on P-selectin and hepatic/renal ischemia and reperfusion injury in rats. World J Gastroenterol 9(7):1563–1566. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Feng L, Ke N, Cheng F, Guo Y, Li S, Li Q, Li Y (2011) The protective mechanism of ligustrazine against renal ischemia/reperfusion injury. J Surg Res 166(2):298–305. CrossRefPubMedGoogle Scholar
  29. 29.
    Sun L, Li Y, Shi J, Wang X, Wang X (2002) Protective effects of ligustrazine on ischemia–reperfusion injury in rat kidneys. Microsurgery 22(8):343–346. CrossRefPubMedGoogle Scholar
  30. 30.
    Liu XH, Li J, Li QX, Ai YX, Zhang L (2008) Protective effects of ligustrazine on cisplatin-induced oxidative stress, apoptosis and nephrotoxicity in rats. Environ Toxicol Pharmacol 26(1):49–55. CrossRefPubMedGoogle Scholar
  31. 31.
    Cheng CY, Sue YM, Chen CH, Hou CC, Chan P, Chu YL, Chen TH, Hsu YH (2006) Tetramethylpyrazine attenuates adriamycin-induced apoptotic injury in rat renal tubular cells NRK-52E. Planta Med 72(10):888–893. CrossRefPubMedGoogle Scholar
  32. 32.
    Juan SH, Chen CH, Hsu YH, Hou CC, Chen TH, Lin H, Chu YL, Sue YM (2007) Tetramethylpyrazine protects rat renal tubular cell apoptosis induced by gentamicin. Nephrol Dial Transpl 22(3):732–739. CrossRefGoogle Scholar
  33. 33.
    Sue YM, Cheng CF, Chang CC, Chou Y, Chen CH, Juan SH (2009) Antioxidation and anti-inflammation by haem oxygenase-1 contribute to protection by tetramethylpyrazine against gentamicin-induced apoptosis in murine renal tubular cells. Nephrol Dial Transpl 24(3):769–777. CrossRefGoogle Scholar
  34. 34.
    Guo LH, Yang C, Wang L, Chen QF, Hu YN, Zhang MZ (2012) Effects of tetramethylpyrazine on cardiac function and mortality rate in septic rats. Chin J Integr Med 18(8):610–615. CrossRefPubMedGoogle Scholar
  35. 35.
    Wang JQ, Zhang L, Tao XG, Wei L, Liu B, Huang LL, Chen YG (2013) Tetramethylpyrazine upregulates the aquaporin 8 expression of hepatocellular mitochondria in septic rats. J Surg Res 185(1):286–293. CrossRefPubMedGoogle Scholar
  36. 36.
    Tao W, Shu YS, Miao QB, Zhu YB (2012) Attenuation of hyperoxia-induced lung injury in rats by adrenomedullin. Inflammation 35(1):150–157. CrossRefPubMedGoogle Scholar
  37. 37.
    Wang F, Yu WL, Jia LL et al (2016) Propofol attenuates kidney injury after liver cold ischemia/reperfusion in rats via inhibiting JAK/STAT signaling activation. Chin J Pathophysiol 32:2026–2030Google Scholar
  38. 38.
    Gao C, Peng H, Wang S, Zhang X (2012) Effects of Ligustrazine on pancreatic and renal damage after scald injury. Burns 38(1):102–107. CrossRefPubMedGoogle Scholar
  39. 39.
    Wang Y, Tong J, Tang R, Dong H, Xu J (2004) Inhibitory effects of ligustrazine, a modulator of thromboxane-prostacycline-nitric oxide balance, on renal injury in rats with passive Heyman nephritis. Nephron Physiol 98(3):p80–p88. CrossRefPubMedGoogle Scholar
  40. 40.
    Doi K, Hu X, Yuen PS, Leelahavanichkul A, Yasuda H, Kim SM, Schnermann J, Jonassen TE, Frokiaer J, Nielsen S, Star RA (2008) AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality. Kidney Int 73(11):1266–1274. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    van Timmeren MM, van den Heuvel MC, Bailly V, Bakker SJ, van Goor H, Stegeman CA (2007) Tubular kidney injury molecule-1 (KIM-1) in human renal disease. J Pathol 212(2):209–217. CrossRefPubMedGoogle Scholar
  42. 42.
    Pennemans V, De Winter LM, Munters E, Nawrot TS, Van Kerkhove E, Rigo JM, Reynders C, Dewitte H, Carleer R, Penders J, Swennen Q (2011) The association between urinary kidney injury molecule 1 and urinary cadmium in elderly during long-term, low-dose cadmium exposure: a pilot study. Environ Health 10:77. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Prozialeck WC, Vaidya VS, Liu J, Waalkes MP, Edwards JR, Lamar PC, Bernard AM, Dumont X, Bonventre JV (2007) Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity. Kidney Int 72(8):985–993. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Lange H (2002) Multiorgan dysfunction syndrome: how water might contribute to its progression. J Cell Mol Med 6(4):653–660. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Wang H, Zhang W, Cheng Y, Zhang X, Xue N, Wu G, Chen M, Fang K, Guo W, Zhou F, Cui H, Ma T, Wang P, Lei H (2018) Design, synthesis and biological evaluation of ligustrazine-flavonoid derivatives as potential anti-tumor agents. Molecules. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Group MR (2016) Global and Chinese ligustrazine phosphate market 2016: industry trends, production, sales, demand, supply, analysis & forecast to 2021Google Scholar
  47. 47.
    Tao Z (2010) Effects of ligustrazine on inflammatory lung injury in septic mice. J Jingchu Univ Technol 2:2010Google Scholar
  48. 48.
    Zhe D, Ying-ju L, Li W, Lu-lu C, Guan-li X (2012) Protective effect of Shenmai combined with ligustrazine injection on liver iniury in sepsis mice. Lishizhen Med Mater Med Res 5Google Scholar
  49. 49.
    Dieterle F, Sistare F, Goodsaid F, Papaluca M, Ozer JS, Webb CP, Baer W, Senagore A, Schipper MJ, Vonderscher J, Sultana S, Gerhold DL, Phillips JA, Maurer G, Carl K, Laurie D, Harpur E, Sonee M, Ennulat D, Holder D, Andrews-Cleavenger D, Gu YZ, Thompson KL, Goering PL, Vidal JM, Abadie E, Maciulaitis R, Jacobson-Kram D, Defelice AF, Hausner EA, Blank M, Thompson A, Harlow P, Throckmorton D, Xiao S, Xu N, Taylor W, Vamvakas S, Flamion B, Lima BS, Kasper P, Pasanen M, Prasad K, Troth S, Bounous D, Robinson-Gravatt D, Betton G, Davis MA, Akunda J, McDuffie JE, Suter L, Obert L, Guffroy M, Pinches M, Jayadev S, Blomme EA, Beushausen SA, Barlow VG, Collins N, Waring J, Honor D, Snook S, Lee J, Rossi P, Walker E, Mattes W (2010) Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium. Nat Biotechnol 28(5):455–462. CrossRefPubMedGoogle Scholar
  50. 50.
    Lan Z, Bi KS, Chen XH (2014) Ligustrazine attenuates elevated levels of indoxyl sulfate, kidney injury molecule-1 and clusterin in rats exposed to cadmium. Food Chem Toxicol 63:62–68. CrossRefPubMedGoogle Scholar
  51. 51.
    Porter AG, Janicke RU (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6(2):99–104. CrossRefPubMedGoogle Scholar
  52. 52.
    Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA (1995) Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-d-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 92(16):7162–7166. CrossRefPubMedGoogle Scholar
  53. 53.
    Kravchick DO, Karpova A, Hrdinka M, Lopez-Rojas J, Iacobas S, Carbonell AU, Iacobas DA, Kreutz MR, Jordan BA (2016) Synaptonuclear messenger PRR7 inhibits c-Jun ubiquitination and regulates NMDA-mediated excitotoxicity. EMBO J 35(17):1923–1934. CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Lotfullina N, Khazipov R (2018) Ethanol and the developing brain: inhibition of neuronal activity and neuroapoptosis. Neuroscientist 24(2):130–141. CrossRefPubMedGoogle Scholar
  55. 55.
    Ding J, Zhou HH, Ma QR, He ZY, Ma JB, Liu YM, Zhang YW, He YQ, Liu J (2017) Expression of NR1 and apoptosis levels in the hippocampal cells of mice treated with MK801. Mol Med Rep 16(6):8359–8364. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jing Ying
    • 1
  • Jin Wu
    • 1
    Email author
  • Yiwei Zhang
    • 1
  • Yangyang Han
    • 1
  • Xinger Qian
    • 1
  • Qiuhong Yang
    • 1
  • Yongjie Chen
    • 1
  • Yijun Chen
    • 1
  • Hao Zhu
    • 1
  1. 1.Department of AnesthesiologyNingbo First HospitalNingboChina

Personalised recommendations