Elsevier

Translational Research

Volume 212, October 2019, Pages 36-53
Translational Research

IMM-H004 therapy for permanent focal ischemic cerebral injury via CKLF1/CCR4-mediated NLRP3 inflammasome activation

https://doi.org/10.1016/j.trsl.2019.05.007Get rights and content

Chemokine-like factor 1 (CKLF1) is a potential target for ischemic stroke therapy. The NOD-like receptor protein 3 (NLRP3) inflammasome has been postulated to mediate inflammatory responses during ischemic/reperfusion (I/R) injury. The compound IMM-H004 is a novel coumarin derivative that can improve cerebral I/R injury. This study aims to investigate the effects of IMM-H004 on ischemia stroke injury and further elucidate the molecular mechanisms. The standard pMCAO model of focal ischemia was used in this paper. Drugs were administered at 6 hours after ischemia, and behavioral assessment, euthanasia, and outcome measures were evaluated at 9 hours after ischemia. The effects of IMM-H004 on ischemic stroke injury were determined using 2,3,5-triphenyltetrazolium chloride (TTC) staining, behavioral tests, enzyme-linked immunosorbent assay (ELISA), and Nissl staining. Immunohistologic staining, immunofluorescence staining, quantitative RT-PCR (qPCR), western blotting, and coimmunoprecipitation (CO-IP) assays were used to elucidate the underlying mechanisms. IMM-H004 treatment provided significant protection against ischemia stroke through a CKLF1-dependent anti-inflammatory pathway in rats. IMM-H004 downregulated the amount of CKLF1 binding with C-C chemokine receptor type 4, further suppressing the activation of NLRP3 inflammasome and the following inflammatory response, ultimately protecting the ischemic brain. This preclinical study established the efficacy of IMM-H004 as a potential therapeutic medicine for permanent cerebral ischemia. These results support further efforts to develop IMM-H004 for human clinical trials in acute cerebral ischemia, particularly for patients who are not suitable for reperfusion therapy.

Section snippets

INTRODUCTION

Stroke, particularly acute ischemic stroke, is a devastating and debilitating disease and is the third leading cause of death in the Western world.1 Currently, thrombolytic therapy remains the common therapy for patients with acute ischemic stroke, and recombinant tissue plasminogen activator (tPA) is the only Food and Drug Administration (FDA)-approved drug for clinical application. However, few patients (2%−7%) benefit from tPA because this drug must be injected within 4.5 hours post onset of

Animals

Male wild-type (WT) adult SD rats (6–8 weeks, 250–280 g) were obtained from VITAL RIVER Laboratories, Beijing, China. CKLF1−/− SD rats (250–280 g) were obtained from the Key Laboratory of Human Disease Comparative Medicine, NHFPC, Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China. All rats were housed under standard conditions. The experimental protocols and animal care were performed according to the National Research

IMM-H004 protects against ischemic stroke-induced brain injury

The protective effects of IMM-H004 on ischemic stroke-induced brain injury were determined by traditional methods. IMM-H004 and urokinase (10,000 UI/kg) treatment pronouncedly reduced the brain infarct size and attenuated the neurologic deficits compared with the pMCAO group (Fig 1, C and D).

Nissl staining indicated a sharp decrease of normal neurons in the CA1 hippocampus, cortex, and striatum after pMCAO operation, with relict neurons manifested by karyopyknosis, anachromasis, nucleoli

DISCUSSION

Cerebral ischemia initiates a complex series of pathophysiological events, which evolve over time and space. These cascade reactions cause progressive and massive neural damage and death, leading to severe functional impairment. Therapeutic strategies that target the acute phase of stroke remain ineffective in patients besides thrombolysis. Thus, it becomes essential to develop new approaches that target new therapeutic windows to significantly improve the processes of functional recovery.

Acknowledgments

Conflicts of Interest: All authors have read the journal's policy on the disclosure of potential conflicts of interest and have no conflicts to declare.

This work was supported by the Hunan University of Chinese Medicine First-class Disciple Construction Project (201803), Project of NDRC and State Administration of Traditional Chinese Medicine (ZYBZH-Y-HUN-24), Hunan Engineering Technology Center of Standardization and Function of Chinese Herbal Decoction Pieces (2016TP2008), Open found of Hunan

References (56)

  • L. Gan et al.

    Protective effect of mitochondrial-targeted antioxidant MitoQ against iron ion (56)Fe radiation induced brain injury in mice

    Toxicol Appl Pharmacol

    (2018)
  • C.Y. Xia et al.

    Corticosterone impairs gap junctions in the prefrontal cortical and hippocampal astrocytes via different mechanisms

    Neuropharmacology

    (2018)
  • X.Y. Song et al.

    IMM-H004, a novel coumarin derivative compound, protects against amyloid beta-induced neurotoxicity through a mitochondrial-dependent pathway

    Neuroscience

    (2013)
  • H.J. Ji et al.

    IMM-H004, a novel courmarin derivative, protects against oxygen-and glucose-deprivation/restoration-induced apoptosis in PC12 cells

    Eur J Pharmacol

    (2014)
  • Y. Yang et al.

    Loss of neuronal CD200 contributed to microglial activation after acute cerebral ischemia in mice

    Neurosci Lett

    (2018)
  • G. Zhang et al.

    Recanalization of occluded large arteries with broadened therapeutic window for acute cerebral infarction

    Clin Neurol Neurosurg

    (2013)
  • M. Huang et al.

    Capsaicin protects cortical neurons against ischemia/reperfusion injury via down-regulating NMDA receptors

    Exp Neurol

    (2017)
  • K.M. Nash et al.

    Development of a reactive oxygen species-sensitive nitric oxide synthase inhibitor for the treatment of ischemic stroke

    Free Radic Biol Med

    (2018)
  • J. Jiang et al.

    Determination of IMM-H004 and its active glucuronide metabolite in rat plasma and Ringer's solution by ultra-performance liquid chromatography-tandem mass spectrometry

    J Chromatogr B Analyt Technol Biomed Life Sci

    (2018)
  • J. Anrather et al.

    Inflammation and stroke: an overview

    Neurotherapeutics

    (2016)
  • L.L. Kong et al.

    Inhibition of chemokine-like factor 1 improves blood-brain barrier dysfunction in rats following focal cerebral ischemia

    Neurosci Lett

    (2016)
  • Q. Zheng et al.

    Reactive oxygen species activated NLRP3 inflammasomes prime environment-induced murine dry eye

    Exp Eye Res

    (2014)
  • M. Lamkanfi et al.

    Manipulation of host cell death pathways during microbial infections

    Cell Host Microbe

    (2010)
  • G. Li et al.

    The chemokine-like factor 1 induces asthmatic pathological change by activating nuclear factor-kappa B signaling pathway

    Int Immunopharmacol

    (2014)
  • E.H. Lo et al.

    Mechanisms, challenges and opportunities in stroke

    Nat Rev Neurosci

    (2003)
  • Thrombolysis 3 to 4.5 hours after acute ischemic stroke

    N Engl J Med

    (2008)
  • M. Fiorelli et al.

    Hemorrhagic transformation within 36 hours of a cerebral infarct: relationships with early clinical deterioration and 3-month outcome in the European Cooperative Acute Stroke Study I (ECASS I) cohort

    Stroke

    (1999)
  • S. Chen et al.

    Homocysteine exaggerates microglia activation and neuroinflammation through microglia localized STAT3 overactivation following ischemic stroke

    J Neuroinflamm

    (2017)
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