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Transl Res. 2019 May 30. pii: S1931-5244(19)30117-3. doi: 10.1016/j.trsl.2019.05.007. [Epub ahead of print]

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

Author information

1
Hunan Engineering Technology Center of Standardization and Function of Chinese Herbal Decoction Pieces and Hunan University of Chinese Medicine First-Class Disciple Construction Project of Chinese Materia Medica, Changsha, China; State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica and Neuroscience Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
2
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica and Neuroscience Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
3
Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China.
4
Key Laboratory of Human Disease Comparative Medicine, NHFPC, Institute of Laboratory Animal Science, Peking Union Medicine College and Chinese Academy of Medical Sciences, Beijing, China.
5
Hunan Engineering Technology Center of Standardization and Function of Chinese Herbal Decoction Pieces and Hunan University of Chinese Medicine First-Class Disciple Construction Project of Chinese Materia Medica, Changsha, China.
6
Tianjin University of Traditional Chinese Medicine, Tianjin, China.
7
School of Basic Medicine, Shanxi University of Traditional Chinese Medicine, Taiyuan, China.
8
Hunan Engineering Technology Center of Standardization and Function of Chinese Herbal Decoction Pieces and Hunan University of Chinese Medicine First-Class Disciple Construction Project of Chinese Materia Medica, Changsha, China; State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica and Neuroscience Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. Electronic address: chennh@imm.ac.cn.

Abstract

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.

KEYWORDS:

ASC = apoptosis-associated speck-like protein containing a caspase activation recruitment domain; BCA = bicinchoninic acid; BSA = bovine serum albumin; CCA = common carotid artery; CCR4 = C-C chemokine receptor type 4; CKLF1 = chemokine-like factor 1; CO-IP = coimmunoprecipitation; ECA = external carotid artery; ELISA = enzyme-linked immunosorbent assay; FDA = Food and Drug Administration; I/R = ischemic/reperfusion; ICA = internal carotid artery; IL-18 = interleukin 18; IL-1β = interleukin 1β; LDH = lactate dehydrogenase; NLRP3 = NOD-like receptor protein 3; PBS = phosphate-buffered saline; SD = standard deviation; TNF-α = tumor necrosis factor α; TTC = 2,3,5-triphenyltetrazolium-chloride; WT = wild-type; pMCAO = permanent middle cerebral artery occlusion; pro-Caspase-1 = precursor Caspase-1; qPCR = quantitative real-time PCR; tMCAO = transient middle cerebral artery occlusion; tPA = tissue plasminogen activator

PMID:
31176667
DOI:
10.1016/j.trsl.2019.05.007

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