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

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.