imm-h004 and Brain-Ischemia

(1) Background: Chemokine-like factor 1 (CKLF1) is a chemokine with potential to be a target for stroke therapy. Compound IMM-H004 is a novel coumarin derivative screened from a CKLF1/C-C chemokine receptor type 4 (CCR4) system and has been reported to improve cerebral ischemia/reperfusion injury. This study aims to investigate the protective effects of IMM-H004 on cerebral ischemia injury and its infectious cardiopulmonary complications in adult and aged rats from the CKLF1 perspective. (2) Methods: The effects of IMM-H004 on the protection was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining, behavior tests, magnetic resonance imaging (MRI) scans, enzyme-linked immunosorbent assay (ELISA), Nissl staining, histo-pathological examination, and cardiopulmonary function detection. Immunohistological staining, immunofluorescence staining, quantitative real-time PCR (qPCR), and western blotting were used to elucidate the underlying mechanisms. (3) Results: IMM-H004 protects against cerebral ischemia induced brain injury and its cardiopulmonary complications, inhibiting injury, and inflammation through CKLF1-dependent anti-inflammation pathway in adult and aged rats. IMM-H004 downregulates the amount of CKLF1, suppressing the followed inflammatory response, and further protects the damaged organs from ischemic injury. (4) Conclusions: The present study suggested that the protective mechanism of IMM-H004 is dependent on CKLF1, which will lead to excessive inflammatory response in cerebral ischemia. IMM-H004 could also be a therapeutic agent in therapy for ischemic stroke and cardiopulmonary complications in the aged population.

Topics: Aging; Animals; Brain Injuries; Brain Ischemia; Chemokines; Coumarins; Inflammation; Lung; Male; MARVEL Domain-Containing Proteins; Myocardium; Neuroprotective Agents; Rats, Sprague-Dawley; Stroke

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.

Topics: Animals; Brain; Brain Injuries; Brain Ischemia; Chemokines; Coumarins; Gene Expression Regulation; Humans; Inflammasomes; Male; MARVEL Domain-Containing Proteins; Molecular Structure; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Rats, Sprague-Dawley; Receptors, CCR4

Compound IMM-H004 (7-hydroxy-5-methoxy-4-methyl-3-[4-methylpiperazin-1-yl]-2H-chromen-2-one) is a new synthetic derivative of coumarin, and previous studies showed that it exhibited antioxidant and neuroprotective roles in focal cerebral ischemia. However, we know little about the compound's function in transient global ischemia. This study is to investigate whether compound IMM-H004 can protect against transient global ischemic injury.. Four-vessel occlusion (4VO) rat model was induced for a 20-min occlusion and different times of reperfusion to mimic transient global cerebral ischemia. IMM-H004 (3, 6, 9 mg/kg) or Edaravone (6 mg/kg) was administered after 30 min of reperfusion. Morris water maze tests were used to estimate the ability of spatial learning and memory. Nissl staining, TUNEL assay and Immunoblot for Bax/Bcl-2 and activated caspase-3 were used to detect hippocampal neuron injury. Immunoblot for PSD-95 and synapsin 1, and electron microscopy were used to observe synaptic function.. Compared with vehicle group, IMM-H004 significantly improved the spatial learning performance and exhibited less CA1 neurons loss. The expressions of Bax/Bcl-2 and activated caspase-3 were decreased. IMM-H004 also ameliorated synaptic structure, decreased PSD-95 and increased synapsin 1 expression.. These findings suggested that IMM-H004 exerted neuroprotective role in global ischemia by reducing apoptosis and maintaining the integrity of synaptic structure.

Topics: Animals; Antipyrine; bcl-2-Associated X Protein; Brain Ischemia; CA1 Region, Hippocampal; Caspase 3; Cell Death; Coumarins; Disease Models, Animal; Disks Large Homolog 4 Protein; Drug Evaluation, Preclinical; Edaravone; Free Radical Scavengers; Intracellular Signaling Peptides and Proteins; Learning Disabilities; Male; Maze Learning; Membrane Proteins; Neurons; Neuroprotective Agents; Nootropic Agents; Random Allocation; Rats, Sprague-Dawley; Spatial Memory; Synapsins

Ischemic stroke is currently treated with thrombolytic therapy with a drawback to induce hemorrhagic transformation (HT) if applied beyond its relatively narrow treatment time window. The present study was designed to examine the role of IMM-H004, a derivative of coumarin, in recombinant tissue plasminogen activator (tPA)-induced HT. Rats subjected to 6 h of thromboembolic occlusion or middle cerebral artery occlusion received tPA with or without IMM-H004. Delayed tPA intervention drastically increased the risk of HT and exaggerated the ischemic injury. To assess the effect of IMM-H004 on delayed treatment of tPA-induced toxicity after ischemia and reperfusion, various approaches were used, including a behavior test, TTC-staining, determination of cerebral hemorrhage, laser speckle imaging, Western blot, gelatin zymogram, immunohistochemistry and immunofluorescence staining. Experiments were also conducted in vitro in human brain microvascular endothelial cells (HBMECs) and PC12 cells to explore the mechanism for the role of IMM-H004. Combination therapy of tPA and IMM-H004 prevented the development of HT, and reduced the mortality rate, infarct volume and brain edema. IMM-H004 also exerted a protective role by decreasing matrix metalloproteinases, the co-localization of matrix metalloproteinase-2 with astrocytes and increasing occludin. Experiments in HBMECs and PC12 revealed an elevation in ATP level and a protein kinase A- and PI3K-dependent activation of Akt by IMM-H004 after tPA administration. These results suggest IMM-H004 as a promising adjuvant to alleviate the detrimental side effects of tPA in clinical therapy of ischemic stroke, and contribute to better understand the mechanism for the beneficial role of this novel remedy.

Topics: Animals; Brain; Brain Ischemia; Cells, Cultured; Cerebral Hemorrhage; Coumarins; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Endothelial Cells; Fibrinolytic Agents; Humans; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; PC12 Cells; Proto-Oncogene Proteins c-akt; Random Allocation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thromboembolism; Time Factors; Tissue Plasminogen Activator

Neuroprotection strategies are of great importance in the treatment of ischemic brain injury. Screening of our in-stock coumarin derivatives identified compound 1 as exhibiting neuroprotective activity. Subsequently, a structural optimization was carried out, which led to the discovery of the potent compound 20. This compound significantly attenuated the damage in a cell line derived from a pheochromocytoma of the rat adrenal medulla induced by oxygen-glucose deprivation in vitro. Furthermore, compound 20 exhibited clear neuroprotection in middle cerebral artery occlusion rats by reducing infarct size and brain-water content, improving neurological function, and suppressing neuronal loss and neuropathological changes in the cortex and hippocampus. Pharmacokinetic evaluation indicated that compound 20 could penetrate the blood-brain barrier of rats.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Coumarins; Male; Molecular Structure; Neuroprotective Agents; Rats; Rats, Sprague-Dawley