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.
Subject(s)
Brain Ischemia/drug therapy , Brain Ischemia/pathology , Chemokines/metabolism , Coumarins/therapeutic use , MARVEL Domain-Containing Proteins/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Receptors, CCR4/metabolism , Animals , Brain/drug effects , Brain/pathology , Brain Injuries/drug therapy , Brain Injuries/pathology , Chemokines/genetics , Coumarins/chemistry , Gene Expression Regulation/drug effects , Humans , Inflammasomes/drug effects , MARVEL Domain-Containing Proteins/genetics , Male , Molecular Structure , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Rats , Rats, Sprague-Dawley , Receptors, CCR4/geneticsABSTRACT
The aims of our study were to investigate into the effect of lithium on smooth muscle contraction and phosphorylation of myosin light chain (MLC20) by MLCK and to find out the clue of its mechanism. Isolated rabbit duodenum smooth muscle strips were used to study the effects of lithium on their contractile activity under the condition of Krebs' solution by means of HW-400S constant temperature smooth muscle trough. Myosin and MLCK were purified from the chicken gizzard smooth muscle. Myosin phosphorylation was determined by Glycerol-PAGE, myosin Mg2+ -ATPase activity was measured by Pi liberation method. Lithium (10-40 mM) inhibited the contraction in duodenum in a dose-related and time-dependent manner. Lithium could also inhibit the extent of myosin phosphorylation in a dose-related and time-dependent manner, whereas it inhibited Mg2+ -ATPase activity in a dose-related manner. Lithium inhibited smooth muscle contraction by inhibition of myosin phosphorylation and Mg2+ -ATPase activity.
