IMM-H004 therapy for permanent focal ischemic cerebral injury and cardiopulmonary complications via CKLF1/CCR4-mediated inflammation pathway

OBJECTIVE To investigate the effects of IMM-H004 on permanent focal cerebral ischemia injury and associated cardiopulmonary complications,further elucidating the molecular mechanisms.METHODS The effects of IMM-H004 were investigated in wild-type(WT) and CKLF1-/-rats.The effects of IMM-H004 on ischemic stroke injury and its cardiopulmonary complications were determined using 2,3,5-triphenyltetrazolium chloride(TTC) staining,behavior tests,magnetic resonance imaging(MRI)scans,enzyme-linked immunosorbent assay(ELISA),Nissl staining,and histo-pathological examination.Multiple molecular experiments including immunohistological staining,immunofluorescence staining,quantitative RT-PCR,Western blotting,and co-immunoprecipitation assays were used to elucidate the underlying mechanisms.RESULTS IMM-H004 treatment provided significant protection against ischemic stroke-induced brain injury and associated cardiopulmonary complications,through CKLF1-depedent-anti-inflammation pathway in rats.IMM-H004 downregulated the amount of CKLF1 and disturbed the combination between CKLF1 and C-C chemokine receptor type 4,suppressing the inflammatory response and protecting the damaged organs in ischemic setting.CONCLUSION This preclinical study established efficacy of IMM-H004 as a potential therapeutic medicine for ischemic stroke and associated cardiopulmonary complications.The protective effects of IMM-H004 may due to its specific mechanism through CKLF1.These results support further efforts to develop IMM-H004 for human clinical trials in acute cerebral ischemia,especially for patients who are not suitable for reperfusion therapy.

化合物IMM-H004对大鼠急性全脑缺血/再灌注损伤的保护作用

目的评价化合物IMM-H004不同剂量(1.5、3、6 mg·kg-1)对大鼠急性全脑缺血/再灌注损伤的保护作用。方法使用四血管结扎法(4VO,20 min)制作大鼠急性全脑缺血/再灌注损伤模型,造模前3天开始尾静脉注射给药(每天1次),参照25分评分法对大鼠进行行为学评分,用尼氏染色方法考察化合物IMM-H004对大鼠海马CA1区、CA3区神经元病理损伤的影响,Western blot检测Bax、Bcl-2表达。结果化合物IMM-H004(1.5、3、6 mg·kg-1)对大鼠急性全脑缺血/再灌注损伤后的行为学有明显的改善作用。假手术组海马CA1区、CA3区神经细胞层次较多,排列紧密,细胞形态完整,核仁清晰,全脑缺血/再灌注明显导致神经元数目减少,细胞皱缩,细胞带分布不均,甚至出现脱失和带中断,而化合物IMM-H004给药组可明显改善神经元形态及数量,逆转缺血/再灌注损伤引起的Bax/Bcl-2比值的升高,并且具有一定剂量依赖性。结论化合物IMM-H004对大鼠急性全脑缺血/再灌注损伤具有保护作用,可作为一种潜在的神经保护剂进行开发。

IMM-H004对小鼠肝脏缺血再灌注损伤的保护作用及其机制

探究香豆素类衍生物IMM-H004对小鼠肝脏缺血再灌注损伤(hepatic ischemia reperfusion injury,HIRI)的保护作用及其机制。本文中所有动物实验都获得中国医学科学院药物研究所伦理学委员会批准。实验分为3组,假手术组、模型组和IMM-H004给药组,检测各组的血清生化指标和病理情况。qPCR检测炎症因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)的mRNA含量,免疫组织化学和免疫荧光检测中性粒细胞浸润的情况,Western blot检测NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)炎症小体通路相关蛋白的表达。IMM-H004可显著降低HIRI过程中谷丙转氨酶(alanine transaminase,ALT)、谷草转氨酶(aspartate transaminase,AST)、乳酸脱氢酶(lactate dehydrogenase,LDH)的含量,病理结果提示IMM-H004给药组肝组织空泡性坏死程度较模型组显著减轻,炎症因子TNF-α、IL-1β、IL-6的mRNA水平与模型组相比显著降低。免疫组化和免疫荧光的结果表明,IMM-H004可显著减少中性粒细胞的聚集。Western blot的结果显示,IMM-H004可显著降低NLRP3、衔接蛋白(apoptosis-associated speck-like protein,ASC)、含半胱氨酸的天冬氨酸蛋白水解酶-1(cysteinyl aspartate specific proteinase-1,caspase-1)、IL-1β的蛋白水平。综上所述,IMM-H004对小鼠HIRI有显著的保护作用,其保护作用与降低HIRI过程中炎症因子mRNA的表达,抑制中性粒细胞的聚集以及抑制NLRP3炎症小体通路的激活相关。