IMM-H004, A New Coumarin Derivative, Improved Focal Cerebral Ischemia via Blood-Brain Barrier Protection in Rats.

IMM-H004 (7-hydroxy-5-methoxy-4-methyl-3-[4-methylpiperazin-1-yl]-2H-chromen-2-one) is a novel coumarin derivative that showed better effect in improving global cerebral ischemia in rats. However, the effects and mechanisms in focal cerebral ischemia were not clear. Blood-brain barrier (BBB) protection is a vital strategy for the treatment of cerebral ischemia. This study is to investigate whether IMM-H004 improves brain ischemia injury via BBB protection. Focal brain ischemia model was induced by middle cerebral artery occlusion for 1 hour and reperfusion (MCAO/R) for 24 hours in rats. IMM-H004 (1.5, 3, 6 mg/kg) and edaravone (positive drug, 6 mg/kg) were administered after 5 minutes of occlusion. Neurological score and TTC staining were used to evaluate the effect of IMM-H004. Evans Blue (EB) staining and electron microscopy were used to assess BBB permeability. Western blot, reverse transcription-polymerase chain reaction, and immunohistochemistry were used to detect the expression of BBB structure-related proteins. Compared with the model group, IMM-H004 in the focal brain ischemia model improved neurological function and reduced cerebral infarction size and edema content. IMM-H004 sharply reduced the EB content and alleviated BBB structure. In addition, IMM-H004 increased the level of zonula occludens (ZO-1) and occluding, decreased the level of aquaporin 4 and matrix metalloproteinase 9, either in cortex or in hippocampus. And all of these changed were related to BBB protection. IMM-H004 improved cerebral ischemia injury via BBB protection. For a potential therapy drug of cerebral ischemia, IMM-H004 merits further study.

Niu F ，Song XY ，Hu JF ，Zuo W ，Kong LL ，Wang XF ，Han N ，Chen NH ... -  《-》

IMM-H004 prevents toxicity induced by delayed treatment of tPA in a rat model of focal cerebral ischemia involving PKA-and PI3K-dependent Akt activation.

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.

Zuo W ，Chen J ，Zhang S ，Tang J ，Liu H ，Zhang D ，Chen N ... -  《-》

Compound IMM-H004, a novel coumarin derivative, protects against CA1 cell loss and spatial learning impairments resulting from transient global ischemia.

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.

Zuo W ，Zhang W ，Han N ，Chen NH ... -  《-》

Hydroquinone Strongly Alleviates Focal Ischemic Brain Injury via Blockage of Blood-Brain Barrier Disruption in Rats.

Hydroquinone (HQ), a major benzene metabolite, occurs naturally in various plants and is manufactured for commercial use. Although HQ displays various biological effects, its neuroprotective effects following ischemic insults have not been investigated. In this study, we first examined neuroprotective effects of HQ in a rat model of transient focal cerebral ischemia. Animals were subjected to transient middle cerebral artery occlusion for 120 min. HQ (50 or 100 mg/kg) or vehicle was intraperitoneally administered once at 30 min after ischemia-reperfusion. Neuroprotection by treatment with 100 mg/kg of HQ was shown using evaluation of neurological deficits, positron-emission tomography (PET) and 2,3,5-triphenyltetrazoliumchloride (TTC) staining. In addition, HQ treatment significantly attenuated ischemia-induced Evans blue dye extravasation from blood vessels and significantly increased immunoreactivities of SMI-71 (an endothelial BBB marker) and glucose transporter-1 (GLUT-1, an endothelial cell marker) in ischemic cortex compared to the vehicle-treated ischemia-operated group. Confocal microscopy and western blot analysis also showed that HQ treatment maintained expressions of tight junction proteins (zonula occludens-1 and occludin) in the ischemic cortex. Post-treatment with HQ protected neurons from transient focal cerebral ischemic injury and the neuroprotective effect of HQ might be closely associated with prevention of BBB disruption via maintaining SMI-71 and GLUT-1 expressions as well as prevention of the degradation of zonula occludens-1 and occludin proteins.

Ha Park J ，Yoo KY ，Hye Kim I ，Cho JH ，Lee JC ，Hyeon Ahn J ，Jin Tae H ，Chun Yan B ，Won Kim D ，Kyu Park O ，Kwon SH ，Her S ，Su Kim J ，Hoon Choi J ，Hyun Lee C ，Koo Hwang I ，Youl Cho J ，Hwi Cho J ，Kwon YG ，Ryoo S ，Kim YM ，Won MH ，Jun Kang I ... -  《-》

Protective Effect of 4-Methoxy Benzyl Alcohol on the Blood-Brain Barrier after Cerebral Ischemia Reperfusion Injury.

Damage of the blood-brain barrier (BBB) during the process of cerebral ischemic injury is a key factor that influences the therapeutic efficacy to the cerebral ischemic injury. This work was designed to investigate the mechanisms underlying the protective effects of 4-methoxy benzyl alcohol (4-MA) on the BBB by developing a cerebral ischemia/reperfusion model of rats (MCAO/R). The MCAO/R was developed through a thread embolism method. The neurologic scales, the brain infarct rate, and the Evans blue (EB) contents of the brains were detected. Meanwhile, the release of nitric oxide (NO) and activities of NO synthase (NOS) in brain tissues were measured. Western blotting analyses were also used to assess the protein expressions of aquaporin-4 (AQP-4), occludin, and claudin-5 in brain tissue. After rats were pretreated with different concentrations of 4-MA, the neurologic scores, the infarct rate, and the EB contents in the brain tissues were significantly decreased. The release of NO and the activities of neuronal NOS and inducible NOS were notably inhibited. Furthermore, the protein expression of AQP-4 was markedly decreased, whereas the protein expressions of claudin-5 and occludin were significantly increased. In conclusion, the 4-MA decreases the permeability of BBB when focal cerebral ischemia occurs. The inhibition of the NOS pathways, the attenuation of the protein expression of AQP-4, and the enhancement of the expressions of the tight junction proteins might contribute to the protective effects of 4-MA on the BBB.

He F ，Duan X ，Dai R ，Li Y ，Lin Q ... -  《-》