Therapeutic effects of JLX001 on cerebral ischemia through inhibiting platelet activation and thrombus formation in rats.

(3ß,5α,16α,20S)-4,4,14-trimethyl-3,20-bis(methylamino)-9,19-cyclopregnan-16-ol-dihydrochloride (JLX001), a derivative of cyclovirobuxine D (CVB-D), is a novel compound from synthesis. This study aims to confirm the therapeutic effect of JLX001 on cerebral ischemia and researchits antiplatelet and antithrombosis activities via thromboxane (TXA2)/phospholipase C-ß-3(PLCß3)/protein kinase C (PKC) pathway suppression. The therapeutic effects of JLX001 was evaluated by infarct sizes, brain edema and neurological scores in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Brain TXA2 and prostacyclin (PGI2) were measured by enzyme-linked immunosorbentassay (ELISA). P-PLCß3and activated PKC were detected by immunohistochemical method. Adenosine diphosphate (ADP) or 9, 11-dieoxy-11α, 9α-epoxymethanoeprostaglandin F2α (U46619) was used as platelet agonist in the in vivo and in vitro platelet aggregation experiments. Clotting time and bleeding time were determined. Besides, two whole-animal experiments including arteriovenous shunt thrombosis and pulmonary thromboembolism model were conducted. Results showed that JLX001 treatment markedly alleviated cerebral infarcts, edema, and neurological scores in permanent middle cerebral artery occlusion (pMCAO) rats. Brain TXA2 level, p-PLCß3and activated PKC were decreased, while PGI2level had no significant change. Besides, JLX001 inhibited platelet aggregation induced by ADP or U46619 and exhibited anti-coagulation effects with a minor bleeding risk. In the two whole-animal experiments, JLX001 inhibited thrombus formation. In summary, JLX001 attenuates cerebral ischemia injury and the underlying mechanisms relate to inhibiting platelet activation and thrombus formation via TXA2/PLCß3/PKC pathway suppression.

Rutin improves functional outcome via reducing the elevated matrix metalloproteinase-9 level in a photothrombotic focal ischemic model of rats.

BACKGROUND: Blood-brain barrier (BBB) disruption mediated by proteases plays a pivotal role in neural tissue damage after acute ischemic stroke. In an animal stroke model, the activation of matrix metalloproteinases (MMPs), especially MMP-9, was significantly increased and it showed potential association with blood-brain barrier (BBB) disruption and cerebral edema. Theoretically, it is expected that early blockade of expression and activation of MMP-9 after ischemic stroke provides neuroprotective effects from secondary neural tissue damage. This study was aimed to determine the ability of rutin to influence MMP-9 expression, activity and BBB disruption using a photothrombotic focal ischemic model in rats. METHODS: Adult male Sprague-Dawley rats, weighing between 250 and 300 g (aged 8 weeks) received focal cerebral ischemia by photothrombosis using Rose Bengal (RB) and cold light. Injured animals were divided into two groups; one group received 50mg/kg of rutin intraperitoneally, starting 1h after injury and at 12h intervals for 3 days, while animals in the control group received weight-adjusted doses of saline vehicle over the same period. In each group, the expressions and activities of MMP-9 were assessed by Western blot and gelatin zymography at 6, 24, 48, and 72 h after photothrombotic insult. The effects of rutin on BBB disruption and functional outcomes were also determined. RESULTS: Western blot and zymographic analysis showed up-regulated MMP-9 expression and activity in the ischemic cortex. The expression and activity of MMP-9 were significantly elevated at 6h after photothrombotic insult, which remained up-regulated for at least until 72 h after injury. In the rutin-treated group, MMP-9 expression and activity were significantly attenuated at 6, 24, and 48 h compared to the control group. Relative to the control group, BBB permeability was significantly reduced in the rutin-treated group. The results of the rotarod test revealed that rutin treatment significantly improved functional outcomes. CONCLUSIONS: Rutin treatment starting 1h after injury attenuated BBB disruption during photothrombotic focal ischemia, which was partly, at least, achieved through inhibitory effects on MMP-9 expression and activity. The results of this study suggest that rutin might be useful in clinical trials aimed to improve the outcome of patients suffering from acute ischemic stroke.

Melatonin reduced the elevated matrix metalloproteinase-9 level in a rat photothrombotic stroke model.

BACKGROUND: The accumulation of toxic free radicals plays a pivotal role in the early molecular cascades of blood-brain barrier (BBB) disruption mediated by matrix metalloproteinases (MMPs) activation in ischemic stroke. Theoretically, it is expected that early blockade of activation of MMPs may provide protective effects from secondary neural tissue damage. The present study was designed to determine the ability of melatonin to influence MMP-9 activity and BBB disruption, in a focal ischemia rat model induced by photothrombosis. METHODS: Adult, male, 8-week Sprague-Dawley rats weighing 230-300 g received focal cerebral ischemia by photothrombosis using Rose Bengal (RB). The injured animals were divided into two groups. One group received 50mg/kg of melatonin intraperitoneally, starting 1h after injury and at 12h intervals for 3 days. The control group received weight-adjusted doses of saline vehicle. In each group, MMP-9 expression and activity were assessed by Western blot and gelatin zymography, respectively, at various times. The effects of melatonin on BBB disruption and brain edema were also determined. RESULTS: MMP-9 activity and expression were significantly elevated at 24h in the ischemic cortex, which remained up-regulated at least until 72 h after injury. Melatonin treatment significantly attenuated MMP-9 activity and expression at 24, 48, and 72 h after ischemic injury. Relative to control group, BBB permeability was significantly reduced in the melatonin-treated group. The water content was decreased by melatonin treatment, although there was no statistically significant difference. CONCLUSIONS: Melatonin treatment starting 1h after injury attenuated BBB disruption during focal ischemia, which is at least partly due to inhibition of MMP-9 activity. Melatonin might have a potential role in clinical trials aimed to improve the outcome of patients suffering cerebral ischemia.