JLX001 Ameliorates Ischemia/Reperfusion Injury by Reducing Neuronal Apoptosis via Down-Regulating JNK Signaling Pathway.

JLX001, a novel compound with similar structure with cyclovirobuxine D (CVB-D), has been proved to exert therapeutical effects on permanent focal cerebral ischemia. However, the protective effects of JLX001 on cerebral ischemia/reperfusion (I/R) injury and its anti-apoptotic effects have not been reported. We investigated the efficacy of JLX001 in two pharmacodynamic tests (pre-treatment test and post-treatment) with rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). The pharmacodynamic tests demonstrated that JLX001 ameliorated I/R injury by reducing infarct sizes and brain edema. The results of Morris water maze, neurological scores, cylinder test and posture reflex test implied that JLX001 improved the learning, memory and motor ability after MCAO/R in the long term. Anti-apoptotic effects of JLX001 and its regulation of cytosolic c-Jun N-terminal Kinases (JNKs) signal pathway were confirmed in vivo by co-immunofluorescence staining and western immunoblotting. Furthermore, primary cortical neuron cultures were prepared and exposed to oxygen glucose deprivation/reoxygenation (OGD/R) for in vitro studies. Cytotoxicity test and mitochondrial membrane potential (MMP) test showed that JLX001 enhanced cell survival rate and maintained MMP. Flow cytometry and TdT-mediated dUTP-X nick end labeling (TUNEL) staining demonstrated the anti-apoptotic effects of JLX001 in vitro. Likewise, JLX001 regulated JNK signal pathway in vivo, which was also confirmed by western immunoblotting. Collectively, this study presents the first evidence that JLX001 exerted protective effects against I/R injury by reducing neuronal apoptosis via down-regulating JNK signaling pathway.

Therapeutic effects of JLX-001 on ischemic stroke by inducing autophagy via AMPK-ULK1 signaling pathway in rats.

(3ß,5α,16α,20S)-4,4,14-trimethyl-3,20-bis(methylamino)-9,19-cyclopregnan-16-ol-dihydrochloride (JLX-001), a structural analogue of cyclovirobuxine D (CVB-D), is a novel compound from synthesis. This study aims to confirm the therapeutic effects of JLX001 on ischemic stroke (IS) and research its induction of autophagy function via 5'-AMP-activated protein kinase (AMPK)-Human Serine/threonine-protein kinase (ULK1) signaling pathway activation. The therapeutic effects of JLX001 were evaluated by infarct sizes, brain edema, neurological scores and proportion of apoptotic neurons in Sprague-Dawley (SD) rats with middle cerebral artery occlusion/reperfusion (MCAO/R). The number of autophagosomes was obtained by transmission electron microscopy. The expression of LC3-II was measured by immunofluorescence. p-AMPK and activated ULK1 were detected by western blots. Results showed that JLX001 treatment markedly alleviated cerebral infarcts, edema, neurological scores and proportion of apoptotic neurons in MCAO/R rats. The number of autophagosomes was increased, accompanying with the increased expressions of LC3-II, p-AMPK and ULK1. In summary, JLX001 attenuates cerebral ischemia injury and the underlying mechanisms may relate to inducing autophagy via AMPK-ULK1 signaling pathway activation.

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.

[Studies on pharmacokinetics of artemether patch in mice].

OBJECTIVE: To establish a HPLC-MS-MS determination method of artemether (ARM) and active derivatives DHA, and compare the pharmacokinetic parameters of ARM after transdermal and oral administration. METHOD: The mice were divided two groups (transdermal and oral) by parallel design. ARM and active derivatives DHA in plasma of mice at different sampling time were determined. The pharmacokinetic parameters were calculated by DAS 2.0 and by statistic analysis. RESULT: compare oral administration, the pharmacokinetic parameters of ARM after transdermal, Cmax Tmax , AUC(0-t) MRT, had significant difference (P < 0.05). CONCLUSION: The artemether patch has long-releasing property.