Novel Caspase-1 inhibitor CZL80 improves neurological function in mice after progressive ischemic stroke within a long therapeutic time-window.

Progressive ischemic stroke (PIS) is featured by progressive neurological dysfunction after ischemia. Ischemia-evoked neuroinflammation is implicated in the progressive brain injury after cerebral ischemia, while Caspase-1, an active component of inflammasome, exaggerates ischemic brain injury. Current Caspase-1 inhibitors are inadequate in safety and druggability. Here, we investigated the efficacy of CZL80, a novel Caspase-1 inhibitor, in mice with PIS. Mice and Caspase-1-/- mice were subjected to photothrombotic (PT)-induced cerebral ischemia. CZL80 (10, 30 mg·kg-1·d-1, i.p.) was administered for one week after PT onset. The transient and the progressive neurological dysfunction (as foot faults in the grid-walking task and forelimb symmetry in the cylinder task) was assessed on Day1 and Day4-7, respectively, after PT onset. Treatment with CZL80 (30 mg/kg) during Day1-7 significantly reduced the progressive, but not the transient neurological dysfunction. Furthermore, we showed that CZL80 administered on Day4-7, when the progressive neurological dysfunction occurred, produced significant beneficial effects against PIS, suggesting an extended therapeutic time-window. CZL80 administration could improve the neurological function even as late as Day43 after PT. In Caspase-1-/- mice with PIS, the beneficial effects of CZL80 were abolished. We found that Caspase-1 was upregulated during Day4-7 after PT and predominantly located in activated microglia, which was coincided with the progressive neurological deficits, and attenuated by CZL80. We showed that CZL80 administration did not reduce the infarct volume, but significantly suppressed microglia activation in the peri-infarct cortex, suggesting the involvement of microglial inflammasome in the pathology of PIS. Taken together, this study demonstrates that Caspase-1 is required for the progressive neurological dysfunction in PIS. CZL80 is a promising drug to promote the neurological recovery in PIS by inhibiting Caspase-1 within a long therapeutic time-window.

Melatonin receptor agonist ramelteon attenuates mouse acute and chronic ischemic brain injury.

Melatonin receptors (MTs) are potential drug targets for stroke therapy. Ramelteon is a selective melatonin receptor agonist used to treat insomnia. In this study we investigated whether ramelteon could attenuate cerebral ischemia in mice. Acute focal cerebral ischemia was induced in mice via middle cerebral artery occlusion (MCAO). We found oral administration of ramelteon (3.0 mg/kg) significantly attenuated ischemic injury even when it was given 4 h after the onset of ischemia. We showed that administration of ramelteon (3.0 mg/kg) displayed comparable protective efficacy and length of effective time window as administration of edaravone (10 mg/kg, i.p.), which was used in clinic to treat ischemic stroke. Chronic ischemic brain injury was induced in mice using photothrombosis. Oral administration of ramelteon (3.0 mg · kg-1 · d-1) for 7 days after ischemia significantly attenuated functional deficits for at least 15 days. The neuroprotection of ramelteon was blocked by 4-P-PDOT, a specific MT antagonist. We further revealed that ramelteon significantly inhibited autophagy in the peri-infarct cortex in both the mouse ischemia models via regulating AMPK/mTOR signaling pathway. Intracerebroventricular injection of rapamycin, an autophagy activator, compromised the neuroprotection of ramelteon, suggesting ramelteon might attenuate ischemic injury by counteracting autophagic cell death. These data demonstrate for the first time the potential benefits of ramelteon in the treatment of both acute and chronic ischemic brain injury and provide the rationale for the application of ramelteon in stroke therapy.

Urolithin A-activated autophagy but not mitophagy protects against ischemic neuronal injury by inhibiting ER stress in vitro and in vivo.

AIM: Mitochondrial autophagy (mitophagy) clears damaged mitochondria and attenuates ischemic neuronal injury. Urolithin A (Uro-A) activates mitophagy in mammal cells and Caenorhabditis elegans. We explored neuroprotection of Uro-A against ischemic neuronal injury. METHODS: Mice were subjected to middle cerebral artery occlusion. The brain infarct and neurological deficit scores were measured. The N2a cells and primary cultured mice cortical neurons were subjected to oxygen-glucose deprivation and reperfusion (OGD/R). Uro-A was incubated during OGD/R, and cell injury was determined by MTT and LDH. Autophagosomes were visualized by transfecting mCherry-microtubule-associated protein 1 light chain 3 (LC3). The protein levels of LC3-II, p62, Translocase Of Inner Mitochondrial Membrane 23 (TIMM23), and cytochrome c oxidase subunit 4 isoform 1 (COX4I1) were detected by Western blot. The ER stress markers, activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), were determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Urolithin A alleviated OGD/R-induced injury in N2a cells and neurons and reduced ischemic brain injury in mice. Uro-A reinforced ischemia-induced autophagy. Furthermore, Uro-A-conferred protection was abolished by 3-methyladenine, suggesting the requirement of autophagy for neuroprotection. However, mitophagy was not further activated by Uro-A. Instead, Uro-A attenuated OGD/R-induced ER stress, which was abolished by 3-methyladenosine. Additionally, neuroprotection was reversed by ER stress inducer. CONCLUSION: Urolithin A protected against ischemic neuronal injury by reinforcing autophagy rather than mitophagy. Autophagy activation by Uro-A attenuated ischemic neuronal death by suppressing ER stress.

Predictive value of thrombospondin-1 for outcomes in patients with acute ischemic stroke.

BACKGROUND: Thrombospondin-1 is a potent regulator of angiogenesis. The expression of cerebral thrombospondin-1 is promoted in a rat model of intracerebral hemorrhage. The current study was designed to investigate the change of plasma thrombospondin-1 concentrations and assess the prognostic value of plasma thrombospondin-1 concentrations for long-term mortality and functional outcome of ischemic stroke patients. METHODS: This study included 192 patients and 150 healthy controls. The plasma thrombospondin-1 concentrations were measured using enzyme-linked immunosorbent assay. An unfavorable outcome was defined as a modified Rankin Scale score >3. The relationships between plasma thrombospondin-1 concentrations and 6-month clinical outcomes were analyzed using multivariate analysis. RESULTS: Compared with healthy controls, plasma thrombospondin-1 concentrations were statistically significantly elevated in patients. Using multivariate analysis, thrombospondin-1 emerged as an independent predictor for 6-month mortality, 6-month unfavorable outcome and 6-month overall survival. Plasma thrombospondin-1 concentrations possessed high predictive values under receiver operating characteristic curve. Their predictive values were similar to those of National Institutes of Health Stroke Scale scores. CONCLUSIONS: Plasma thrombospondin-1 concentrations are elevated obviously and are highly associated with long-term outcome of ischemic stroke.

Prognostic value of neuropeptide proenkephalin A in patients with severe traumatic brain injury.

High plasma proenkephalin A levels have been associated with poor clinical outcome of aneurysmal subarachnoid hemorrhage. This prospective observatory study was designed to investigate the relationship between plasma proenkephalin A levels and 1-week mortality, 6-month mortality and 6-month unfavorable outcome (defined as Glasgow Outcome Scale score of 1-3) in patients with severe traumatic brain injury. This study recruited 128 patients and 128 sex- and age-matched healthy controls. Plasma proenkephalin A levels, as measured by chemoluminescence sandwich immunoassay, were statistically significantly higher in patients than in healthy controls (239.1±93.0 pmol/L vs.81.3±22.1 pmol/L; P<0.001) and were correlated with Glasgow Coma Scale scores (r=-0.540, P<0.001). It was identified as an independent prognostic predictor of 1-week mortality [odds ratio (OR), 1.214; 95% confidence interval (CI), 1.103-1.425; P<0.001], 6-month mortality (OR, 1.162; 95% CI, 1.101-1.372; P<0.001) and 6-month unfavorable outcome (OR, 1.116; 95% CI, 1.097-1.281; P<0.001). Moreover, it had high predictive value for 1-week mortality [area under curve (AUC), 0.852; 95% CI, 0.778-0.908], 6-month mortality (AUC, 0.841; 95% CI, 0.766-0.899) and 6-month unfavorable outcome (AUC, 0.830; 95% CI, 0.754-0.891). Furthermore, its predictive value was similar to Glasgow Coma Scale score's (all P>0.05). Yet, a combined logistic-regression model did not show that it statistically significantly improved the predictive value of Glasgow Coma Scale score (all P>0.05). Thus, it was proposed that enhanced plasma proenkephalin A could be a useful, complementary tool to predict short- or long-term clinical outcome after severe traumatic brain injury.

Cytotoxic triterpenoid saponins from Vaccaria segetalis.

By the guidance of bioassay, one new cytotoxic triterpenoid saponin, 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] quillaic acid 28-O-beta-D-glucopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-fucopyranosyl-(1-->4)]-beta-D-fucopyranoside (1), and five known cytotoxic triterpenoid saponins, vaccaroside E (2), vaccaroside G (3), vaccaroside B (4), segetoside H (5) and segetoside I (6), were isolated from Vaccaria segetalis. Their structures were established on the basis of ESI-MS, IR, extensive NMR ((1)H NMR, (13)C NMR, TOCSY, (1)H-(1)H COSY, DEPT, HMQC, HMBC and ROESY) analyses, chemical degradation, and by comparing with previously reported data. Compounds 1-6 showed moderate cytotoxic activities against LNcap, P-388 and A-549 cell lines with IC(50) values in the range 0.1-12.9 microM.

Semisynthesis and antitumor activities of new styryl-lactone derivatives.

Nineteen new derivatives of the naturally occurring compound, goniothalamin, were prepared by chemical modification and semi-synthetic methods. The antitumor activities of these derivatives and goniothalamin were evaluated in vitro against human tumor cell lines, and most of them showed an inhibitory effect against HL-60 cancer cells. The derivatives 10-nitro-goniothalamin and 10-amino-goniothalamin gave selective inhibition concentration (IC50) of 1.10 and 1.14 microg/ml, respectively, against human stomach cancer SGC-7901 cells, while that of etoposide (vp-16) as the positive control was 6.07 microg/ml. Finally, the partition coefficients, logP (pi values), of these derivative molecules, were evaluated by calculating the additive approximate organic fragment logP value.

Constituents from the stems of Goniothalamus griffithii.

A new cyclopeptide, grifficyclocin A (1), and a new aporphine alkaloid, griffinin (2) were isolated together with two known styryllactones from the stems of Goniothalamus griffithii. Their structures were identified spectroscopically and chemically. Among them, the griffinin (2) was isolated as the enol form in two tautomers, and the two known styryllactones, goniothalamin (3) and 8- O-acetylgoniotriol (4), showed selective in vitro antitumor activities.