Clematichinenoside protects blood brain barrier against ischemic stroke superimposed on systemic inflammatory challenges through up-regulating A20.

Suppression of excessive inflammation can ameliorate blood brain barrier (BBB) injury, which shows therapeutic potential for clinical treatment of brain injury induced by stroke superimposed on systemic inflammatory diseases. In this study, we investigated whether and how clematichinenoside (AR), an anti-inflammatory triterpene saponin, protects brain injury from stroke superimposed on systemic inflammation. Lipopolysaccharide (LPS) was intraperitoneally injected immediately after middle cerebral artery occlusion (MCAO) in rats. Rat microvessel endothelial cells (rBMECs) were exposed to hypoxia/reoxygenation (H/R) coexisting with LPS. The results revealed that AR suppressed the excessive inflammation, restored BBB dysfunction, alleviated brain edema, decreased neutrophil infiltration, lessened neurological dysfunction, and decreased infarct rate. Further study demonstrated that the expression of nucleus nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) and interlukin-1ß (IL-1ß) were suppressed by AR via zinc finger protein A20. Besides, AR increased in vitro BBB integrity through A20. In conclusion, AR alleviated cerebral inflammatory injury through A20-NF-κB signal pathway, offering an alternative medication for stroke associated with systemic inflammatory diseases.

Platelet activating factor induces transient blood-brain barrier opening to facilitate edaravone penetration into the brain.

The blood-brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra-performance liquid chromatograph combined with a hybrid quadrupole time-of-flight mass spectrometer (UPLC-MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 µM PAF for 1 h significantly increased monolayer permeability to (125)I-albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule-1, matrix-metalloproteinase-9 and P-glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain. Platelet activating factor (PAF) transiently induces BBB dysfunction and increases BBB permeability, which may be due to vessel contraction and a temporary decline of regional cerebral blood flow (rCBF) triggered by PAF. More importantly, the PAF induced transient BBB opening facilitates neuroprotectant edaravone penetration into brain. The results of this study may provide a new approach to improve drug delivery into the brain.

XQ-1H Suppresses Neutrophils Infiltration and Oxidative Stress Induced by Cerebral Ischemia Injury Both In Vivo and In Vitro.

Cerebral ischemia/reperfusion injury plays an important role in the development of tissue injury after acute stroke, including neutrophils adhesion and infiltration, inflammation and oxidative stress. 10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H) is a novel ginkdolide B derivative. In this study, we investigated the anti-inflammatory and anti-oxidative activities of XQ-1H in vivo and vitro. In our study, rats were treating with XQ-1H (31.2, 15.6 and 7.8 mg/kg) after middle cerebral artery occlusion surgery. Primary cultured cortical rat neurons were treated with Na2S2O4 for 1.5 h to mimic hypoxia and reoxygenation injury in vitro. Cortical neurons were preincubated with XQ-1H (100, 10, 1 µM) 24 h before hypoxic injury. Brain edema was evaluated by brain water content. Neutrophil infiltration was determined by fluorescence imaging method and myeloperoxidase assay. Intercellular adhesion molecule 1 (ICAM-1) and matrix metallopeptidase 9 (MMP-9) expressions were examined by immunohistochemistry analysis. Neuronal injury was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, lactate dehydrogenase releasing and lactic acid content. The anti-oxidative effects of XQ-1H were evaluated by superoxide dismutase (SOD) activity and malondialdehyde content in ischemic brain and neuron cultures subjected to hypoxia/reoxygenation procedure. Results showed that XQ-1H reduced neutrophils infiltration to ischemic brain, which might result from down regulation of inflammatory mediators, such as ICAM-1 and MMP-9. In addition, an antioxidative effect of XQ-1H was observed in cortical neuron and brain homogenates by enhancing SOD activity and inhibiting lipid peroxidation. These results indicated that XQ-1H possessed a protective effect against cerebral ischemia, especially on neutrophil infiltration and oxidative stress.

Clematichinenoside attenuates myocardial infarction in ischemia/reperfusion injury both in vivo and in vitro.

Clematichinenoside is a triterpenoid saponin isolated from the roots of Clematis chinensis. Oxidative stress and excessive nitric oxide production are thought to play considerable roles in ischemia/reperfusion injury that impairs cardiac function. The present study investigated the protective effect of clematichinenoside on regional and global ischemia/reperfusion injury and ventricular myocytes. In vivo, regional myocardial ischemia/reperfusion injury of rats was induced by the occlusion of the left anterior descending coronary artery, and isolated guinea pigs heart using Langendorff apparatus served as a global ischemia/reperfusion injury model ex vivo. Primary cultured neonatal ventricular myocytes were further applied to explore the anti-ischemia/reperfusion injury property in vitro. Infarct size was measured with TTC stain; enzyme activities such as lactate dehydrogenase, creatine kinase, superoxide dismutase, malondialdehyde, and nitric oxide were analyzed with assay kits; inducible nitric oxide synthase and endothelial nitric oxide synthase expressions were determined by Western blot. Clematichinenoside attenuated infarct size, decreased lactate dehydrogenase, creatine kinase, and malondialdehyde levels and enhanced superoxide dismutase activity. Clematichinenoside improved hemodynamics indexes, such as left ventricular developed pressure, maximum left ventricular developed pressure, and increase/decrease rate (± dp/dtmax) in the isolated guinea pig heart after reperfusion. Clematichinenoside also inhibited excessive production of nitric oxide through downregulating inducible nitric oxide synthase as well as upregulating endothelial nitric oxide synthase during ischemia/reperfusion injury. Clematichinenoside attenuates ischemia/reperfusion injury in vivo, ex vivo, and in vitro via an antioxidant effect and by restoring the balance between inducible nitric oxide synthase and endothelial nitric oxide synthase.

Therapeutic neuroprotective effects of XQ-1H in a rat model of permanent focal cerebral ischemia.

Cerebral ischemia is one of the leading causes for death and severe disabilities in the world. XQ-1H exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after transient focal cerebral ischemia. The present study evaluated the dose effectiveness and therapeutic time window of neuroprotection of XQ-1H by behavioral and histological measures in rats subjected to permanent middle cerebral artery occlusion (pMCAO). Neurological deficits, TTC stain, brain water content, necrosis neuron counts, and Evans-Blue extravasation were used to quantify brain damage and blood-brain barrier dysfunction. Our results demonstrated that postischemic treatment with XQ-1H at a dose of 31.2 mg/kg produced a significant reduction in neurological scores when treatment was initiated within 2 h of pMCAO. A similar improvement was also observed in infarct volume, brain water content, Evans-Blue extravasation, and neuronal necrosis when treatment was initiated within 1 h of pMCAO. Treatment with XQ-1H at the dose of 15.6 mg/kg within 1 h also produced significant improvement in ischemia deficit. In conclusion, the therapeutic time window of XQ-1H extends for up to 1 h after pMCAO, and treatment with XQ-1H exhibits potent neuroprotection that may be of value for the design of stroke therapies.

Attenuated Blood-Brain Barrier Dysfunction by XQ-1H Following Ischemic Stroke in Hyperlipidemic Rats.

Following ischemic stroke, blood-brain barrier (BBB) is disrupted and is further aggravated with the corresponding incidence of hyperlipidemia. BBB breakdown promotes inflammation infiltration into the brain, which exacerbates cerebral ischemic injury as a result. Here, we report that 10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H), a novel analog of ginkgolide B, alleviates BBB breakdown in hyperlipidemic rats and protects endothelial cells against inflammatory response. Middle cerebral artery occlusion (MCAO) modeled ischemic stroke in rats. Before surgery, these rats were fed a cholesterol-rich diet to induce an experimental hyperlipidemic condition. Additionally, lipopolysaccharide (LPS) incubation with rat brain microvessel endothelial cells (rBMECs) was applied to mimic hyperlipidemia-induced inflammatory injury of BBB. The results indicated more severe infarct size, increased BBB permeability, excessive secretion of pro-inflammatory cytokines, and exaggerated inflammation infiltration of the brain in hyperlipidemic rats following MCAO when compared to rats fed with normal diet. XQ-1H protected BBB integrity, lessoned brain edema and inflammation penetration, downregulated MMP-9 and VCMA-1 expressions, and extenuated ischemic infarction. XQ-1H alleviated LPS-induced inflammatory response in rBMECs, characterized by promoting cell viability, inhibiting TNF-α, IL-1ß, and IL-6 releasing, and downregulating NF-κB inflammatory signal and downstream proteins, such as VCAM-1 and iNOS. In conclusion, the present study shows that XQ-1H stabilizes BBB function following ischemic stroke in hyperlipidemic rats, and the possible mechanisms may be related to inflammation inhibition.

Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation.

BACKGROUND: Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented. AIMS: This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro. METHODS: Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na(2)S(2)O(4) induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and (125)I-albumin were used to quantify BBB dysfunction; UPLC-MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp. RESULTS: Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and (125)I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury. CONCLUSION: These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.

Therapeutic neuroprotective effects of ginkgolide B on cortex and basal ganglia in a rat model of transient focal ischemia.

Cerebral ischemia and reperfusion is one of the leading causes for death and severe disabilities in the world and often lead to irreversible brain damage over later lifespan. The aim of this study was to investigate the evolution of pathological damage in cerebral cortex and basal ganglia following ischemia and to evaluate the therapeutic neuroprotective effect of ginkgolide B in a rat model of stroke induced by middle cerebral artery occlusion (MCAO). TTC stain, brain water content and Evans-Blue extravasation were used to quantify brain damage. Our results demonstrated that basal ganglia undergo progressive pathological damage earlier following MCAO, and injury was stable and irreversible after 5 h following ischemia. However, onset of ischemia injury in cerebral cortex appeared later than basal ganglia and became evident about 3 h following MCAO, and injury was stable and irreversible after 6 h following ischemia. Blood brain barrier opened progressively, and it seemed to be significantly destroyed after 4 h following MCAO comparing with 0 h. Post-ischemic treatment with ginkgolide B improved neurological function and reduced infarct size in basal ganglia within 3 h and cerebral cortex within 5 h following MCAO. The therapeutic effect of ginkgolide B on extenuate brain edema and decrease blood brain barrier permeability were extended for 5h after ischemia, and more evident reversal effect were observed when administrated at earlier time.