Hung-Yi Chen

Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats.

We examined the neuroprotective action of a standardized extract of Ginkgo biloba leaves (EGb 761) in permanent and transient middle cerebral artery (MCA) occlusion models in Sprague‐Dawley rats. Forty‐four animals were given either EGb 761 (50–200 mg/kg) or vehicle intraperitoneally, 1 hr before permanent MCA occlusion, to evaluate the dose‐response effects. An additional 58 animals received EGb 761 (200 mg/kg) or vehicle, 0.5– 4 hr after permanent MCA occlusion, for establishing the therapeutic window. Delayed treatment was also employed in 110 animals treated with either EGb 761 (100–200 mg/kg) or vehicle at 2–3 hr following transient focal cerebral ischemia induced by MCA occlusion for 2 hr. Neurobehavioral scores were determined 22–24 hr after permanent MCA occlusion and either 3 or 7 days after transient MCA occlusion, and brain infarction volumes were measured upon sacrifice. Local cortical blood flow (LCBF) was serially measured in a subset of animals receiving EGb 761 (100–200 mg/kg) or vehicle, 0.5 hr and 2 hr after permanent and transient MCA occlusion, respectively. Relative to vehicle‐treated controls, rats pretreated with EGb761 (100 and 200 mg/kg) had significantly reduced infarct volumes, by 36% and 49%, respectively, and improved sensory behavior (P < 0.05). Delayed treatment with EGb 761 also significantly reduced brain infarction, by 20–29% and 31%, when given up to 2 and 3 hr following transient and permanent MCA occlusion, respectively, whereas improved neurobehavioral scores were noted up to 2 hr after the onset of MCA occlusion (P < 0.05). LCBF was significantly improved in the ipsilateral cortex following the EGb 761 treatment, whereas a higher dose showed a more sustained effect. In conclusion, EGb 761 protected against transient and permanent focal cerebral ischemia and was effective after a prolonged reperfusion period even when therapy is delayed up to 2 hr. This neuroprotection may be at least partially attributed to the beneficial effects of selectively improved LCBF in the area at risk of infarction.

Melatonin attenuates gray and white matter damage in a mouse model of transient focal cerebral ischemia

Abstract:  We have previously shown that melatonin reduces infarct volumes and enhances neurobehavioral and electrophysiological recoveries following transient middle cerebral artery (MCA) occlusion in rats. In the study, we examined whether melatonin would display neuroprotection against neuronal, axonal and oligodendrocyte pathology after 24 hr of reperfusion following 1 hr of MCA occlusion in mice. Melatonin (5 mg/kg) or vehicle was given intraperitoneally at the commencement of reperfusion. Neurological deficits were assessed 24 hr after ischemia. Gray matter damage was evaluated by quantitative histopathology. Axonal damage was determined with amyloid precursor protein and microtubule‐associated protein tau‐1 immunohistochemistry to identify postischemic disrupted axonal flow and oligodendrocyte pathology, respectively. Oxidative damage was assessed by 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) and 4‐hydroxynonenal (4‐HNE) immunohistochemistry. Relative to controls, melatonin‐treated animals not only had a significantly reduced volume of gray matter infarction by 42% (P < 0.001), but also exhibited a decreased score of axonal damage by 42% (P < 0.001) and a reduction in the volume of oligodendrocyte pathology by 58% (P < 0.005). Melatonin‐treated animals also had significantly reduced immunopositive reactions for 8‐OHdG and 4‐HNE by 53% (P < 0.001) and 49% (P < 0.001), respectively. In addition, melatonin improved sensory and motor neurobehavioral outcomes by 47 and 30%, respectively (P < 0.01). Thus, delayed (1 hr) treatment with melatonin reduced both gray and white matter damage and improved neurobehavioral outcomes following transient focal cerebral ischemia in mice. The finding of reduced oxidative damage observed with melatonin suggests that its major mechanisms of action are mediated through its antioxidant and radical scavenging activity.

Intravenous administration of melatonin reduces the intracerebral cellular inflammatory response following transient focal cerebral ischemia in rats

Abstract:  We have previously shown that exogenous melatonin improves the preservation of the blood–brain barrier (BBB) and neurovascular unit following cerebral ischemia–reperfusion. Recent evidence indicates that postischemic microglial activation exaggerates the damage to the BBB. Herein, we explored whether melatonin mitigates the cellular inflammatory response after transient focal cerebral ischemia for 90 min in rats. Melatonin (5 mg/kg) or vehicle was given intravenously at reperfusion onset. Immunohistochemistry and flow cytometric analysis were used to evaluate the cellular inflammatory response at 48 hr after reperfusion. Relative to controls, melatonin‐treated animals did not have significantly changed systemic cellular inflammatory responses in the bloodstream (P > 0.05). Melatonin, however, significantly decreased the cellular inflammatory response by 41% (P < 0.001) in the ischemic hemisphere. Specifically, melatonin effectively decreased the extent of neutrophil emigration (Ly6G‐positive/CD45‐positive) and macrophage/activated microglial infiltration (CD11b‐positive/CD45‐positive) by 51% (P < 0.01) and 66% (P < 0.01), respectively, but did not significantly alter the population composition of T lymphocyte (CD3‐positive/CD45‐positive; P > 0.05). This melatonin‐mediated decrease in the cellular inflammatory response was accompanied by both reduced brain infarction and improved neurobehavioral outcome by 43% (P < 0.001) and 50% (P < 0.001), respectively. Thus, intravenous administration of melatonin upon reperfusion effectively decreased the emigration of circulatory neutrophils and macrophages/monocytes into the injured brain and inhibited focal microglial activation following cerebral ischemia–reperfusion. The finding demonstrates melatonins inhibitory ability against the cellular inflammatory response after cerebral ischemia–reperfusion, and further supports its pleuripotent neuroprotective actions suited either as a monotherapy or an add‐on to the thrombolytic therapy for ischemic stroke patients.

Cantharidin induces G2/M phase arrest and apoptosis in human colorectal cancer colo 205 cells through inhibition of CDK1 activity and caspase-dependent signaling pathways

Cantharidin (CTD) is a traditional Chinese medicine and an effective component isolated from blister beetle, and it has been demonstrated to have anticancer, antibiotic, antivirus activities and immune-regulated functions. It has been reported that CTD induces cell cycle arrest and apoptosis in many cancer cell types. However, there are no reports showing that CTD would induce cell cycle arrest and apoptosis in human colorectal cancer colo 205 cells. In this study, we studied colo 205 cells which were treated with CTD and demonstrated its molecular mechanisms in apoptosis. CTD induced growth inhibition, G2/M phase arrest and apoptosis in colo 205 cells. The IC50 is 20.53 µM in CTD-treated colo 205 cells. DAPI/TUNEL double staining and Annexin V assays were used to confirm the apoptotic cell death in colo 205 cells after CTD exposure. CTD caused G2/M arrest, down-regulated CDK1 activity, decreased Cyclin A, Cyclin B, CDK1 and increased CHK1 and p21 protein levels. Colorimetric assays also indicated that CTD triggered activities of casapse-8, -9 and -3 in colo 205 cells. Moreover, CTD increased ROS production and decreased the level of mitochondrial membrane potential (ΔΨm) in colo 205 cells. Consequently, CTD-induced growth inhibition was significantly attenuated by N-acetylcysteine (NAC, a scavenger). CTD stimulated the protein levels of Fas/CD95, the caspase-3 active form, cytochrome c and Bax, but suppressed the protein levels of pro-caspase-8, pro-caspase-9 and Bcl-2, determined by Western blot analysis. Based on our observations, we suggest that CTD is able to induce G2/M phase arrest and apoptosis in colo 205 cells through inhibition of CDK1 activity and caspase-dependent signaling pathways.

MELATONIN DECREASES NEUROVASCULAR OXIDATIVE/NITROSATIVE DAMAGE AND PROTECTS AGAINST EARLY INCREASES IN THE BLOOD–BRAIN BARRIER PERMEABILITY AFTER TRANSIENT FOCAL CEREBRAL ISCHEMIA IN MICE

Abstract:  We have recently shown that melatonin decreases the late (24 hr) increase in blood–brain barrier (BBB) permeability and the risk of tissue plasminogen activator‐induced hemorrhagic transformation following ischemic stroke in mice. In the study, we further explored whether melatonin would reduce postischemic neurovascular oxidative/nitrosative damage and, therefore, improve preservation of the early increase in the BBB permeability at 4 hr after transient focal cerebral ischemia for 60 min in mice. Melatonin (5 mg/kg) or vehicle was given intraperitoneally at the beginning of reperfusion. Hydroethidine (HEt) in situ detection and immunohistochemistry for nitrotyrosine were used to evaluate postischemic accumulation in reactive oxygen and nitrogen species, respectively, in the ischemic neurovascular unit. BBB permeability was evaluated by spectrophotometric and microscopic quantitation of Evans Blue leakage. Relative to controls, melatonin‐treated animals not only had a significantly reduced superoxide accumulation in neurovascular units in boundary zones of infarction, by reducing 35% and 54% cytosolic oxidized HEt in intensity and cell‐expressing percentage, respectively (P < 0.001), but also exhibited a reduction in nitrotyrosine by 52% (P < 0.01). Additionally, melatonin‐treated animals had significantly reduced early postischemic disruption in the BBB permeability by 53% (P < 0.001). Thus, melatonin reduced postischemic oxidative/nitrosative damage to the ischemic neurovascular units and improved the preservation of BBB permeability at an early phase following transient focal cerebral ischemia in mice. The findings further highlight the ability of melatonin in anatomical and functional preservation for the ischemic neurovascular units and its relevant potential in the treatment of ischemic stroke.

Melatonin attenuates the postischemic increase in blood-brain barrier permeability and decreases hemorrhagic transformation of tissue-plasminogen activator therapy following ischemic stroke in mice

Abstract:  Melatonin protects against transient middle cerebral artery (MCA) occlusion and may be suited as an add‐on therapy of tissue plasminogen activator (t‐PA) thrombolysis. Herein, we examined whether melatonin would reduce postischemic increase in the blood–brain barrier (BBB) permeability and, therefore, attenuate the risk of hemorrhagic transformation after t‐PA therapy in experimental stroke. Twelve mice were subjected to transient occlusion of the MCA for 1 hr, followed by 24 hr of reperfusion. Melatonin (5 mg/kg, i.p.) or vehicle was given at the beginning of reperfusion. BBB permeability was evaluated by quantitation of Evans Blue leakage. An additional 32 mice underwent photothrombotic occlusion of the distal MCA, and were administered vehicle or t‐PA (10 mg/kg, i.v.), alone or in combination with melatonin (5 mg/kg, i.p.), at 6 hr postinsult. The animals were then killed after 24 hr for the determination of infarct and hemorrhage volumes. Relative to controls, melatonin‐treated animals had significantly reduced BBB permeability (by 52%; P < 0.001). Additionally, we found that at 6 hr after photo‐irradiation, either t‐PA or melatonin, or a combined administration of t‐PA plus melatonin, did not significantly affect brain infarction (P > 0.05), compared with controls. Mice treated with t‐PA alone, however, had significantly increased hemorrhagic formation (P < 0.05), and the event was effectively reversed by co‐treatment with melatonin (P < 0.05). Thus, melatonin improved postischemic preservation of the BBB permeability and a decreased risk of adverse hemorrhagic transformation after t‐PA therapy for ischemic stroke. The findings further highlight melatonins potential role in the field of thrombolytic treatment for ischemic stroke patients.

Gallic acid inhibits the migration and invasion of A375.S2 human melanoma cells through the inhibition of matrix metalloproteinase-2 and Ras

Melanoma is one of the most common cancers worldwide and its incidence has been increasing over the past few decades. Gallic acid (GA) can inhibit the growth of human cancer cells in vitro and in vivo. However, there is no available information to address the effects of GA on migration and invasion of human skin cancer cells. Matrix metalloproteinases (MMPs), zinc-dependent proteolytic enzymes, play an important role in the invasion, metastasis, and angiogenesis of cancer cells. Therefore, MMPs are one of the targets for agents to suppress and that could inhibit the migration and invasion of cancer cells. GA affected the viable A375.S2 cells by propidium iodide exclusion and flow cytometric analysis. Cell migration and invasion were investigated by Boyden chamber assay and we also determined the levels of protein and mRNA expression cell migration and invasion by gelatin zymography, western blotting, and real-time PCR assays. In this study, we examined the influence of GA on the protein levels and gene expression of MMP-2 and MMP-9 and in-vitro migration and invasiveness of human melanoma cells. GA decreases the MMPs and associated signal pathway protein and MMPs mRNA levels in A375.S2 human melanoma cells. Our findings suggest that GA has antimetastatic potential by decreasing invasiveness of cancer cells. Moreover, this action of GA was involved in the Ras, p-ERK signaling pathways resulting in inhibition of MMP-2 in A375.S2 human melanoma cells. These data, therefore, provide evidence for the role of GA as a potential cancer chemotherapeutic agent, which can markedly inhibit the invasive capacity of melanoma cells.

Melatonin decreases matrix metalloproteinase‐9 activation and expression and attenuates reperfusion‐induced hemorrhage following transient focal cerebral ischemia in rats

Abstract:  We have previously shown that melatonin reduces postischemic rises in the blood–brain barrier (BBB) permeability and improves neurovascular dysfunction and hemorrhagic transformation following ischemic stroke. It is known that activation of the matrix metalloproteinases (MMPs) plays a crucial role in the pathogenesis of brain edema and hemorrhagic transformation after ischemic stroke. We, herein, investigated whether melatonin would ameliorate MMP‐2 and MMP‐9 activation and expression in a rat model of transient focal cerebral ischemia. Adult male Sprague–Dawley rats were subjected to a 90‐min middle cerebral artery (MCA) occlusion using an intraluminal filament. Melatonin (5 mg/kg) or vehicle was intravenously injected upon reperfusion. Brain infarction and hemorrhage within infarcts were measured, and neurological deficits were scored. The activity and expression of MMP‐2 and MMP‐9 were determined by zymography, in situ zymography and Western immunoblot analysis. Cerebral ischemia–reperfusion induced increased pro‐MMP‐9 and MMP‐9 activity and expression 24 hr after reperfusion onset. Relative to controls, melatonin‐treated animals, however, had significantly reduced levels in the MMP‐9 activity and expression (P < 0.01), in addition to reduced brain infarct volume and hemorrhagic transformation as well as improved sensorimotor neurobehavioral outcomes. No significant change in MMP‐2 activity was observed throughout the course experiments. Our results indicate that the melatonin‐mediated reductions in ischemic brain damage and reperfusion‐induced hemorrhage are partly attributed to its ability to reduce postischemic MMP‐9 activation and increased expression, and further support the fact that melatonin is a suitable as an add‐on to thrombolytic therapy for ischemic stroke patients.

Melatonin inhibits postischemic matrix metalloproteinase-9 (MMP-9) activation via dual modulation of plasminogen/plasmin system and endogenous MMP inhibitor in mice subjected to transient focal cerebral ischemia

Abstract:  We have shown that melatonin attenuated matrix metalloproteinase‐9 (MMP‐9) activation and decreased the risk of hemorrhagic transformation following cerebral ischemia‐reperfusion. Herein, we investigate the possible involvement of the plasminogen/plasmin system and endogenous MMPs inhibitor underlying the melatonin‐mediated MMP‐9 inhibition. Mice were subjected to 1‐hr ischemia and 48‐hr reperfusion of the right middle cerebral artery. Melatonin (5 mg/kg) or vehicle was intravenously injected upon reperfusion. Brain infarction and hemorrhagic transformation were measured. Extracellular matrix damage was determined by Western immunoblot analysis for laminin protein. The activity and expression of MMP‐2 and MMP‐9 were determined by gelatin zymography, in situ zymography, and Western immunoblot analysis. In addition, the activities of tissue and urokinase plasminogen activators (tPA and uPA) were evaluated by plasminogen‐dependent casein zymography. Endogenous plasminogen activator inhibitor (PAI) and tissue inhibitors of MMP (TIMP‐1) were investigated using enzyme‐linked immunosorbent assay (ELISA) and Western immunoblot analysis, respectively. Cerebral ischemia‐reperfusion induced increased MMP‐9 activity and expression at 12–48 hr after reperfusion onset. Relative to controls, melatonin‐treated animals had significantly decreased MMP‐9 activity and expression (P < 0.05), in addition to reduced brain infarction and hemorrhagic transformation as well as improved laminin protein preservation. This melatonin‐mediated MMP‐9 inhibition was accompanied by reduced uPA activity (P < 0.05), as well as increased TIMP‐1 expression and PAI activity (P < 0.05, respectively). These results demonstrate the melatonin’s pluripotent mechanisms for attenuating postischemic MMP‐9 activation and neurovascular damage, and further support it as an add‐on to thrombolytic therapy for ischemic stroke patients.

The roles of endoplasmic reticulum stress and mitochondrial apoptotic signaling pathway in quercetin-mediated cell death of human prostate cancer PC-3 cells

Prostate cancer has its highest incidence and is becoming a major concern. Many studies have shown that traditional Chinese medicine exhibited antitumor responses. Quercetin, a natural polyphenolic compound, has been shown to induce apoptosis in many human cancer cell lines. Although numerous evidences show multiple possible signaling pathways of quercetin in apoptosis, there is no report to address the role of endoplasmic reticulum (ER) stress in quercetin‐induced apoptosis in PC‐3 cells. The purpose of this study was to investigate the effects of quercetin on the induction of the apoptotic pathway in human prostate cancer PC‐3 cells. Cells were treated with quercetin for 24 and 48 h and at various doses (50–200 μM), and cell morphology and viability decreased significantly in dose‐dependent manners. Flow cytometric assay indicated that quercetin at 150 μM caused G0/G1 phase arrest (31.4–49.7%) and sub‐G1 phase cells (19.77%) for 36 h treatment and this effect is a time‐dependent manner. Western blotting analysis indicated that quercetin induces the G0/G1 phase arrest via decreasing the levels of CDK2, cyclins E, and D proteins. Quercetin also stimulated the protein expression of ATF, GRP78, and GADD153 which is a hall marker of ER stress. Furthermore, PC‐3 cells after incubation with quercetin for 48 h showed an apoptotic cell death and DNA damage which are confirmed by DAPI and Comet assays, leading to decrease the antiapoptotic Bcl‐2 protein and level of ΔΨm, and increase the proapoptotic Bax protein and the activations of caspase‐3, ‐8, and ‐9. Moreover, quercetin promoted the trafficking of AIF protein released from mitochondria to nuclei. These data suggest that quercetin may induce apoptosis by direct activation of caspase cascade through mitochondrial pathway and ER stress in PC‐3 cells.