Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1ß Transcription.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1ß, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1ß transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1ß mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1ß, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1ß siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1ß transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

Clinacanthus nutans Mitigates Neuronal Apoptosis and Ischemic Brain Damage Through Augmenting the C/EBPß-Driven PPAR-γ Transcription.

Clinacanthus nutans Lindau (C. nutans) is a traditional herbal medicine widely used in Asian countries for treating a number of remedies including snake and insect bites, skin rashes, viral infections, and cancer. However, the underlying molecular mechanisms for its action and whether C. nutans can offer protection on stroke damage in brain remain largely unknown. In the present study, we demonstrated protective effects of C. nutans extract to ameliorate neuronal apoptotic death in the oxygen-glucose deprivation model and to reduce infarction and mitigate functional deficits in the middle cerebral artery occlusion model, either administered before or after hypoxic/ischemic insult. Using pharmacological antagonist and siRNA knockdown approaches, we demonstrated ability for C. nutans extract to protect neurons and ameliorate ischemic injury through promoting the anti-apoptotic activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ), a stress-induced transcription factor. Reporter and chromatin immunoprecipitation promoter analysis further revealed C. nutans extract to selectively increase CCAAT/enhancer binding protein (C/EBP)ß binding to specific C/EBP binding site (-332~-325) on the PPAR-γ promoter to augment its transcription. In summary, we report a novel transcriptional activation involving C/EBPß upregulation of PPAR-γ expression to suppress ischemic neuronal apoptosis and brain infarct. Recognition of C. nutans to enhance the C/EBPߠ→ PPAR-γ neuroprotective signaling pathway paves a new way for future drug development for prevention and treatment of ischemic stroke and other neurodegenerative diseases.

Expression of DHA-Metabolizing Enzyme Alox15 is Regulated by Selective Histone Acetylation in Neuroblastoma Cells.

The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) is enriched in neural membranes of the CNS, and recent studies have shown a role of DHA metabolism by 15-lipoxygenase-1 (Alox15) in prefrontal cortex resolvin D1 formation, hippocampo-prefrontal cortical long-term-potentiation, spatial working memory, and anti-nociception/anxiety. In this study, we elucidated epigenetic regulation of Alox15 via histone modifications in neuron-like cells. Treatment of undifferentiated SH-SY5Y human neuroblastoma cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate significantly increased Alox15 mRNA expression. Moreover, Alox15 expression was markedly upregulated by Class I HDAC inhibitors, MS-275 and depsipeptide. Co-treatment of undifferentiated SH-SY5Y cells with the p300 histone acetyltransferase (HAT) inhibitor C646 and TSA or sodium butyrate showed that p300 HAT inhibition modulated TSA or sodium butyrate-induced Alox15 upregulation. Differentiation of SH-SY5Y cells with retinoic acid resulted in increased neurite outgrowth and Alox15 mRNA expression, while co-treatment with the p300 HAT inhibitor C646 and retinoic acid modulated the increases, indicating a role of p300 HAT in differentiation-associated Alox15 upregulation. Increasing Alox15 expression was found in primary murine cortical neurons during development from 3 to 10 days-in-vitro, reaching high levels of expression by 10 days-in-vitro-when Alox15 was not further upregulated by HDAC inhibition. Together, results indicate regulation of Alox15 mRNA expression in neuroblastoma cells by histone modifications, and increasing Alox15 expression in differentiating neurons. It is possible that one of the environmental influences on the immature brain that can affect cognition and memory, may take the form of epigenetic effects on Alox15 and metabolites of DHA.

Clinacanthus nutans Extracts Modulate Epigenetic Link to Cytosolic Phospholipase A2 Expression in SH-SY5Y Cells and Primary Cortical Neurons.

Clinacanthus nutans Lindau (C. nutans), commonly known as Sabah Snake Grass in southeast Asia, is widely used in folk medicine due to its analgesic, antiviral, and anti-inflammatory properties. Our recent study provided evidence for the regulation of cytosolic phospholipase A2 (cPLA2) mRNA expression by epigenetic factors (Tan et al. in Mol Neurobiol. doi: 10.1007/s12035-015-9314-z , 2015). This enzyme catalyzes the release of arachidonic acid from glycerophospholipids, and formation of pro-inflammatory eicosanoids or toxic lipid peroxidation products such as 4-hydroxynonenal. In this study, we examined the effects of C. nutans ethanol leaf extracts on epigenetic regulation of cPLA2 mRNA expression in SH-SY5Y human neuroblastoma cells and mouse primary cortical neurons. C. nutans modulated induction of cPLA2 expression in SH-SY5Y cells by histone deacetylase (HDAC) inhibitors, MS-275, MC-1568, and TSA. C. nutans extracts also inhibited histone acetylase (HAT) activity. Levels of cPLA2 mRNA expression were increased in primary cortical neurons subjected to 0.5-h oxygen-glucose deprivation injury (OGD). This increase was significantly inhibited by C. nutans treatment. Treatment of primary neurons with the HDAC inhibitor MS-275 augmented OGD-induced cPLA2 mRNA expression, and this increase was modulated by C. nutans extracts. OGD-stimulated increase in cPLA2 mRNA expression was also reduced by a Tip60 HAT inhibitor, NU9056. In view of a key role of cPLA2 in the production of pro-inflammatory eicosanoids and free radical damage, and the fact that epigenetic effects on genes are often long-lasting, results suggest a role for C. nutans and phytochemicals to inhibit the production of arachidonic acid-derived pro-inflammatory eicosanoids and chronic inflammation, through epigenetic regulation of cPLA2 expression.

Clinacanthus nutans Protects Cortical Neurons Against Hypoxia-Induced Toxicity by Downregulating HDAC1/6.

Many population-based epidemiological studies have unveiled an inverse correlation between intake of herbal plants and incidence of stroke. C. nutans is a traditional herbal medicine widely used for snake bite, viral infection and cancer in Asian countries. However, its role in protecting stroke damage remains to be studied. Despite of growing evidence to support epigenetic regulation in the pathogenesis and recovery of stroke, a clear understanding of the underlying molecular mechanisms is still lacking. In the present study, primary cortical neurons were subjected to in vitro oxygen-glucose deprivation (OGD)-reoxygenation and hypoxic neuronal death was used to investigate the interaction between C. nutans and histone deacetylases (HDACs). Using pharmacological agents (HDAC inhibitor/activator), loss-of-function (HDAC siRNA) and gain-of-function (HDAC plasmid) approaches, we demonstrated an early induction of HDAC1/2/3/8 and HDAC6 in neurons after OGD insult. C. nutans extract selectively inhibited HDAC1 and HDAC6 expression and attenuated neuronal death. Results of reporter analysis further revealed that C. nutans suppressed HDAC1 and HDAC6 transcription. Besides ameliorating neuronal death, C. nutans also protected astrocytes and endothelial cells from hypoxic-induced cell death. In summary, results support ability for C. nutans to suppress post-hypoxic HDACs activation and mitigate against OGD-induced neuronal death. This study further opens a new avenue for the use of herbal medicines to regulate epigenetic control of brain injury.

Anti-apoptotic actions of PPAR-gamma against ischemic stroke.

Stroke is a leading cause of adult disability and mortality. Diabetes is a major risk factor for stroke. Patients with diabetes have a higher incidence of stroke and a poorer prognosis after stroke. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-modulated transcriptional factor and a therapeutic target for treating type II diabetes. It is well-documented that activation of PPAR-gamma can also attenuate postischemic inflammation and damage. In this review, we focus on the newly revealed anti-apoptotic actions of PPAR-gamma against cerebral ischemia. PPAR-gamma, by increasing superoxide dismutase/catalase and decreasing nicotinamide adenine dinucleotide phosphate oxidase levels, attenuated ischemia-induced reactive oxygen species and subsequently alleviated the postischemic degradation of Bcl-2, Bcl-xl, and Akt. The preserved Akt phosphorylated Bad. Meanwhile, PPAR-gamma also promotes the transcription of 14-3-3epsilon. Elevated 14-3-3epsilon binds and sequesters p-Bad and prevents Bad translocation to neutralize the anti-apoptotic function of Bcl-2. This review further supports the notion that PPAR-gamma may serve as a potential therapeutic target for treating ischemic stroke.

Ligand-activated peroxisome proliferator-activated receptor-gamma protects against ischemic cerebral infarction and neuronal apoptosis by 14-3-3 epsilon upregulation.

BACKGROUND: Thiazolidinediones have been reported to protect against ischemia-reperfusion injury. Their protective actions are considered to be peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-dependent; however, it is unclear how PPAR-gamma activation confers resistance to ischemia-reperfusion injury. METHODS AND RESULTS: We evaluated the effects of rosiglitazone or PPAR-gamma overexpression on cerebral infarction in a rat model and investigated the antiapoptotic actions in the N2-A neuroblastoma cell model. Rosiglitazone or PPAR-gamma overexpression significantly reduced infarct volume. The protective effect was abrogated by PPAR-gamma small interfering RNA. In mice with knock-in of a PPAR-gamma dominant-negative mutant, infarct volume was enhanced. Proteomic analysis revealed that brain 14-3-3epsilon was highly upregulated in rats treated with rosiglitazone. Upregulation of 14-3-3epsilon was abrogated by PPAR-gamma small interfering RNA or antagonist. Promoter analysis and chromatin immunoprecipitation revealed that rosiglitazone induced PPAR-gamma binding to specific regulatory elements on the 14-3-3epsilon promoter and thereby increased 14-3-3epsilon transcription. 14-3-3epsilon Small interfering RNA abrogated the antiapoptotic actions of rosiglitazone or PPAR-gamma overexpression, whereas 14-3-3epsilon recombinant proteins rescued brain tissues and N2-A cells from ischemia-induced damage and apoptosis. Elevated 14-3-3epsilon enhanced binding of phosphorylated Bad and protected mitochondrial membrane potential. CONCLUSIONS: Ligand-activated PPAR-gamma confers resistance to neuronal apoptosis and cerebral infarction by driving 14-3-3epsilon transcription. 14-3-3epsilon Upregulation enhances sequestration of phosphorylated Bad and thereby suppresses apoptosis.

Rosiglitazone and PPAR-gamma overexpression protect mitochondrial membrane potential and prevent apoptosis by upregulating anti-apoptotic Bcl-2 family proteins.

To determine the involvement of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in cytoprotection, we subjected N2-A cells to oxygen-glucose deprivation followed by reoxygenation (H-R). Following H-R insults, H(2)O(2) production was increased while cell viability declined, which was accompanied by loss of mitochondrial membrane potential (MMP), cytochrome c release, caspases 9 and 3 activation, poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis. Rosiglitazone up to 5 microM protected cell viability, normalized MMP, and prevented apoptotic signals. The protective effect of rosiglitazone was abrogated by GW9662, a PPAR-gamma antagonist, or a specific PPAR-gamma small interference RNA (siRNA) but not a control scRNA. PPAR-gamma overexpression alone was effective in maintaining MMP and preventing apoptosis and its protective effect was also abrogated by PPAR-gamma siRNA or GW9662. To elucidate the mechanism by which PPAR-gamma protects MMP and prevents apoptosis, we analyzed Bcl-2, Bcl-xl, and phosphorylated Bad (p-Bad). H-R suppressed them. Rosiglitazone or PPAR-gamma overexpression restored them via PPAR-gamma. Rosiglitazone or PPAR-gamma overexpression preserved phosphorylated Akt and 3-phosphoinositide-dependent kinase-1 (PDK-1) in a PPAR-gamma dependent manner. These results indicate that ligand-activated PPAR-gamma protects N2-A cells against H-R damage by enhancing Bcl-2/Bcl-xl and maintaining p-Bad via preservation of p-Akt.

15d-prostaglandin J2 protects brain from ischemia-reperfusion injury.

OBJECTIVE: Brain expresses abundant lipocalin-type prostaglandin (PG) D2 (PGD2) synthase but the role of PGD2 and its metabolite, 15-deoxy-Delta(12,14) PGJ2 (15d-PGJ2) in brain protection is unclear. The aim of this study is to assess the effect of 15d-PGJ2 on neuroprotection. METHODS AND RESULTS: Adenoviral transfer of cyclooxygenase-1 (Adv-COX-1) was used to amplify the production of 15d-PGJ2 in ischemic cortex in a rat focal infarction model. Cortical 15d-PGJ2 in Adv-COX-1-treated rats was increased by 3-fold over control, which was correlated with reduced infarct volume and activated caspase 3, and increased peroxisome proliferator activated receptor-gamma (PPARgamma) and heme oxygenase-1 (HO-1). Intraventricular infusion of 15d-PGJ2 resulted in reduction of infarct volume, which was abrogated by a PPARgamma inhibitor. Rosiglitazone infusion had a similar effect. 15d-PGJ2 and rosiglitazone at low concentrations suppressed H2O2-induced rat or human neuronal apoptosis and necrosis and induced PPARgamma and HO-1 expression. The anti-apoptotic effect was abrogated by PPARgamma inhibition. CONCLUSIONS: 15d-PGJ2 suppressed ischemic brain infarction and neuronal apoptosis and necrosis in a PPARgamma dependent manner. 15d-PGJ2 may play a role in controlling acute brain damage induced by ischemia-reperfusion.

Induction of prostacyclin/PGI2 synthase expression after cerebral ischemia-reperfusion.

Prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet aggregation and leukocyte activation, is crucial in vascular diseases such as stroke. Prostacyclin synthase (PGIS) is the key enzyme for PGI2 synthesis. Although expression of PGIS was noted in the brain, its role in ischemic insult remains unclear. Here we reported the temporal and spatial expression of PGIS mRNA and protein after 60-min transient ischemia. Northern blot and in situ hybridization revealed a delayed increase of PGIS mRNA in the ischemic cortex at 24- to 72-h after ischemia; PGIS was detected mainly in the ipsilateral penumbra area, pyriform cortex, hippocampus, and leptomeninges. Western blot and immunohistochemical analysis revealed that PGIS proteins were expressed temporally and spatially similar to PGIS mRNA. PGIS was heavily colocalized with PECAM-1 to endothelial cells at the leptomeninges, large and small vessels, and localized to neuronal cells, largely at the penumbra area. A substantial amount of PGIS was also detected in the macrophage and glial cells. To evaluate its role against ischemic infarct, we overexpressed PGIS by adenoviral gene transfer. When infused 72 h before ischemia (- 72 h), Adv-PGIS reduced infarct volume by approximately 50%. However, it had no effect on infarct volume when infused immediately after ischemia (0 h). Eicosanoid analysis revealed selective elevation of PGI2 at - 72 h while PGI2 and TXB2 were both elevated at 0 h, altering the PGI2/thromboxane A2 (TXA2) ratio from 10 to 4. These findings indicate that PGIS protects the brain by enhancing PGI2 synthesis and creating a favorable PGI2/TXA2 ratio.

Mediating of caspase-independent apoptosis by cadmium through the mitochondria-ROS pathway in MRC-5 fibroblasts.

Cadmium (Cd) is an environmental pollutant of global concern with a 10-30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 microM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 microM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd.

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells.

Cadmium, a well-known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress-induced cell death. However, the molecular signaling underlying cadmium-induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC-5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 microM cadmium. Pretreatment with Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro-caspase-3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC-5 cells is caspase-independent. JC-1 staining also indicated that mitochondrial depolarization is a prelude to cadmium-induced apoptosis, which was accompanied by a translocation of caspase-independent pro-apoptotic factor apoptosis-inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase-independent apoptotic pathway through mitochondria-mediated AIF translocation into the nucleus.