Effects of isosakuranetin on cerebral infarction and blood brain barrier damage from cerebral ischemia/reperfusion injury in a rat model.

This study investigated the effects of isosakuranetin (5,7-dihydroxy-4'-methoxyflavanone) on cerebral infarction and blood brain barrier (BBB) damage in cerebral ischemia and reperfusion (I/R) in a rat model. The right middle cerebral artery was occluded for 2 h followed by reperfusion. The experimental rats were divided into five groups: a sham, or control group; vehicle group; and 5 mg/kg, 10 mg/kg, and 20 mg/kg bodyweight isosakuranetin-treated I/R groups. After 24 h of reperfusion, the rats were tested using a six-point neurological function score. The percentage of cerebral infarction was evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining. BBB leakage was determined by Evan Blue injection assay and brain morphology changes were observed under light microscopy following staining with hematoxylin and eosin (H&E). The results of neurological function score revealed that isosakuranetin reduced the severity of neurological damage. A dose of 10 and 20 mg/kg bodyweight of isosakuranetin significantly decreased the infarct volume. All three doses of isosakuranetin significantly decreased Evan Blue leakage. The penumbra area of the I/R brains revealed the characteristics of apoptotic cell death. Therefore, isosakuranetin-treated I/R attenuated the brain damage from cerebral I/R injury and further investigation of the mechanisms warrant further investigation to assist in the development of protective strategies against cerebral I/R injury in clinical trials.Communicated by Ramaswamy H. Sarma.

5,6,7,4'-Tetramethoxyflavanone attenuates NADPH oxidase 1/4 and promotes sirtuin-1 to inhibit cell stress, senescence and apoptosis in Aß25-35-mediated SK-N-SH dysfunction.

Amyloidogenesis is a fundamental step of amyloid beta (Aß) generation-induced toxicity that is commonly reported to disrupt neuronal circuits, function and survival in Alzheimer's disease (AD). The neuroprotective effect of 5,6,7,4'-tetramethoxyflavanone (TMF) from Chormolaela odorata extract on brain degeneration and amyloidogenesis has previously been demonstrated. However, the mechanistic evidence for TMF's effects is still unclear. In this study, we evaluated the neuroprotective effect of TMF in Aß25-35-induced toxicity in SK-N-SH neuroblastoma cells. Herein, we demonstrated that TMF exhibited potent antioxidant activity and significantly increased cell viability and decreased ROS production in a dose-dependent manner. Moreover, TMF reversed the effect of Aß25-35, which caused energy deprivation and apoptosis, by decreasing the ratio of Bax/Bcl-xL and reducing mitochondrial membrane potential (Δψm), caspase-3 expression, apoptotic cells, and attenuating glucose transporter (Glut-3) expression. In addition, TMF protected against Aß25-35-induced cellular senescence by attenuating ß-galactosidase, p-21 and p-53 expression and promoted the expression of Sirt-1 and p-Rb. In addition, the effects of TMF on Aß25-35 toxicity were related to the upregulation of phase II antioxidant and nuclear factor erythroid 2-related factor-2 (Nrf2) signaling, including superoxide dismutase (SOD), heme oxygenase (HO)-1, and nuclear translocation of Nrf2. Finally, we also found that TMF attenuated Aß25-35-reduced synaptic plasticity by increasing the expression of synaptophysin and PSD-95, which was correlated with a decrease in acetylcholine esterase (AChE). Importantly, we found that the protective effects of TMF on Aß25-35 were bidirectional, including marked inhibition of NADPH oxidase (NOX)-4 activity and partial activation of Sirt-1, which occurred prior to a reduction in the negative responses. Therefore, TMF may be useful for treating Aß toxicity in AD.

The antiaging property of aqueous extract of Millingtonia hortensis flowers in aging neuron.

Cellular senescence is the key mediator of cellular dysfunction before undergoing degenerative disease such as Alzheimer's disease. The aging process was mainly by the overactivation of senescence associated ß-galactosidase (SA-ß-gal) enzyme before mediated several negative responses, including intracellular reactive oxygen species (ROS) production, cellular senescence regulation, and death prior encourage synaptic loss. Thus, in the recent work, we evaluated the in vitro effects of aqueous extract of Millingtonia hortensis L. (MH) from flower in hydrogen peroxide (H2O2)-induced senescence in SK-N-SH cells. Herein, we demonstrated that MH significantly increased cell viability and decreased both of apoptotic cells and ROS production in a dose-dependent manner comparing to aging group (P < 0.01) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, and ROS assay. Furthermore, the number of SA-ß-gal-positive cells was also reduced in MH treatment (P < 0.01) together with the promotion of Sirt-1 protein. Importantly, MH also promoted the synaptic plasticity by decreased acetylcholinesterase activity and increased synaptophysin expression in aging neurons comparing to aging group (P < 0.01). Hispidulin (the active ingredient in MH) was also revealed the similarly effects to MH. Therefore, we suggested that MH might be beneficially for neurodegenerative disease that caused by aging.

Phonopheresis Associated with Nanoparticle Gel from Phyllanthus amarus Relieves Pain by Reducing Oxidative Stress and Proinflammatory Markers in Adults with Knee Osteoarthritis / 中国结合医学杂志

OBJECTIVE@#To determine the changes in serum levels of inflammatory biomarkers and antioxidant levels among the knee osteoarthritis (OA) patients after treatment with Phyllanthus amarus (PP) by nanoparticle gel phonophoresis.@*METHODS@#This study was a randomized, double-blind, placebo-control, parallel-group, clinical trial involving 30 subjects with mild-to-moderate degree of knee OA. The patients were allocated to two groups using a computer-generated random numbers, and received conventional ultrasound therapy (control group, 15 cases) and PP (treatment group, 15 cases) once daily for 10 sessions. The pain was evaluated by visual analogue scale (VAS). Serum levels of tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbnent assay (ELISA). Nitric oxide (NO) was determined by modified Griess reagent. The antioxidant effects, including superoxide dismutase (SOD) and total antioxidant capacity (TAC), were also measured by ELISA assay.@*RESULTS@#The VAS score was significantly decreased in the treatment group compared with the control group after treatment (P<0.01). The serum concentrations of TNF-α and NO were significantly reduced in the treatment group compared with the control group (P<0.01) after treatment. However, the serum concentrations of SOD and TAC in the treatment group were significantly higher after treatment compared with the control group (P<0.01).@*CONCLUSION@#PP could alleviate knee pain and significantly reduce systemic anti-inflammatory effects in knee OA patients.

Cyanidin attenuates Aß25-35-induced neuroinflammation by suppressing NF-κB activity downstream of TLR4/NOX4 in human neuroblastoma cells.

Cyanidin is polyphenolic pigment found in plants. We have previously demonstrated that cyanidin protects nerve cells against Aß25-35-induced toxicity by decreasing oxidative stress and attenuating apoptosis mediated by both the mitochondrial apoptotic pathway and the ER stress pathway. To further elucidate the molecular mechanisms underlying the neuroprotective effects of cyanidin, we investigated the effects of cyanidin on neuroinflammation mediated by the TLR4/NOX4 pathway in Aß25-35-treated human neuroblastoma cell line (SK-N-SH). SK-N-SH cells were exposed to Aß25-35 (10 µmol/L) for 24 h. Pretreatment with cyanidin (20 µmol/L) or NAC (20 µmol/L) strongly inhibited the NF-κB signaling pathway in the cells evidenced by suppressing the degradation of IκBα, translocation of the p65 subunit of NF-κB from the cytoplasm to the nucleus, and thereby reducing the expression of iNOS protein and the production of NO. Furthermore, pretreatment with cyanidin greatly promoted the translocation of the Nrf2 protein from the cytoplasm to the nucleus; upregulating cytoprotective enzymes, including HO-1, NQO-1 and GCLC; and increased the activity of SOD enzymes. Pretreatment with cyanidin also decreased the expression of TLR4, directly improved intracellular ROS levels and regulated the activity of inflammation-related downstream pathways including NO production and SOD activity through TLR4/NOX4 signaling. These results demonstrate that TLR4 is a primary receptor in SK-N-SH cells, by which Aß25-35 triggers neuroinflammation, and cyanidin attenuates Aß-induced inflammation and ROS production mediated by the TLR4/NOX4 pathway, suggesting that inhibition of TLR4 by cyanidin could be beneficial in preventing neuronal cell death in the process of Alzheimer's disease.

Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells.

Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood-brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH.

Melatonin suppresses methamphetamine-triggered endoplasmic reticulum stress in C6 cells glioma cell lines.

Methamphetamine (METH) is a neurotoxic drug that causes brain damage by inducing neuronal and glial cell death together with glial cell hyperactivity-mediated progressive neurodegeneration. Previous studies have shown that METH induced glial cell hyperactivity and death via oxidative stress, the inflammatory response, and endoplasmic reticulum stress (ER stress) mechanisms, and melatonin could reverse these effects. However, the exact mechanism of the protective role of melatonin in METH-mediated ER stress has not been understood. This study investigated the protective effect of melatonin against METH toxicity-mediated ER stress in glial cells. Our study demonstrated that METH increased glial cell toxicity related to METH-induced ER stress by stimulating the unfolded protein response (UPR) to activate the expression of ER stress transducers, including phosphorylated double-stranded RNA-activated protein kinase (PKR)-like ER kinase (p-PERK), activating transcription factor (ATF6), and phosphorylated inositol-requiring enzyme 1 (p-IRE1). Moreover, the expression of binding immunoglobulin protein (Bip), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, phosphorylated eukaryotic translation initiation factor 2 alpha (p-eIF2α) and spliced X-box-binding protein-1 (XBP-1) mRNA were also increased. Melatonin reduced ER stress induced by METH toxicity by reducing the expression of ER stress response genes and proteins in a concentration-dependent manner. In addition, melatonin promoted the expression of Bip chaperone in a concentration-dependent manner. Taken together, our findings suggest that melatonin can protect against ER stress-induced glial cell death induced by METH.

Inhibitory effect of melatonin on cerebral endothelial cells dysfunction induced by methamphetamine via NADPH oxidase-2.

Melatonin is a hormone that mostly produced from the pineal gland, and it performs as a strong neuroprotectant to both neuron and glial cells against methamphetamine (METH)-induced neurotoxicity. Recently, it has been found that METH also damages the blood brain barrier (BBB) structure and function. However, the protective mechanism of melatonin on the BBB impairment caused by METH has not been investigated. In this study, the primary rat brain microvascular endothelium cells (BMVECs) isolated from neonatal rats was used to investigate the protective effect of melatonin on METH-induced BBB impairment and the underlying mechanism. The results demonstrated that melatonin decreased the level of reactive oxygen species (ROS), reactive nitrogen species (RNS), and apoptosis induced by METH via NADPH oxidase (NOX)-2 since apocynin, a NOX-2 inhibitor abolished those changes. In addition, melatonin was found to improve cell integrity by increasing the transendothelial electric resistance (TEER) values, and up-regulate the tight junction proteins ZO-1, occludin, and claudin-5, thereby decreasing the paracellular permeability caused by METH mediated by NOX-2. Our data suggest that METH induces BBB impairment by mediating NOX-2 activity, and then induces oxidative and nitrative stress, as well as apoptosis, which causes the impairment of cell integrity, and that melatonin reduces these negative effects of METH by mediating via MT1/2 receptors.

WITHDRAWN: Melatonin improves methamphetamine-induced blood brain barrier impairment through NADPH oxidase-2 in primary rat brain microvascular endothelium cells.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in 〈BRES, 1646 (2016) 182-192〉, 10.1016/j.brainres.2016.05.049. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Melatonin promotes blood-brain barrier integrity in methamphetamine-induced inflammation in primary rat brain microvascular endothelial cells.

Melatonin is a neurohormone and has high potent of antioxidant that is widely reported to be active against methamphetamine (METH)-induced toxicity to neuron, glial cells, and brain endothelial cells. However, the role of melatonin on the inflammatory responses which are mostly caused by blood-brain barrier (BBB) impairment by METH administration has not been investigated. This study used the primary rat brain microvascular endothelial cells (BMVECs) to determine the protective mechanism of melatonin on METH-induced inflammatory responses in the BBB via nuclear factor-ĸB (NF-κB) and nuclear factor erythroid 2-related factor-2 (Nrf2) signaling. Herein, we demonstrated that melatonin reduced the level of the inflammatory mediators, including intercellular adhesion molecules (ICAM)-1, vascular cell adhesion molecules (VCAM)-1, matrix metallopeptidase (MMP)-9, inducible nitric oxide synthase (iNOS), and nitric oxide (NO) caused by METH. These responses were related to the decrease of the expression and translocation of the NF-κB p65 subunit and the activity of NADPH oxidase (NOX)-2. In addition, melatonin promoted the antioxidant processes, modulated the expression and translocation of Nrf2, and also increased the level of heme oxygenase (HO)-1, NAD (P) H: quinone oxidoreductase (NQO)-1, γ-glutamylcysteine synthase (γ-GCLC), and the activity of superoxide dismutase (SOD) through NOX2 mechanism. In addition, we found that the protective role of melatonin in METH-induced inflammatory responses in the BBB was mediated through melatonin receptors (MT1/2). We concluded that the interaction of melatonin with its receptor prevented METH-induced inflammatory responses by suppressing the NF-κB signaling and promoting the Nrf2 signaling before BBB impairment.

Association of Neuroprotective Effect of Di-O-Demethylcurcumin on Aß25-35-Induced Neurotoxicity with Suppression of NF-κB and Activation of Nrf2.

Amyloid-ß peptides (Aß), a major component of senile plaques, play an important role in the development and progression of Alzheimer's disease. Several lines of evidence have demonstrated that Aß-induced neuronal death is mediated by oxidative stress. The present study aimed to evaluate the potential involvement of di-O-demethylcurcumin, an analog of curcuminoid, on Aß-induced neurotoxicity in culture neuroblastoma cells (SK-N-SH cells) through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and the suppression of nuclear factor-κB (NF-κB) signaling pathway and their downstream targets. The results showed that pretreatment with di-O-demethylcurcumin elevated cell viability and decreased the level of reactive oxygen species. Moreover, treatment with di-O-demethylcurcumin promoted the translocation of Nrf2 protein from the cytoplasm to the nucleus, increased the expression of Nrf2-ARE pathway-related downstream proteins including heme oxygenase (HO-1), NAD(P)H:quinone oxidoreductase 1 and glutamate-cysteine ligase catalytic subunit, and increased the activity of superoxide dismutase enzymes. On the other hand, di-O-demethylcurcumin suppressed the degradation of IκBα, translocation of the p65 subunit of NF-κB from cytoplasm to nucleus and thereby, attenuated the expression of inducible nitric oxide synthase protein and nitric oxide production. Taken together, these results suggest that neuroinflammatory effect of di-O-demethylcurcumin might potentially be due to inhibit NF-κB and activate Nrf2 signaling pathways induced by Aß25-35.

Melatonin Protects Methamphetamine-Induced Neuroinflammation Through NF-κB and Nrf2 Pathways in Glioma Cell Line.

Methamphetamine (METH) is known as a toxin for neuronal and glial cells. Previous studies have found that METH-induced glial cell death and inflammation is mediated by oxidative stress. However, the exact mechanisms of the inflammatory response remain unclear. Therefore, we hypothesized that the activation of nuclear factor-κB (NF-κB) signaling, a key mediator of inflammation, and the inhibition of nuclear factor erythroid 2-related factor-2 (Nrf2) signaling, a regulator of the antioxidant response, would be significant events occurring in response to METH-induced inflammation in a rat glioma cell line (C6 cells). Our results show that METH increased the production of nitric oxide (NO) and up-regulated the expression of its main regulatory protein, inducible nitric oxide synthase (iNOS). METH also induced NF-κB activation by increasing inhibitory κBα (IκBα) degradation and translocation of the NF-κB (p65) subunit into the nucleus. Additionally, METH inhibited the activation of the Nrf2 pathway by decreasing the translocation of Nrf2 into the nucleus and also by suppressing the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO-1), and glutamate-cysteine ligase catalytic subunit (γ-GCLC), resulting in the suppression of superoxide dismutase (SOD) activity. Pretreatment with melatonin effectively promoted Nrf2 activation and reversed the METH-induced NF-κB response. Melatonin increased the expression of HO-1, NQO-1, and γ-GCLC, resulting in increased SOD activity. In addition, melatonin also decreased IκBα degradation, translocation of the p65 subunit, and expression of iNOS, resulting in decreased NO production. Taken together, our results indicate that melatonin diminishes the proinflammatory mediator in METH-stimulated C6 cells by inhibiting NF-κB activation and inducing Nrf2-mediated HO-1, NQO-1, and γ-GCLC expression.

Protective effect of melatonin on methamphetamine-induced apoptosis in glioma cell line.

Methamphetamine (METH) is a highly addictive drug causing neurodegenerative diseases. METH has been known to be neurotoxic by inducing oxidative stress, free radical, and pro-inflammatory cytokines. Previous studies have shown that METH could induce neuron and glial cell death, especially inducing glial cell-mediated neurotoxicity that plays a critical role in stress-induced central nervous system damage. Therefore, the aim of the present study is to explore the mechanisms of METH-induced cell death in the glial cell. METH-induced glial cells death is mediated via mitochondrial damage pathway. METH activates the upregulation of the Bax, cytochrome c, cleavage caspase 9 and 3 proteins, and downregulation of Bcl-XL protein in cascade. Pretreatment with melatonin, a neurohormone secreted by the pineal gland, effectively reduced glial cell death. Moreover, melatonin increased the Bcl-XL/Bax ratio but reduced the level of cytochrome c, cleavage caspase 9 and 3 proteins. Therefore, these results demonstrated that melatonin could reduce the cytotoxic effect of METH by decreasing the mitochondrial death pathway activation in glial cells. This outcome suggests that melatonin might be beneficial as the neuroprotection in neurodegenerative diseases caused by METH or other pathogens.