Fortilin interacts with TGF-ß1 and prevents TGF-ß receptor activation.

Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-ß1 and prevents it from activating the TGF-ß1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-ß1 and its isoforms. The modified ELISA assay shows that TGF-ß1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-ß1. The SPR analysis also reveals that fortilin and the TGF-ß receptor II (TGFßRII) compete for TGF-ß1. Both luciferase and secreted alkaline phosphatase reporter assays show that fortilin prevents TGF-ß1 from activating Smad3 binding to Smad-binding element. Fortilin inhibits the phosphorylation of Smad3 in both quantitative western blot assays and ELISA. Finally, fortilin inhibits TGFß-1-induced differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. A computer-assisted virtual docking reveals that fortilin occupies the pocket of TGF-ß1 that is normally occupied by TGFßRII and that TGF-ß1 can bind either fortilin or TGFßRII at any given time. These data support the role of extracellular fortilin as a negative regulator of the TGF-ß1 signaling pathway.

GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy.

Cisplatin (CDDP) induces senescence characterized by senescence-associated secretory phenotypes (SASP) and the unfolded protein response (UPR). In this study, we investigated the proteins related to the UPR during the senescence cell fate. Strikingly, we found that one of the critical ER-resident proteins, GRP78/BiP, was significantly altered. Here we show that GRP78 levels differentially expressed depending on non-small lung cancer subtypes. GRP78 indeed regulates the evasion of senescence in adenocarcinoma A549 cells, in which the increased GRP78 levels enable them to re-proliferate after CDDP removal. Conversely, GRP78 is downregulated in the senescence H460 cells, making them lacking senescence evasion capability. We observed that the translational regulation critically contributed to the GRP78 protein levels in CDDP-induces senescence. Furthermore, the increased GRP78 level during senescence confers resistance to senolytic drug, Bortezomib, as observed by a twofold increase in IC50 in A549 senescence cells compared to the wild-type. This observation is also consistent in the cells that have undergone genetic manipulation by transfection with pcDNA3.1(+)-GRP78/BiP plasmids and pSpCas9(BB)-2A-Puro containing guide RNA sequence targeting GRP78 exon 3 to induce the overexpression and downregulation of GRP78 in H460 cells, respectively. Our findings reveal a unique role of GRP78 on the senescence evasion cell fate and senolytic drug resistance after cisplatin-based chemotherapy.

Fortilin inhibits p53, halts cardiomyocyte apoptosis, and protects the heart against heart failure.

Heart failure (HF) has reached epidemic proportions in developed countries, affecting over 20 million people worldwide. Despite modern medical and device therapies, 60-70% of HF patients still die within 5 years of diagnosis as it relentlessly progresses through pervasive apoptotic loss of cardiomyocytes. Although fortilin, a 172-amino-acid anti-p53 molecule, is one of the most expressed proteins in the heart, its precise role there has remained unknown. Also unclear is how cardiomyocytes are protected against apoptosis. Here, we report that failing human hearts express less fortilin than do non-failing hearts. We also found that mice lacking fortilin in the heart (fortilinKO-heart) die by 9 weeks of age due to extensive cardiomyocyte apoptosis and severe HF, which suggests that fortilin sustains cardiomyocyte viability. The lack of fortilin is also associated with drastic upregulation of p53 target genes in the hearts. The heart-specific deletion of p53 in fortilinKO-heart mice extends their life spans from 9 to 18 weeks by mitigating cardiomyocyte apoptosis. Our data suggest that fortilin is a novel cardiac p53 inhibitor and that its inadequate expression in failing hearts and subsequent overactivation of the p53 apoptosis pathway in cardiomyocytes exacerbates HF.

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.

Phonophoresis of Phyllanthus amarus nanoparticle gel improves functional capacity in individuals with knee osteoarthritis: A randomized controlled trial.

OBJECTIVE: This study examined the effects of treatment with Phyllanthus amarus nanoparticle gel applied by phonophoresis (PP) and ultrasound therapy (UT) in patients with symptomatic knee osteoarthritis (OA) using a randomized, double-blind, controlled trial. METHODS: Patients with knee OA (n = 40; mean age ±â€¯SD, 64.30 ±â€¯9.71 years), who had visual analogue scale (VAS) scores for knee pain intensity of 68.00 ±â€¯9.58 (UT group) and 71.00 ±â€¯8.74 (PP group, respectively) before treatment, were randomly allocated into two groups. Both groups were treated with an ultrasound program in continuous mode, 1.0 W/cm2, 10 min per session, for 10 sessions. Nanoparticles of P. amarus were used in the PP group, whereas a nondrug coupling gel was used in the UT group. The 6-min walk test (6-MWT) was performed to evaluate functional capacity. The VAS and the 6-MWT were evaluated before and after 10 treatment sessions in both groups using a double-blind procedure. RESULTS: VAS and 6-MWT showed significant improvement after treatment in both groups (p < 0.05). The PP group showed more significant effects than the UT group, in terms of both reducing the VAS pain score (p < 0.05) and improving 6-MWT (p < 0.05). CONCLUSIONS: PP is suggested as an effective method for the treatment of symptomatic knee OA for reducing pain and improving functional capacity.

Ticagrelor induces paraoxonase-1 (PON1) and better protects hypercholesterolemic mice against atherosclerosis compared to clopidogrel.

Ticagrelor (TIC), a P2Y purinoceptor 12 (P2Y12)-receptor antagonist, has been widely used to treat patients with acute coronary syndrome. Although animal studies suggest that TIC protects against atherosclerosis, it remains unknown whether it does so through its potent platelet inhibition or through other pathways. Here, we placed hypercholesterolemic Ldlr-/-Apobec1-/- mice on a high-fat diet and treated them with either 25 mg/kg/day of clopidogrel (CLO) or 180 mg/kg/day of TIC for 16 weeks and evaluated the extent of atherosclerosis. Both treatments equally inhibited platelets as determined by ex vivo platelet aggregation assays. The extent of atherosclerosis, however, was significantly less in the TIC group than in the CLO group. Immunohistochemical staining and ELISA showed that TIC treatment was associated with less macrophage infiltration to the atherosclerotic intima and lower serum levels of CCL4, CXCL10, and TNFα, respectively, than CLO treatment. Treatment with TIC, but not CLO, was associated with higher serum activity and tissue level of paraoxonase-1 (PON1), an anti-atherosclerotic molecule, suggesting that TIC might exert greater anti-atherosclerotic activity, compared with CLO, through its unique ability to induce PON1. Although further studies are needed, TIC may prove to be a viable strategy in the prevention and treatment of chronic stable human atherosclerosis.

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.

Simple artificial training device for respiratory muscle strength and lung volumes in healthy young male and female subjects: A pilot study.

OBJECTIVE: The aim of this study was to evaluate the efficiency of a simple artificial device for respiratory muscle strength training and lung volumes using either combined or non-combined exercise with elastic bands in healthy young participants. METHODS: Forty healthy young participants (20 male and 20 female) aged 19-24 years old were randomized into two main experiments with four sub-groups; (1) artificial device (n = 10) & standard device (n = 10) training, and (2) artificial device training combined with elastic band (EB) exercise (n = 10) & standard device training combined with EB (n = 10) exercise. Respiratory muscle strength with maximal peak inspiratory pressure (PImax), and lung volumes; tidal volume (TV), inspiratory reserve volume (IRV), expiratory reserve volume (ERV) and vital capacity (VC) were evaluated before and after training once daily for 3 weeks. Moreover, the peak dyspnea score and vital sign parameters were compared between the experimental groups after final training. RESULTS: All parameters had no statistical differences (p > 0.5) between the training devices alone and those combined with EB exercise prior to any experiments. Results from the first experiment showed that training with an artificial device increased all parameters (PImax, VC, IRV, ERV) significantly (p < 0.05), except for TV, when compared to pre training results, which were the same as those in the standard device training group. No statistical difference was shown between these groups after the training period had been performed. Furthermore, results of applying artificial device training combined with EB exercise showed a significant increase in all parameters, except for TV, and they were the same as the increased results in training with the standard device combined with EB exercise. There was no significant difference of data between these groups after the training period. Finally, the results of peak dyspnea score and all vital sign parameters from using the artificial device, with or without EB exercise, showed no statistical difference when compared to use of the standard device. CONCLUSION: This study proposed that a simple artificial device can be used to train the respiratory muscle with or without elastic band exercise in healthy young subjects.

Fortilin: A Potential Target for the Prevention and Treatment of Human Diseases.

Fortilin is a highly conserved 172-amino-acid polypeptide found in the cytosol, nucleus, mitochondria, extracellular space, and circulating blood. It is a multifunctional protein that protects cells against apoptosis, promotes cell growth and cell cycle progression, binds calcium (Ca2+) and has antipathogen activities. Its role in the pathogenesis of human and animal diseases is also diverse. Fortilin facilitates the development of atherosclerosis, contributes to both systemic and pulmonary arterial hypertension, participates in the development of cancers, and worsens diabetic nephropathy. It is important for the adaptive expansion of pancreatic ß-cells in response to obesity and increased insulin requirement, for the regeneration of liver after hepatectomy, and for protection of the liver against alcohol- and ER stress-induced injury. Fortilin is a viable surrogate marker for in vivo apoptosis, and it plays a key role in embryo and organ development in vertebrates. In fish and shrimp, fortilin participates in host defense against bacterial and viral pathogens. Further translational research could prove fortilin to be a viable molecular target for treatment of various human diseases including and not limited to atherosclerosis, hypertension, certain tumors, diabetes mellitus, diabetic nephropathy, hepatic injury, and aberrant immunity and host defense.

Effects of a simple prototype respiratory muscle trainer on respiratory muscle strength, quality of life and dyspnea, and oxidative stress in COPD patients: a preliminary study.

BACKGROUND: The aim of this study was to evaluate the efficiency of a simple prototype device for training respiratory muscles in lung function, respiratory muscle strength, walking capacity, quality of life (QOL), dyspnea, and oxidative stress in patients with COPD. METHODS: Thirty COPD patients with moderate severity of the disease were randomized into three groups: control (n=10, 6 males and 4 females), standard training (n=10, 4 males and 6 females), and prototype device (n=10, 5 males and 5 females). Respiratory muscle strength (maximal inspiratory pressure [PImax] and maximal expiratory pressure [PEmax]), lung function (forced vital capacity [FVC], percentage of FVC, forced expiratory volume in 1 second [FEV1], percentage of FEV1 [FEV1%], and FEV1/FVC), 6-minute walking distance (6MWD), QOL, and oxidative stress markers (total antioxidant capacity [TAC]), glutathione (GSH), malondialdehyde (MDA), and nitric oxide (NO) were evaluated before and after 6 weeks of training. Moreover, dyspnea scores were assessed before; during week 2, 4, and 6 of training; and at rest after training. RESULTS: All parameters between the groups had no statistical difference before training, and no statistical change in the control group after week 6. FVC, FEV1/FVC, PImax, PEmax, QOL, MDA, and NO showed significant changes after 6 weeks of training with either the standard or prototype device, compared to pre-training. FEV1, FEV1%, 6MWD, TAC, and GSH data did not change statistically. Furthermore, the results of significant changes in all parameters were not statistically different between training groups using the standard and prototype device. The peak dyspnea scores increased significantly in week 4 and 6 when applying the standard or prototype device, and then lowered significantly at rest after 6 weeks of training, compared to pre-training. CONCLUSION: This study proposes that a simple prototype device can be used clinically in COPD patients as a standard device to train respiratory muscles, improving lung function and QOL, as well as involving MDA and NO levels.

Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death.

The endoplasmic reticulum, the cytoplasmic organelle that matures a massive amount of nascent secretory polypeptides, is particularly sensitive to stress. Endoplasmic reticulum stress causes unfolded proteins to populate the organelle, eliciting the unfolded protein response. During the unfolded protein response, GRP78-an endoplasmic reticulum master stress regulator-detaches from three endoplasmic reticulum stress sensors (IRE1α, PERK, and ATF6) and allows them to activate the apoptotic signaling pathway. Fortilin, a pro-survival molecule, is known to inhibit apoptosis by binding and inhibiting p53, but its role in endoplasmic reticulum stress-induced apoptosis remains unknown. Here, we report that fortilin directly interacts with the cytoplasmic domain of IRE1α, inhibits both kinase and endoribonuclease (RNase) activities of the stress sensor, and protects cells against apoptotic cell death at both cellular and whole animal levels. Our data support a role of fortilin in the unfolded protein response and its potential participation in human diseases caused by unfolded protein response.IRE1α is an ER stress sensor, whose activity induces apoptosis. Here, the authors report that fortilin, a pro-survival factor, with yet unknown roles in ER stress, interacts with active IRE1α, inhibits both its kinase end RNase activities, and protects cells from apoptosis both in vitro and in vivo.

Short-Term Pulmonary Rehabilitation for a Female Patient with Chronic Scleroderma under a Single-Case Research Design.

Although previously proposed that chronic scleroderma should be cared for clinically and early rehabilitation should be performed in hospital by a chest physical therapist, little evidence is currently available on its benefits. Therefore, this study demonstrated the benefits of short-term pulmonary rehabilitation during hospitalization in a female patient with chronic scleroderma. The aim of rehabilitation was to improve ventilation and gas exchange by using airway clearance, chest mobilization, and breathing-relearning techniques, including strengthening the respiratory system and the muscles of the limbs by using the BreathMax® device and elastic bands. Gross motor function and activities of daily life were regained by balancing, sitting, and standing practices. Data on minimal chest expansion, high dyspnea, high respiratory rate, and low maximal inspiratory mouth pressure were recorded seven days before rehabilitation or at the baseline period. But there was a clinically significant improvement in dyspnea, chest expansion, maximal inspiratory mouth pressure, and respiratory rate, when compared to baseline data, which were recorded by a chest physical therapist during seven days of rehabilitation. Furthermore, physicians decided to stop using a mechanical ventilator, and improvement in functional capacity was noted. Therefore, in the case of chronic and stable scleroderma, short-term rehabilitation during hospitalization for chest physical therapy possibly shows clinical benefits by improving both pulmonary function and physical performance.

A preliminary study on the effects of star fruit consumption on antioxidant and lipid status in elderly Thai individuals.

OBJECTIVE: The aims of this preliminary study were to evaluate the antioxidant and lipid status before and after star fruit juice consumption in healthy elderly subjects, and the vitamins in star fruit extracts. METHODS: A preliminary designated protocol was performed in 27 elderly individuals with a mean (±SD) age of 69.5±5.3 years, by planning a 2-week control period before 4 weeks of consumption of star fruit twice daily. Oxidative stress parameters such as total antioxidant capacity, glutathione, malondialdehyde, protein hydroperoxide, multivitamins such as l-ascorbic acid (Vit C), retinoic acid (Vit A), and tocopherol (Vit E), and the lipid profile parameters such as cholesterol, triglyceride, high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were analyzed. Moreover, Vit C, Vit A, and Vit E levels were evaluated in the star fruit extracts during the 4-week period. RESULTS: In the 2-week control period, all parameters showed no statistically significant difference; after 4 weeks of consumption, significant improvement in the antioxidant status was observed with increased total antioxidant capacity and reduced malondialdehyde and protein hydroperoxide levels, as well as significantly increased levels of Vit C and Vit A, when compared to the two-time evaluation during the baseline periods. However, glutathione and Vit E showed no statistical difference. In addition, the HDL-C level was higher and the LDL-C level was significantly lower when compared to both baseline periods. But the levels of triglyceride and cholesterol showed no difference. Vit C and Vit A were identified in small quantities in the star fruit extract. CONCLUSION: This preliminary study suggested that consumption of star fruit juice twice daily for 1 month improved the elderly people's antioxidant status and vitamins, as well as improved the lipoproteins related to Vit C and Vit A in the star fruit extract.

Consumption of star fruit juice on pro-inflammatory markers and walking distance in the community dwelling elderly.

PURPOSE: This study aimed to evaluate the effect of star fruit juice supplementation on tumor necrosis factor-alpha (TNF-α), interleukin-23 (IL-23) and interleukin-2 (IL-2), nitric oxide (NO), and 6 min walking distance (6MWD) in a group of elderly individuals. METHODS: Twenty-nine individuals (20 males, 9 females) with a mean age of 72.4±8.3 years completed this study. A two-week control period was followed by four weeks of 100g fresh star fruit juice consumption twice per day after meals. RESULTS: Plasma TNF-α, IL-23, IL-2, NO and the 6MWD were evaluated twice during the control period (weeks 0 and 2) and once after the star fruit juice consumption (week 6). RESULTS: The results showed that all parameters in the blood did not change significantly during the control period. After 4 weeks of star fruit juice consumption, a significant reduction in NO, TNF-α and IL-23 was found; however, there was no change in IL-2. Moreover, the 6MWD increased significantly at week 6, when compared to that at week 0 and 2. Furthermore, the results also showed a significantly positive and negative correlation of NO and TNF-α to the 6MWD, but no correlation of IL-23 and IL-2. CONCLUSION: This preliminary study concluded that consumption of star fruit juice at 100g twice daily for one month can significantly depress the pro-inflammation cytokines: TNF-α, IL-23, and NO, while increasing walking distance. Low TNF-α and high NO also present a significant correlation to walking capacity in elderly individuals.

Fortilin potentiates the peroxidase activity of Peroxiredoxin-1 and protects against alcohol-induced liver damage in mice.

Fortilin, a pro-survival molecule, inhibits p53-induced apoptosis by binding to the sequence-specific DNA-binding domain of the tumor suppressor protein and preventing it from transcriptionally activating Bax. Intriguingly, fortilin protects cells against ROS-induced cell death, independent of p53. The signaling pathway through which fortilin protects cells against ROS-induced cell death, however, is unknown. Here we report that fortilin physically interacts with the antioxidant enzyme peroxiredoxin-1 (PRX1), protects it from proteasome-mediated degradation, and keeps it enzymatically active by blocking its deactivating phosphorylation by Mst1, a serine/threonine kinase. At the whole animal level, the liver-specific overexpression of fortilin reduced PRX1 phosphorylation in the liver, enhanced PRX1 activity, and protected the transgenic animals against alcohol-induced, ROS-mediated, liver damage. These data suggest the presence of a novel oxidative-stress-handling pathway where the anti-p53 molecule fortilin augments the peroxidase PRX1 by protecting it against degradation and inactivation of the enzyme. Fortilin-PRX1 interaction in the liver could be clinically exploited further to prevent acute alcohol-induced liver damage in humans.

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.

Di-O-demethylcurcumin protects SK-N-SH cells against mitochondrial and endoplasmic reticulum-mediated apoptotic cell death induced by Aß25-35.

Alzheimer's disease (AD) is a neurodegenerative and progressive disorder. The hallmark of pathological AD is amyloid plaque which is the accumulation of amyloid ß (Aß) in extracellular neuronal cells and neurofibrillary tangles (NFT) in neuronal cells, which lead to neurotoxicity via reactive oxygen species (ROS) generation related apoptosis. Loss of synapses and synaptic damage are the best correlates of cognitive decline in AD. Neuronal cell death is the main cause of brain dysfunction and cognitive impairment. Aß activates neuronal death via endoplasmic reticulum (ER) stress and mitochondria apoptosis pathway. This study investigated the underlying mechanisms and effects of di-O-demethylcurcumin in preventing Aß-induced apoptosis. Pretreatment with di-O-demethylcurcumin for 2 h, which was followed by Aß25-35 (10 µM) in human neuroblastoma SK-N-SH cells improved cell viability by using MTS assay and decreased neuronal cell apoptosis. Pretreatment with di-O-demethylcurcumin attenuated the number of nuclear condensations and number of apoptotic cells in Aß25-35-induced group in a concentration-dependent manner by using transmission electron microscope (TEM) and flow cytometry, respectively. Di-O-demethylcurcumin also increased the ratio of Bcl-XL/Bax protein, and reduced intracellular ROS level, cytochrome c protein expression, cleaved caspase-9 protein expression, and cleaved caspase-3 protein expression. Additionally, di-O-demethylcurcumin treatment also reduced the expression of ER stress protein markers, including protein kinase RNA like endoplasmic reticulum kinase (PERK) phosphorylation, eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation, inositol-requiring enzyme 1 (IRE1) phosphorylation, X-box-binding protein-1 (XBP-1), activating transcription factor (ATF6), C/EBP homologous protein (CHOP), and cleaved caspase-12 protein. CHOP and cleaved caspase-12 protein are the key mediators of apoptosis. Our data suggest that di-O-demethylcurcumin is a candidate protectant against neuronal death through its suppression of the apoptosis mediated by mitochondrial death and ER stress pathway.

Neuroprotective effect of purple rice extract and its constituent against amyloid beta-induced neuronal cell death in SK-N-SH cells.

This study evaluated the protective effects of purple rice (Oryza sativa L.) extract (PRE) and its major constituent, cyanidin, and their underlying mechanisms against Aß 25-35-induced neuronal cell death in SK-N-SH cells. Aß 25-35-induced neuronal toxicity is characterized by decrease in cell viability, the release of lactate dehydrogenase (LDH), decrease superoxide dismutase (SOD) activity, increase in reactive oxygen species (ROS) production, morphological alteration, and activation of mitochondrial death pathway. Pretreatment with PRE and cyanidin significantly attenuated Aß 25-35-induced loss of cell viability, apoptosis, and increase in ROS and RNS production in a dose-dependent manner. In addition, PRE and cyanidin also helped to bring about the downregulation of the expression of Bax, cytochrome c, cleavage caspase-9, and cleavage caspase-3 proteins, and the upregulation of the Bcl-XL protein in cascade. Therefore, it is evident that PRE and its major constituent, cyanidin, were successful in protecting from the cytotoxic effect of Aß 25-35 through attenuation ROS and RNS production and modulation of mitochondrial death pathway in SK-N-SH cells. This result suggests that PRE and its major constituent, cyanidin, might be beneficial as potential therapeutic agents in preventing neurodegenerative diseases.

Elevation of serum fortilin levels is specific for apoptosis and signifies cell death in vivo.

BACKGROUND: Billions of cells undergo apoptosis each day in the average normal adult. The ability to readily assess the degree of apoptosis in human diseases is hampered by the lack of sensitive and specific serum biomarkers of apoptosis. Fortilin is a novel prosurvival molecule that protects cells against various noxious stimuli. While fortilin is secreted into the extracellular space under certain conditions, the relationship between the serum concentration of fortilin and the presence and extent of apoptosis in vivo remains unknown. METHODS & RESULTS: Using a newly developed fortilin ELISA system, we show here that fortilin exists in the normal human and mouse circulation. We further demonstrate that fortilin serum levels are significantly elevated in patients with solid cancer, in response to anti-cancer chemo- or radiation therapy. The elevation of fortilin serum levels is more robust and sensitive than that of such previously-reported serum biomarkers of apoptosis as fragmented cytokeratin-18, cytochrome c, and nucleosomal DNA. In addition, targeted apoptotic liver damage induced by Jo2 anti-Fas (CD95) antibody consistently and significantly increased serum fortilin levels in C57BL/6J mice. Finally, when challenged by anti-human-Fas IgM antibody, Jurkat leukemic T cells apoptosed and released fortilin into the medium before plasma membrane integrity was compromised. CONCLUSIONS: Taken together, these data suggest that serum fortilin levels reflect the degree and extent of apoptosis occurring in vivo. GENERAL SIGNIFICANCE: Fortilin is a viable serum biomarker of in vivo apoptosis and can be utilized to noninvasively assess the status of in vivo apoptosis in humans.