Superoxide-mediated oxidative stress accelerates skeletal muscle atrophy by synchronous activation of proteolytic systems.

The maintenance of skeletal muscle mass depends on the overall balance between the rates of protein synthesis and degradation. Thus, age-related muscle atrophy and function, commonly known as sarcopenia, may result from decreased protein synthesis, increased proteolysis, or simultaneous changes in both processes governed by complex multifactorial mechanisms. Growing evidence implicates oxidative stress and reactive oxygen species (ROS) as an essential regulator of proteolysis. Our previous studies have shown that genetic deletion of CuZn superoxide dismutase (CuZnSOD, Sod1) in mice leads to elevated oxidative stress, muscle atrophy and weakness, and an acceleration in age-related phenotypes associated with sarcopenia. The goal of this study is to determine whether oxidative stress directly influences the acceleration of proteolysis in skeletal muscle of Sod1-/- mice as a function of age. Compared to control, Sod1-/- muscle showed a significant elevation in protein carbonyls and 3-nitrotyrosine levels, suggesting high oxidative and nitrosative protein modifications were present. In addition, age-dependent muscle atrophy in Sod1-/- muscle was accompanied by an upregulation of the cysteine proteases, calpain, and caspase-3, which are known to play a key role in the initial breakdown of sarcomeres during atrophic conditions. Furthermore, an increase in oxidative stress-induced muscle atrophy was also strongly coupled with simultaneous activation of two major proteolytic systems, the ubiquitin-proteasome and lysosomal autophagy pathways. Collectively, our data suggest that chronic oxidative stress in Sod1-/- mice accelerates age-dependent muscle atrophy by enhancing coordinated activation of the proteolytic systems, thereby resulting in overall protein degradation.

Engineered Heterochronic Parabiosis in 3D Microphysiological System for Identification of Muscle Rejuvenating Factors.

Exposure of aged mice to a young systemic milieu revealed remarkable rejuvenation effects on aged tissues, including skeletal muscle. Although some candidate factors have been identified, the exact identity and the underlying mechanisms of putative rejuvenating factors remain elusive, mainly due to the complexity of in vivo parabiosis. Here, we present an in vitro muscle parabiosis system that integrates young- and old-muscle stem cell vascular niche on a three-dimensional microfluidic platform designed to recapitulate key features of native muscle stem cell microenvironment. This innovative system enables mechanistic studies of cellular dynamics and molecular interactions within the muscle stem cell niche, especially in response to conditional extrinsic stimuli of local and systemic factors. We demonstrate that vascular endothelial growth factor (VEGF) signaling from endothelial cells and myotubes synergistically contribute to the rejuvenation of the aged muscle stem cell function. Moreover, with the adjustable on-chip system, we can mimic both blood transfusion and parabiosis and detect the time-varying effects of anti-geronic and pro-geronic factors in a single organ or multi-organ systems. Our unique approach presents a complementary in vitro model to supplement in vivo parabiosis for identifying potential anti-geronic factors responsible for revitalizing aging organs.

Critical Limb Ischemia Induces Remodeling of Skeletal Muscle Motor Unit, Myonuclear-, and Mitochondrial-Domains.

Critical limb ischemia, the most severe form of peripheral artery disease, leads to extensive damage and alterations to skeletal muscle homeostasis. Although recent research has investigated the tissue-specific responses to ischemia, the role of the muscle stem cell in the regeneration of its niche components within skeletal muscle has been limited. To elucidate the regenerative mechanism of the muscle stem cell in response to ischemic insults, we explored cellular interactions between the vasculature, neural network, and muscle fiber within the muscle stem cell niche. Using a surgical murine hindlimb ischemia model, we first discovered a significant increase in subsynaptic nuclei and remodeling of the neuromuscular junction following ischemia-induced denervation. In addition, ischemic injury causes significant alterations to the myofiber through a muscle stem cell-mediated accumulation of total myonuclei and a concomitant decrease in myonuclear domain size, possibly to enhance the transcriptional and translation output and restore muscle mass. Results also revealed an accumulation of total mitochondrial content per myonucleus in ischemic myofibers to compensate for impaired mitochondrial function and high turnover rate. Taken together, the findings from this study suggest that the muscle stem cell plays a role in motor neuron reinnervation, myonuclear accretion, and mitochondrial biogenesis for skeletal muscle regeneration following ischemic injury.

The elevation training mask induces modest hypoxaemia but does not affect heart rate variability during cycling in healthy adults.

This study examined the acute effects of the elevation training mask (ETM) on haemodynamics and heart rate variability (HRV) at rest, during cycling, and during recovery in healthy adults. Fifteen healthy male (N=9) and female (N=6) adults (27.0 ± 1.14 years) completed two trials with the mask (MASK) and without the mask (CON). The 40-minute cycling exercise protocol included 10-minute phases of (1) rest, (2) 50% of VO2peak cycling, (3) 70% of VO2peak cycling, and (4) recovery. Blood pressure and pulse oximetry saturation (SPO2) were measured at each phase. An Actiwave-Cardio ECG monitor (CamNtech, UK) was used to measure HRV variables including time and frequency domains. A greater response in systolic blood pressure (p=.035) was observed at rest while SPO2 (p=.033) was lower during high-intensity cycling (70% of VO2peak) in the MASK trial. The HRV indices were not different between trials during cycling. However, heart rate (p=.047) was greater while inter-beat interval and sympathovagal balance (the ratio between low-frequency and high-frequency components; ln LF/HF, p=.01) were lower in the MASK than the CON trials during recovery. Wearing an ETM during high-intensity cycling (70% of VO2peak) induces modest hypoxaemia. Although this device did not affect HRV changes during cycling, it seems to delay the cardiac-autonomic recovery from exercise. Healthy adults may be required to perform high-intensity exercise with an ETM to simulate a hypoxic environment, but future studies are needed to determine whether repeated exposure to this condition provides similar benefits as altitude training.

Acute effects of Kinesio taping on muscle function and self-perceived fatigue level in healthy adults.

This study investigated the acute effects of Kinesio taping (KT) on muscular power, strength, endurance, and self-perceived fatigue level. This is a randomized, partial double-blind, crossover trial. Eighteen healthy adults (7 males [23.86 ± 1.68 years] and 11 females [24.82 ± 3.71 years]) were enrolled in this study. All subjects underwent three different trials which included no tap (NT), placebo tap (PT), and KT. Idividuals were assessed for peak and mean power, muscular strength and endurance, and self-perceived fatigue after each condition. The results revealed no significant differences in all variables (p > 0.05) except muscular endurance (F = 5.775, p = 0.007). Muscular endurance in the NT (58.28 ± 12.18 reps/min) condition was significantly higher than that in the KT (52.83 ± 11.76 reps/min) condition. These results suggest that KT on rectus femoris and the patella of the lower limb does not improve muscular function and self-perceived fatigue level. KT is unlikely to enhance exercise performance capacity in healthy adults.

Metabolites distinguishing visceral fat obesity and atherogenic traits in individuals with overweight.

OBJECTIVE: To screen the metabolomes of both overweight subjects with low visceral fat area (LFO) and high visceral fat area (HFO) to identify potential metabolites that are associated with the different metabolic characteristics. METHODS: The metabolic characteristics of 112 overweight (25 kg/m2 ≤ BMI < 30 kg/m2 ) Korean individuals aged 30 to 65 years were examined. Plasma metabolomic profiling of HFO [visceral fat area (VFA) at L4 ≥ 100 cm2 ] and LFO (L4 VFA <100 cm2 ) individuals matched for age, gender, and BMI was performed. RESULTS: HFO subjects showed higher VFA at L1 and L4 than LFO subjects. The HFO group showed higher blood pressure, lipid profile, high-sensitivity C-reactive protein, malondialdehyde, oxidized low-density lipoprotein (LDL), and homeostasis model assessment-insulin resistance and lower high-density lipoprotein-cholesterol levels. In plasma metabolite identification, the HFO group showed significantly higher levels of long-chain (C14:1, C16:1, C16) acylcarnitines (ACs), medium-chain (C12:1, C12) ACs, urobilinogen, docosahexaenoic acid (C22:6ω3), lysoPE (22:6), lysoPC (22:6), lysoPC (22:5), methoxybenzenepropanoic acid, and isodesmosine. All five ACs correlated positively with VFA and oxidized LDL levels and negatively with high-density lipoprotein-cholesterol levels and LDL particle size. CONCLUSIONS: Twelve major metabolites, including three long-chain fatty acids and two medium-chain ACs, are important for distinguishing HFO and LFO. Chronic lipid surplus from visceral fat in HFO is likely associated with substantial increases in plasma medium-chain ACs and long-chain fatty acids, which are closely related to atherogenic traits.

Protective effects of TES trioleate, an inhibitor of phospholipase A2, on reactive oxygen species and UVA-induced cell damage.

2-[Tris(oleoyloxymethyl)methylamino]-1-ethane sulfonic acid (TES trioleate) is an inhibitor of phospholipase A2 (PLA2), which hydrolyzes cell membrane phospholipids to produce arachidonic acid (AA) and lysophospholipids (LysoPLs). Here, we investigated the protective effects of TES trioleate on cell damage caused by ultraviolet A (UVA) light and reactive oxygen species (ROS). Pre-incubation with 250-1000µM TES trioleate resulted in concentration-dependent protection from UVA-induced damage in HaCaT cells. Additionally, 25-1000µM TES trioleate provided protection against H2O2 in a concentration-dependent manner. In human erythrocytes treated with 1O2, 10-100µM TES trioleate showed concentration-dependent protective effects, similar to but stronger than the controls, 4-BPB and lipophilic antioxidant (+)-α-tocopherol at 100µM. TES trioleate did not have detectable radical scavenging activity. Moreover, compared with (+)-α-tocopherol and rutin, TES trioleate showed low ROS scavenging activity. Thus, although TES trioleate showed cell protective effects against UVA, H2O2, and 1O2-induced damages, these effects were not caused by the scavenging ability of the radical or ROS. Finally, pretreatment of HaCaT cells and human erythrocytes with l-α-lysophosphatidylcholine produced by PLA2 promoted increased cell damage at low concentrations. Thus, the protective effects of TES trioleate on cellular damage by UVA and ROS may be associated with inhibition of PLA2-dependent cell damage rather than ROS scavenging.

Red Ginseng as an Ergogenic Aid: A Systematic Review of Clinical Trials.

Purpose: This systematic review was performed to summarize clinical trials assessing the effect of Red Ginseng (RG) supplementation on exercise performance and fatigue recovery. Methods: Two English databases (PUBMED, MEDLINE) and two Korean databases (KISS, RISS) were used as systematic searching engines. We included only articles written in the English and Korean languages. Clinical trials, which evaluated exercise performance and recovery variables with RG supplementation, were included in this review. The methodological quality of all studies was assessed using the Cochrane Risk of Bias tool. Analysis was conducted with Comprehensive Meta-Analysis Version 3. Results: In total, 135 potentially relevant studies were identified, and 14 studies were included. Overall, the aerobic capacity (VO2max, heart rate, time to exhaustion, shuttle run, and anaerobic threshold) exhibited no improvement with RG supplementation. In anaerobic capacity (peak power, mean power, and 30 m dash run), no significant improvements with RG supplementation was described in most of the studies. The antioxidant function predominantly measured by levels of superoxide dismutase (SOD) and malondialdehyde (MDA) showed mixed results. Red Ginseng's effects on fatigue recovery were evaluated using lactate as a main outcome. Two studies observed significant effects while other 5 studies showed no significant effects. Conclusion: The clinical effects of RG have been assessed in various conditions. Although the number of studies presented in this review is small and results of studies are mixed, it is hypothesized that this review article may provide useful guideline to design and conduct future studies investigating efficacy of RG supplementation on exercise performance and fatigue recovery in human trials.

Terrein reduces age-related inflammation induced by oxidative stress through Nrf2/ERK1/2/HO-1 signalling in aged HDF cells.

This study investigated whether multiple bioactivity of terrein such as anti-inflammatory and anti-oxidant inhibits age-related inflammation by promoting an antioxidant response in aged human diploid fibroblast (HDF) cells. HDF cells were cultured serially for in vitro replicative senescence. To create the ageing cell phenotype, intermediate stage (PD31) HDF cells were brought to stress-induced premature senescence (SIPS) using hydrogen peroxide (H2 O2). Terrein increased cell viability even with H2O2 stress and reduced inflammatory molecules such as intracellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (COX-2), interleukin-1beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α). Terrein reduced also phospho-extracellular kinase receptor1/2 (p-EKR1/2) signalling in aged HDF cells. SIPS cells were attenuated for age-related biological markers including reactive oxygen species (ROS), senescence associated beta-galactosidase (SA ß-gal.) and the aforementioned inflammatory molecules. Terrein induced the induction of anti-oxidant molecules, copper/zinc-superoxide defence (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and heme oxygenase-1 (HO-1) in SIPS cells. Terrein also alleviated reactive oxygen species formation through the Nrf2/HO-1/p-ERK1/2 pathway in aged cells. The results indicate that terrein has an alleviative function of age-related inflammation characterized as an anti-oxidant. Terrein might be a useful nutraceutical compound for anti-ageing.

Propyl gallate inhibits adipogenesis by stimulating extracellular signal-related kinases in human adipose tissue-derived mesenchymal stem cells.

Propyl gallate (PG) used as an additive in various foods has antioxidant and anti-inflammatory effects. Although the functional roles of PG in various cell types are well characterized, it is unknown whether PG has effect on stem cell differentiation. In this study, we demonstrated that PG could inhibit adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells (hAMSCs) by decreasing the accumulation of intracellular lipid droplets. In addition, PG significantly reduced the expression of adipocyte-specific markers including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein 2 (aP2). PG inhibited adipogenesis in hAMSCs through extracellular regulated kinase (ERK) pathway. Decreased adipogenesis following PG treatment was recovered in response to ERK blocking. Taken together, these results suggest a novel effect of PG on adipocyte differentiation in hAMSCs, supporting a negative role of ERK1/2 pathway in adipogenic differentiation.

Davallialactone reduces inflammation and repairs dentinogenesis on glucose oxidase-induced stress in dental pulp cells.

INTRODUCTION: The chronic nature of diabetes mellitus (DM) raises the risk of oral complication diseases. In general, DM causes oxidative stress to organs. This study aimed to evaluate the cellular change of dental pulp cells against glucose oxidative stress by glucose oxidase with a high glucose state. The purpose of this study was to test the antioxidant character of davallialactone and to reduce the pathogenesis of dental pulp cells against glucose oxidative stress. METHODS: The glucose oxidase with a high glucose concentration was tested for hydroxy peroxide (H2O2) production, cellular toxicity, reactive oxygen species (ROS) formation, induction of inflammatory molecules and disturbance of dentin mineralization in human dental pulp cells. The anti-oxidant effect of Davallilactone was investigated to restore dental pulp cells' vitality and dentin mineralization via reduction of H2O2 production, cellular toxicity, ROS formation and inflammatory molecules. RESULTS: The treatment of glucose oxidase with a high glucose concentration increased H2O2 production, cellular toxicity, and inflammatory molecules and disturbed dentin mineralization by reducing pulp cell activity. However, davallialactone reduced H2O2 production, cellular toxicity, ROS formation, inflammatory molecules, and dentin mineralization disturbances even with a long-term glucose oxidative stress state. CONCLUSIONS: The results of this study imply that the development of oral complications is related to the irreversible damage of dental pulp cells by DM-induced oxidative stress. Davallialactone, a natural antioxidant, may be useful to treat complicated oral disease, representing an improvement for pulp vital therapy.

The antioxidant property of pachymic acid improves bone disturbance against AH plus-induced inflammation in MC-3T3 E1 cells.

INTRODUCTION: The cytotoxicity of resin-based sealer is influential on the inflammatory reaction and cell survival for oral periapical cells. In this study, pachymic acid as an antioxidant was investigated for the improvement of bone disturbance against AH Plus (Dentsply DeTrey GmbH, Konstanz, Germany)-induced inflammation in MC-3T3 E1 cells. METHODS: AH Plus was prepared according to the manufacturer's instructions. Using mouse osteoblast cells (MC-3T3 E1), a specimen of AH Plus was eluted with the culture medium for 1 day and was diluted by 30%. The cellular cytotoxicity and reactive oxygen species formation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2',7'-dichlorodihydrofluorescein diacetate with fluorescence-activated cell sorting. The secretion of proinflammatory cytokines was determined by an enzyme-linked immunosorbent assay, and the expression of inflammatory and osteogenic molecules was determined by immunoblotting. RESULTS: Cells with AH Plus elutes showed a decrease of cell viability and ALP activity. However, pachymic acid and N-acetyl-L-cysteine (control antioxidant) restored cell viability and ALP activity damaged by AH plus. The secretion of nitric oxide, tumor necrosis factor α, and interleukin-1ß were increased in AH Plus-stimulated MC-3T3 E1 cells, but pachymic acid suppressed its production. Furthermore, pachymic acid reduced the receptor activator of nuclear factor-κB ligand, cyclooxygenase-2, matrix metalloproteinase-2 and -9, increased bone morphogenetic protein-2 and -7, and runt-related transcription factor 2 despite AH Plus stimuli. In addition, pachymic acid affected the removal effect of reactive oxygen species formation as did N-acetyl-L-cysteine. More importantly, pachymic acid inhibited nuclear factor-κB translocation. CONCLUSIONS: The property of pachymic acid can mitigate the unfavorable conditions induced by AH Plus stimuli. Therefore, the use of pachymic acid is suggested to prevent the complications of oral diseases such as inflammation and alveolar destruction of the oral cavity.

Aging of in vitro pulp illustrates change of inflammation and dentinogenesis.

INTRODUCTION: Dental pulp functions include pulp cell activity involvement in dentin formation. In this study we investigated the age-related changes in dental pulp cells that may influence pulp cell activity for restoring pulp function. METHODS: Human dental pulp cells (HDPCs) were serially subcultured until spontaneously arrested. Altered expression of chronic inflammatory molecules and age-related molecules were determined by Western blotting. Odontogenic functions impaired by senescence were assayed by Western blotting, reverse transcriptase polymerase chain reaction, alkaline phosphatase activity, and alizarin red S staining. To understand the mechanism of aging process by stress-induced premature senescence (SIPS), the cells were treated with H(2)O(2). Replicative senescence and SIPS were also compared. RESULTS: Replicative senescence of HDPCs was characterized by senescence-associated ß-galactosidase activity and reactive oxygen species formation. These cells exhibited altered expression of chronic inflammatory molecules such as intracellular adhesion molecule-1, vascular cell adhesion molecule-1, peroxisome proliferator activated receptor-gamma, and heme oxygenase-1 and age-related molecules such as p53, p21, phosphorylated-extracellular signal-regulated kinase, and c-myb. SIPS cell results were similar to replicative senescence. Furthermore, HDPCs decreased odontogenic markers such as dentin sialophosphoprotein and dentin matrix-1 and osteogenic markers such as bone morphogenetic protein-2 and -7, runt-related transcription factor-2, osteopontin, alkaline phosphatase activity, and mineralized nodule formation by replicative senescence and SIPS. CONCLUSIONS: This study suggests that development of aging-related molecules in pulp cells offers understanding of cellular mechanisms and biological events responsible for tooth preservation and maintenance strategies for healthy teeth across the life span.

The survival role of peroxisome proliferator-activated receptor gamma induces odontoblast differentiation against oxidative stress in human dental pulp cells.

INTRODUCTION: Peroxisome proliferator-activated receptor gamma (PPARγ) has well-known anti-inflammatory action in human dental pulp cells (HDPCs). The purpose of this study was to investigate whether the anti-inflammatory action of PPARγ involves in cellular cytoprotection and supports odontoblast differentiation under oxidative stress in HDPCs. METHODS: To simulate long-term oxidative stress, pulp cells were treated with 150 µmol hydrogen peroxide (H(2)O(2)) for 12 days. The replication deficiency adenovirus (adenovirus PPARγ) was introduced for PPARγ overexpression in pulp cells. The cellular cytotoxicity and reactive oxygen species formation by H(2)O(2) were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2',7'-dichlorodihydrofluorescein diacetate with fluorescence-activated cell sorting assay. To determine the roles of PPARγ, several molecules of odontogenic/osteogenic and signal pathway were analyzed by reverse-transcription polymerase chain reaction and Western hybridization. Dentin mineralization was determined by alizarin red stain and alkaline phosphatase activity assay. RESULTS: Pulp cells treated with long-term H(2)O(2) showed high reactive oxygen species formation, low cell viability, down-expression of antioxidant molecules (Cu/Zn and Mn superoxide dismutase), and odontogenic/osteogenic markers (eg, dentin sialophosphoprotein, dentin matrix protein-1, osteopontin, bone sialoprotein, Runx-2, and bone morphogenetic protein 2 and 7). In addition, pulp cells with oxidative stress underwent the activation of ERK1/2, activator protein-1, and nuclear factor-κB translocation to the nucleus. However, the PPARγ-overexpressed cells gave opposite results although under oxidative stress. Furthermore, PPARγ and its agonist rosiglitazone exhibited an induction of dentin mineralization under oxidative stress. CONCLUSIONS: PPARγ in pulp cells increases cell viability, odontoblastic differentiation, and dentin mineralization under oxidative stress. These results offer new insights into the potential antioxidative activity of PPARγ and its agonist for therapeutic agents for pulp vitality in HDPCs.

PPARγ inhibits inflammation and RANKL expression in epoxy resin-based sealer-induced osteoblast precursor cells E1 cells.

OBJECTIVES: The AH26 of epoxy resin-based sealer is used widely owing to its excellent physical characteristics but it induces oxidative stress and cytotoxicity at the periapical tissues. AH26 exhibited cytotoxicity towards MC-3T3-E1 cells, which resulted in mitochondria-mediated apoptosis. Peroxisome proliferator-activated receptor (PPARγ) has an anti-inflammatory effect in several tissue and cells, but its action of AH26-related inflammation is not completely understood. The aim of this study is to investigate the anti-inflammatory and anti-osteoclastic mechanisms of PPARγ in AH26-induced MC-3T3 E1 cells. METHODS: AH26 was prepared according to the manufacturer's instructions. The 1-day extraction sample, which was diluted by 30%, was tested in this experiment. Recombinant deficiency adenoviral PPARγ (Ad/PPARγ) was used to examine PPARγ over-expression in MC-3T3 E1 cells. AH26-induced reactive oxygen species (ROS) formation was analysed using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) with fluorescence-activated cell sorting (FACS), and the expression of receptor activator of nuclear factor-κB ligand (RANKL) and inflammatory molecules was determined by immunoblotting. The anti-inflammatory and anti-osteoclastic mechanisms of the PPARγ-involved signal pathway was examined by immunoblotting. RESULTS: The AH26 elutes induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), RANKL expression and ROS formation. In addition, the AH26 elutes suppressed the expression of PPARγ. However, the recovery of PPARγ expression with Ad/PPARγ resulted in the inhibition of iNOS, COX-2, RANKL and ROS formation despite the AH26 treatment in MC-3T3 E1 cells. The mechanism of PPARγ was confirmed by the blocking of nuclear factor kappa B (NF-κB) translocation to the nucleus after the suppression of ERK1/2, SAPK/JNK and AP-1 in AH26-induced MC-3T3 E1 cells. CONCLUSION: From this result, PPARγ acts to inhibit bone destruction in AH26-induced bone cells. Therefore, the anti-inflammatory and anti-osteoclastic character of PPARγ might be applicable for healing periapical lesions more rapidly or reducing the induction of cellular inflammation caused by some endodontic sealers.

PPARγ delivered by Ch-GNPs onto titanium surfaces inhibits implant-induced inflammation and induces bone mineralization of MC-3T3E1 osteoblast-like cells.

OBJECTIVES: To deliver the efficacy and safety of Ch-GNPs (Chitosan gold nanoparticles) conjugated anti-inflammatory molecules peroxisome proliferator activated receptor gamma (PPARγ) on implant surface titanium (Ti) to reduce implant-induced inflammation. MATERIALS AND METHODS: The Ch-GNPs were conjugated with the PPARγ cDNA through a coacervation process. Conjugation was cast over Ti surfaces by dipping, and cells were seeded on different sizes (6 × 6 × 0.1 cm and 1 × 1 × 0.1 cm; n = 3) of Ti surfaces. The size of Ch-GNPs and surface characterization of Ti was performed using UV-vis spectroscopy, TEM (Transmission electron microscopy) and EDX (energy-dispersive X-ray). The DNA conjugation and transfection capacity of Ch-GNPs were simultaneously confirmed by agarose gel electrophoresis, ß-galactosidase staining, and immunoblotting. RESULTS: The Ch-GNPs were well dispersed and spherical in shape, with average size around 10-20 nm. Ti surfaces coated with Ch-GNPs/LacZ, as transfection efficacy molecule, showed strong ß-galactosidase staining in MC-3T3 E1 cells. Cells cultured on Ch-GNPs/PPARγ-coated Ti surfaces were able to inhibit implant-induced inflammation by simultaneously suppressing the expression of tumor necrosis factor- alpha (TNF-α), interleukin-1 beta (IL-1ß), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-2 (MMP-2). The inhibition mechanism of Ch-GNPs/PPARγ was due to inhibition of both reactive oxygen species (ROS) and nitric oxide (NO) secretion (n = 3; P < 0.05). In addition, Ch-GNPs/PPARγ was able to increase expression of bone morphogenetic protein (BMP-7) and runt-related transcription factor-2 (RUNX-2). Furthermore, alkaline phosphatase activity (ALP) was also increased than that in control (n = 3; P < 0.01). Whereas, expression of receptor activator of NF-κB ligand (RANKL) was decreased. CONCLUSIONS: The novel gene delivery materials, like Ch-GNPs, can carry the PPARγ cDNA into the required areas of the implant surfaces, thus aiding to inhibit inflammation and promote osteoblast function. Thus, the PPARγ on implant surfaces may promote its clinical application on peri-implantitis or periodontitis like diseases.

Fomitoside-K from Fomitopsis nigra induces apoptosis of human oral squamous cell carcinomas (YD-10B) via mitochondrial signaling pathway.

In this study, a new lanostane triterpene glycoside (fomitoside-K) having biologically active molecules was isolated from a mushroom Fomitopsis nigra to test its anticancer activity on human oral squamous cell carcinomas (YD-10B). We focused on the effect of fomitoside-K on apoptosis, the mitochondria-mediated death pathway and the accumulation of reactive oxygen species (ROS) in YD-10B cells. Fomitoside-K could induce a dose and time-dependent apoptosis in YD-10B cells as characterized by cell morphology, cell cycle arrest, inhibition of survivin, activation of poly(ADP-ribose) polymerase (PARP), caspase-3, -9 and an increased expression ratio of Bax/Bcl-2. The mitochondria membrane potential loss and cytochrome c (Cyt C) release from mitochondria to cytosol were observed during the induction. Moreover, fomitoside-K caused dose-dependent elevation of intracellular ROS level and increase phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in YD-10B cells. To further investigate the mechanisms, we examined the effects of ROS scavenger N-acetyl-L-cysteine (NAC) and selective inhibitors for mitogen activated protein kinase (MAPK) pathways on the cell death. The fomitoside-K induced cell death by ROS was significantly inhibited by NAC, ERK (PD98059) and JNK inhibitor (SP600125). In addition, fomitoside-K has a synergistic effect with adriamycin in suppressing the growth of YD-10B cells. These data suggest that fomitoside-K induces apoptosis in YD-10B cells through the ROS-dependent mitochondrial dysfunction pathway and provides a mechanistic framework for further exploring the use of fomitoside-K against the proliferation of human oral cancer.

Anti-inflammatory mechanism of PPARγ on LPS-induced pulp cells: role of the ROS removal activity.

OBJECTIVES: PPARγ has an anti-inflammatory effect on LPS-induced pulpal inflammation by decreasing the expression of MMPs, ICAM-1 and VCAM-1. However, the anti-inflammatory mechanism of PPARγ on the cell adhesion molecules and their upper signal pathways has not been clarified in pulp cells. The aim of this study is to investigate the anti-inflammatory mechanism of PPARγ in pulpal inflammation. METHODS: Human dental pulp cells (HDPCs) were isolated from freshly extracted third molar and cultured. The over-expression of PPARγ was used by adenoviral PPARγ (Ad/PPARγ). The formation of ROS was analysed using DCFH-DA with FACS, and NO was analysed using colorimetric bioassay. The expression of inflammatory molecules and inflammatory mechanism of PPARγ involved signal pathway were determined by immunoblotting. RESULTS: LPS-induced HDPC decreased PPARγ expression gradually and strongly activated the ERK1/2 signals amongst the MAPK, and induced NF-κB translocation from the cytosol to the nucleus. On the other hand, the cells to restore PPARγ with Ad/PPARγ were inhibited ERK1/2 despite being stimulated with LPS. In addition, the cells treated with rosiglitazone (PPARγ agonist) also were inhibited ERK1/2 activation, and the expression of ICAM-1, VCAM-1 and NF-κB translocation under LPS stimulation. The GW9667 (PPARγ antagonist)-treated HDPC did not affect the adhesion molecules and signal activation. LPS-induced HDPC produced significant NO and ROS levels, but their production was attenuated in the PPARγ over-expressed cells. Overall, the PPARγ effect under LPS stimulation is due to the removal activity of cellular NO and ROS formation. CONCLUSION: These results suggest that anti-inflammatory mechanism of PPARγ is due to the removal activity of NO and ROS, and its removal effect suppressed ERK1/2 signal activation and NF-κB translocation. Therefore, the NO and ROS removal activity of PPARγ suggests major anti-inflammatory mechanism in HDPC, and it might offer us a possible molecule for various types of inflammatory inhibition.

c-myb mediates inflammatory reaction against oxidative stress in human breast cancer cell line, MCF-7.

Emerging evidence suggests that oncogenes play an important role in the inflammatory reactions in cancer cells, but the precise molecular and cellular mechanisms linking the oncogenes to inflammation is unclear. This study examined the contribution of proto-oncogene c-myb to inflammation in MCF-7 breast cancer cells. An inflammatory response was elicited directly by the cells using an in vitro culture system whereby the cells were exposed to H(2) O(2) . Upon exposure to H(2) O(2) , the cells showed a local inflammatory response, as evidenced by matrix metalloproteinases (MMPs) and ICAM-1 expression. Significant up-regulation of the proto-oncogene c-myb also was observed under inflammatory conditions. c-myb, overexpressed in the cells by transducing with Ad/c-myb, showed an increase in MMPs and ICAM-1 expression under H(2) O(2) stimulation. Despite H(2) O(2) stimulation, the c-myb down-regulated cells by c-myb siRNA inhibit the expression of MMPs and ICAM-1. Among the MAPKs, ERK1/2 and SAPK/JNK were activated by the H(2) O(2) treatment. Interestingly, the H(2) O(2) -induced activation of ERK1/2 and SAPK/JNK was inhibited by siRNA c-myb. These results suggest that breast cancer cells may play a significant role in sustaining and amplifying the inflammation process through the activation of c-myb, which results in the activation of the ERK1/2 and SAPK/JNK pathway. This condition highlights the potential link between inflammation and its involvement in promoting breast cancer proliferation.

Reactive oxygen species removal activity of davallialactone reduces lipopolysaccharide-induced pulpal inflammation through inhibition of the extracellular signal-regulated kinase 1/2 and nuclear factor kappa b pathway.

INTRODUCTION: Davallialactone, hispidin analogues derived from the mushroom Inonotus xeranticus, has antioxidant properties. This study examined whether the reactive oxygen species (ROS) removal activity of davallialactone affects the lipopolysaccharide (LPS)-induced anti-inflammatory activity in human dental pulp cells. METHODS: The LPS-induced formation of ROS was analyzed by using dichlorofluorescein diacetate with fluorescence-activated cell sorter, and the expression of inflammatory molecules in primary cultured human dental pulp cells was determined by immunoblotting. The inflammatory mechanism of the davallialactone-involved signal pathway was examined by immunoblotting. RESULTS: Davallialactone acted as an antioxidant to confirm the elimination of ROS formation and elevation of Cu/Zn superoxide dismutase and Mn superoxide dismutase expression in LPS-induced pulp cells. The antioxidant activity of davallialactone leads to inhibition of LPS-induced inflammation by blocking the extracellular signal-regulated kinase (ERK1/2) and nuclear factor kappa B (NF-κB) pathway, which decreases the expression of inflammatory molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, matrix metalloproteinase-2, matrix metalloproteinase-9, inducible nitric oxide synthase, and cyclooxygenase-2. The character of davallialactone was more effective in comparison with N-acetylcysteine as the control antioxidant in this study. CONCLUSIONS: Davallialactone has antioxidant activity and anti-inflammatory effects in LPS-induced human dental pulp cells through the suppression of ERK1/2 activation followed by blockage of NF-κB translocation from cytosol into nuclear. Therefore, the good anti-inflammatory capacity of davallialactone might be used for oral diseases such as pulpitis and periodontitis.