Thymosin Beta 4 Inhibits LPS and ATP-Induced Hepatic Stellate Cells via the Regulation of Multiple Signaling Pathways.

Risk signals are characteristic of many common inflammatory diseases and can function to activate nucleotide-binding oligomerization (NLR) family pyrin domain-containing 3 (NLRP3), the innate immune signal receptor in cytoplasm. The NLRP3 inflammasome plays an important role in the development of liver fibrosis. Activated NLRP3 nucleates the assembly of inflammasomes, leading to the secretion of interleukin (IL)-1ß and IL-18, the activation of caspase-1, and the initiation of the inflammatory process. Therefore, it is essential to inhibit the activation of the NLRP3 inflammasome, which plays a vital role in the immune response and in initiating inflammation. RAW 264.7 and LX-2 cells were primed with lipopolysaccharide (LPS) for 4 h and subsequently stimulated for 30 min with 5 mM of adenosine 5'-triphosphate (ATP) to activate the NLRP3 inflammasome. Thymosin beta 4 (Tß4) was supplemented to RAW264.7 and LX-2 cells 30 min before ATP was added. As a result, we investigated the effects of Tß4 on the NLRP3 inflammasome. Tß4 prevented LPS-induced NLRP3 priming by inhibiting NF-kB and JNK/p38 MAPK expression and the LPS and ATP-induced production of reactive oxygen species. Moreover, Tß4 induced autophagy by controlling autophagy markers (LC3A/B and p62) through the inhibition of the PI3K/AKT/mTOR pathway. LPS combined with ATP significantly increased thee protein expression of inflammatory mediators and NLRP3 inflammasome markers. These events were remarkably suppressed by Tß4. In conclusion, Tß4 attenuated NLRP3 inflammasomes by inhibiting NLRP3 inflammasome-related proteins (NLRP3, ASC, IL-1ß, and caspase-1). Our results indicate that Tß4 attenuated the NLRP3 inflammasome through multiple signaling pathway regulations in macrophage and hepatic stellate cells. Therefore, based on the above findings, it is hypothesized that Tß4 could be a potential inflammatory therapeutic agent targeting the NLRP3 inflammasome in hepatic fibrosis regulation.

Thymosin Beta 4 Protects Hippocampal Neuronal Cells against PrP (106-126) via Neurotrophic Factor Signaling.

Prion protein peptide (PrP) has demonstrated neurotoxicity in brain cells, resulting in the progression of prion diseases with spongiform degenerative, amyloidogenic, and aggregative properties. Thymosin beta 4 (Tß4) plays a role in the nervous system and may be related to motility, axonal enlargement, differentiation, neurite outgrowth, and proliferation. However, no studies about the effects of Tß4 on prion disease have been performed yet. In the present study, we investigated the protective effect of Tß4 against synthetic PrP (106-126) and considered possible mechanisms. Hippocampal neuronal HT22 cells were treated with Tß4 and PrP (106-126) for 24 h. Tß4 significantly reversed cell viability and reactive oxidative species (ROS) affected by PrP (106-126). Apoptotic proteins induced by PrP (106-126) were reduced by Tß4. Interestingly, a balance of neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor) and receptors (nerve growth factor receptor p75, tropomyosin related kinase A and B) were competitively maintained by Tß4 through receptors reacting to PrP (106-126). Our results demonstrate that Tß4 protects neuronal cells against PrP (106-126) neurotoxicity via the interaction of neurotrophic factors/receptors.

Antioxidation and anti-inflammatory effects of gamma-irradiated silk sericin and fibroin in H2O2-induced HaCaT Cell.

Oxidative stress in skin cells can induce the formation of reactive oxygen species (ROS), which are critical for pathogenic processes such as immunosuppression, inflammation, and skin aging. In this study, we confirmed improvements from gamma-irradiated silk sericin (I-sericin) and gamma-irradiated silk fibroin (I-fibroin) to skin cells damaged by oxidative stress. We found that I-sericin and I-fibroin effectively attenuated oxidative stress-induced ROS generation and decreased oxidative stress-induced inflammatory factors COX-2, iNOS, tumor necrosis factor-α, and interleukin-1ß compared to the use of non-irradiated sericin or fibroin. I-sericin and I-fibroin effects were balanced by competition with skin regenerative protein factors reacting to oxidative stress. Taken together, our results indicated that, compared to non-irradiated sericin or fibroin, I-sericin, and I-fibroin had anti-oxidation and anti-inflammation activity and protective effects against skin cell damage from oxidative stress. Therefore, gamma-irradiation may be useful in the development of cosmetics to maintain skin health.

Protective Effects of Phosphatidylcholine against Hepatic and Renal Cell Injury from Advanced Glycation End Products.

Background and Objectives: Receptors of the advanced glycation products (RAGE) are activated to promote cell death and contributes to chronic diseases such as diabetes and inflammation. Advanced glycation end products (AGEs), which interact with RAGE are complex compounds synthesized during diabetes development and are presumed to play a significant role in pathogenesis of diabetes. Phosphatidylcholine (PC), a polyunsaturated fatty acid found in egg yolk, mustard, and soybean, is thought to exert anti-inflammatory activity. We investigated the effects of PC on AGEs-induced hepatic and renal cell injury. Materials and Methods: In this study, we evaluated cytokine and NF-κB/MAPK signal pathway activity in AGEs induced human liver (HepG2) cells and human kidney (HK2) cells with and without PC treatment. Results: PC reduced RAGE expression and attenuated levels of inflammatory cytokines and NF-kB/MAPK signaling. Moreover, cells treated with PC exhibited a significant reduction in cytotoxicity, oxidative stress, and inflammatory factor levels. Conclusions: These findings suggest that PC could be an effective functional material for hepatic and renal injury involving with oxidative stress caused by AGEs during diabetic conditions.

Sortilin-related receptor 1 interacts with amyloid precursor protein and is activated by 6-shogaol, leading to inhibition of the amyloidogenic pathway.

Sortilin-related receptor 1 (SORL1) is a neuronal sorting protein that reduces amyloid precursor protein (APP) trafficking to secretases that generate amyloid beta (Aß). Although 6-shogaol, a constituent of ginger, has been reported to have anti-inflammatory and anti-oxidant effects on neuronal cells, research regarding the activation of SORL1 has not yet been reported. Here, we aimed to investigate whether 6-shogaol contributes to the increases in SORL1 that are related to Alzheimer's disease (AD). To clarify the effect of 6-shogaol as a possible activator of SORL1, we used SORL1 siRNA as a blockade of SORL1 in hippocampal neuronal cells (HT22). We found that SORL1 siRNA treatment naturally inhibited SORL1 and led to increases in ß-secretase APP cleaving enzyme (BACE), secreted APP-ß (sAPPß) and Aß. In contrast, 6-shogaol-mediated activation of SORL1 significantly downregulated BACE, sAPPß, and Aß in both in vitro HT22 cells and in vivo APPSw/PS1-dE9 Tg mice. Therefore, SORL1 activation by 6-shogaol provides neuronal cell survival through the inhibition of Aß production. These results indicate that 6-shogaol should be regarded as an SORL1 activator and a potential preventive agent for the treatment of AD.

Rutin protects rat articular chondrocytes against oxidative stress induced by hydrogen peroxide through SIRT1 activation.

The progressive degeneration and ossification of articular chondrocytes are main symptoms in the pathogenesis of osteoarthritis (OA). Several flavonoids may provide an adjunctive alternative for the management of moderate OA in humans. Rutin, a natural flavone derivative (quercetin-3-rhamnosylglucoside), is well known for its potent anti-inflammatory and anti-oxidant properties against oxidative stress. However, the protective function of rutin related to OA, which is characterized by deterioration of articular cartilage, remains unclear. The present study investigated the protective effects of rutin, an activator of silent information regulator 1 (SIRT1), involved in the inhibition of NF-κB/MAPK signaling pathway in hydrogen peroxide (H2O2)-induced oxidative stress in rat chondrocytes. SIRT1 activation by rutin attenuated levels of inflammatory cytokines and NF-κB/MAPK signaling, whereas the inhibition of SIRT1 by sirtinol counteracted the beneficial effects of rutin in H2O2-treated chondrocytes. The findings of these studies suggested the potential involvement of SIRT1 in the pathogenesis of OA, and indicated that rutin is a possible therapeutic option for OA.

6-Shogaol has anti-amyloidogenic activity and ameliorates Alzheimer's disease via CysLT1R-mediated inhibition of cathepsin B.

Although 6-shogaol, a constituent of ginger, has been reported to have anti-inflammatory and anti-oxidant effects on neuronal cells, the effects of 6-shogaol on Alzheimer's disease (AD) have not yet been investigated. Here we aimed to determine whether 6-shogaol exerts neuroprotective effects against AD. Specifically, we investigated the effects of 6-shogaol on the cysteinyl leukotriene 1 receptor (CysLT1R), a major factor in AD pathogenesis. Moreover, we clarified the relationship between CysLT1R and cathepsin B, a cysteine protease. We used in vitro and in vivo models to determine whether 6-shogaol inhibits CysLT1R/cathepsin B in an amyloid-beta (Aß; 1-42)-induced model of neurotoxicity. We first confirmed that CysLT1R and cathepsin B are upregulated by Aß (1-42) and that CysLT1R activation induces cathepsin B. In contrast, we found that 6-shogaol-mediated inhibition of CysLT1R downregulates cathepsin B in both in vitro and in vivo models. Furthermore, we found that 6-shogaol-mediated inhibition of CysLT1R/cathepsin B reduces Aß deposition in the brain and ameliorates behavioral deficits in APPSw/PS1-dE9 Tg mice. Our results indicate that 6-shogaol is a CysLT1R/cathepsin B inhibitor and is a novel potential therapeutic agent for the treatment of various neurodegenerative diseases, including AD.

Actin cytoskeletal rearrangement and dysfunction due to activation of the receptor for advanced glycation end products is inhibited by thymosin beta 4.

KEY POINTS: Thymosin beta 4 (Tß4 ) attenuates the vascular cellular toxicity induced by advanced glycation end products (AGEs) in human umbilical vein endothelial cells (HUVECs). Tß4 reduces expression of both the receptor of AGEs (RAGE) and the filamentous actin (F-actin) to globular actin (G-actin) ratio. RAGE expression was regulated by actin cytoskeleton involved in Tß4 . Tß4 attenuates the vascular cellular toxicity induced by AGEs via remodelling of the actin cytoskeleton. AGEs attenuate vascular-like tube formation of HUVECs, which is reversed by Tß4 via remodelling of the actin cytoskeleton. ABSTRACT: The receptor of advanced glycation end products (RAGE) is a cell-surface receptor that is a key factor in the pathogenesis of diabetic complications, including vascular disorders. Dysfunction of the actin cytoskeleton contributes to disruption of cell membrane repair in response to various type of endothelial cell damage. However, mechanism underlying RAGE remodelling of the actin cytoskeleton, by which globular actin (G-actin) forms to filamentous actin (F-actin), remains unclear. In this study we examined the role of thymosin beta 4 (Tß4 ) - which binds to actin, blocks actin polymerization, and maintains the dynamic equilibrium between G-actin and F-actin in human umbilical vein endothelial cells (HUVECs) - in the response to RAGE. Tß4 increased cell viability and decreased levels of reactive oxygen species in HUVECs incubated with AGEs. Tß4 reduced the expression of RAGE, consistent with a down-regulation of the F-actin to G-actin ratio. The effect of remodelling of the actin cytoskeleton on RAGE expression was clarified by adding Phalloidin, which stabilizes F-actin. Moreover, small interfering RNA was used to determine whether intrinsic Tß4 regulates RAGE expression in the actin cytoskeleton. The absence of intrinsic Tß4 in HUVECs evoked actin cytoskeleton disorder and increased RAGE expression. These findings suggest that regulation of the actin cytoskeleton by Tß4 plays a pivotal role in the RAGE response to AGEs.

Thymosin beta 4 improves dermal burn wound healing via downregulation of receptor of advanced glycation end products in db/db mice.

BACKGROUND: The delay of dermal burn wound healing caused by vascular disorders is a critical problem for many diabetic patients. Thymosin ß4 (Tß4), identified by subtractive cloning of endothelial cells on plastic versus basement membrane substrates, has been found to promote angiogenesis and dermal wound repair in rats, aged mice, and db/db diabetic mice. However, previous studies involving the role of Tß4 in wound repair were limited to mechanical damage and dermal impairment. Thus, this study aimed to evaluate the improvement of healing of burn wounds by Tß4 in relation to advanced glycation end products (AGE), which are pathological factors in diabetes. METHODS: We adapted a dermal burn wound in vivo model in which the dorsal skin of db/db mice was exposed for 10s to 100°C heated water to produce a deep second-degree burn 10mm in diameter. Five mg/kg of Tß4 was then injected intradermally near the burn wound twice a week for 2weeks. RESULTS: After treatment, Tß4 improved wound healing markers such as wound closure, granulation, and vascularization. Interestingly, Tß4 reduced levels of receptor of AGE (RAGE) during the wound healing period. CONCLUSIONS: Tß4 exerts effects to remedy burn wounds via downregulation of RAGE. GENERAL SIGNIFICANCE: Our results suggest the potential importance of Tß4 as a new therapy for impaired burn wound healing that is associated with diabetes.

Hepatoprotective effect of phosphatidylcholine against carbon tetrachloride liver damage in mice.

It has been shown that phosphatidylcholine (PC) extracted from egg yolk possesses a variety of biological activities, such as anti-inflammatory and anti-oxidant effects, and prevents oxidative stress. The aim of this study was to evaluate the hepatoprotective effects of PC against carbon tetrachloride (CCl4), which is a well-known hepatotoxicant that causes extensive oxidative liver damage, and to investigate the mechanisms involved in this protective effect. Mice were treated with PC (0.1 ml, 10 or 100 mg/kg, orally) once daily for 5 consecutive days prior to CCl4 administration (0.1 ml, 20 mg/kg, intraperitoneally). The experimental data show that pretreatment with PC significantly prevented increases of serum aspartate transaminase, alanine transaminase, and alkaline phosphatase, and reduced reactive oxygen species levels. Histopathological evaluation of the liver also revealed that PC effectively ameliorated CCl4-induced hepatic injury and fibrosis. In addition, PC significantly counteracted the increase in glutathione levels and glutathione-S-transferase activity induced by CCl4. Concordantly, PC significantly decreased CCl4-induced upregulation of apoptotic proteins in the liver. These results suggest that PC exerts its protective effects against CCl4-induced hepatotoxicity via its activities as an anti-oxidant and free radical scavenger.

Rutin upregulates neurotrophic factors resulting in attenuation of ethanol-induced oxidative stress in HT22 hippocampal neuronal cells.

BACKGROUND: Alcoholism, which refers to the excessive consumption of alcohol, has deleterious effects on personal and social health worldwide. Oxidative stress evoked by ethanol plays an important role in the pathogenesis of neurodegenerative diseases. Rutin is a bioflavonoid that has been demonstrated to scavenge superoxide radicals. However, the effects of rutin on neuronal toxicity following ethanol-induced oxidative stress have not previously been investigated. Thus we investigated the antioxidant effect of rutin in hippocampal neuronal cells (HT22 cells) exposed to ethanol. RESULTS: We found that rutin pretreatment prevented the ethanol-induced decrease in protein level expression of nerve growth factor, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in HT22 cells. Cell viability as analyzed by the MTT method revealed a significant increase in cell viability in the rutin-treated group compared with the ethanol-only treated group. Antioxidant effect of rutin was confirmed to be due to reduction of intracellular reactive oxidative species production in ethanol-treated HT22 cells. Moreover, rutin significantly increased the level of the antioxidant glutathione, and the activities of the antioxidant enzymes superoxide dismutase and catalase. CONCLUSION: These findings indicate that rutin has potential as a therapeutic agent to treat alcohol-related neurodegenerative disorders.

COMP-Ang1 inhibits apoptosis as well as improves the attenuated osteogenic differentiation of mesenchymal stem cells induced by advanced glycation end products.

BACKGROUND: In the present study, we have investigated the possibility that cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), important factor in angiogenesis, osteogenesis and the survival of mesenchymal stem cells (MSCs) through the Ang1/Tie2 pathway has beneficial effects on osteogenic differentiated cells (ODCs) from MSCs treated by advanced glycation end products (AGE), which are pathological factors of diabetes. METHODS: Primary culture of MSCs was used. For comparison analysis of AGE and COMP-Ang1 effects, we performed cell viability assay with each treated variety concentration for 24h. Apoptosis rate and Caspase-3 activity were measured by each ELISA assay. To make sure with Ang1/Tie2 pathway, we performed small interfering RNA transfected to MSCs. Real-time RT-PCR was performed to identify ODCs marker genes. Immunoblotting was used to evaluate the expression of Tie2, AKT, p38 and ERK. RESULTS: Our results clearly demonstrate that COMP-Ang1 upregulates the phosphorylation of AKT and p38 by activating the Ang1/Tie2 signaling pathway, indicating that COMP-Ang1 affects both AGE-induced apoptosis and the attenuated osteogenic differentiation of MSCs through the p38/MAPK and PI3K/AKT pathways. CONCLUSIONS: COMP-Ang1 improves cell viability and differentiation function of ODCs against AGE via Ang/Tie2 signaling pathway. GENERAL SIGNIFICANCE: Our results suggest the potential importance of COMP-Ang1 as a new therapy for impaired bone formation that is associated with diabetes and advanced age.

COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

Rutin alleviates prion peptide-induced cell death through inhibiting apoptotic pathway activation in dopaminergic neuronal cells.

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and accumulation of the abnormal form of the scrapie prion protein (PrP). Rutin is a flavonoid that occurs naturally in plant-derived beverages and foods and is used in traditional and folkloric medicine worldwide. In the present study, we evaluated the protective effects of rutin against PrP fragment (106-126)-induced neuronal cell death. Rutin treatment blocked PrP (106-126)-mediated increases in reactive oxygen species production and nitric oxide release and helped slowing the decrease of neurotrophic factors that results from PrP accumulation. Rutin attenuated PrP (106-126)-associated mitochondrial apoptotic events by inhibiting mitochondrial permeability transition and caspase-3 activity and blocking expression of the apoptotic signals Bax and PARP. Additionally, rutin treatment significantly decreased the expression of the death receptor Fas and its ligand Fas-L. Overall, our results demonstrated that rutin protects against the neurodegenerative effects of prion accumulation by increasing production of neurotropic factors and inhibiting apoptotic pathway activation in neuronal cells. These results suggested that rutin may have clinical benefits for prion diseases and other neurodegenerative disorders.

Comparative analysis of superovulated versus uterine-embryo synchronized recipients for embryo transfer in cynomolgus monkeys (Macaca fascicularis).

Introduction: Assisted reproductive technologies (ARTs), such as intracytoplasmic sperm injection and embryo transfer, are essential for generating genetically edited monkeys. Despite their importance, ARTs face challenges in recipient selection in terms of time and the number of animals required. The potential of superovulated monkeys, commonly used as oocyte donors, to serve as surrogate mothers, remains underexplored. The study aimed to compare the efficacy of superovulated and uterine-embryo synchronized recipients of embryo transfer in cynomolgus monkeys (Macaca fascicularis). Methods: This study involved 23 cynomolgus monkeys divided into two groups-12 superovulated recipients and 11 synchronized recipients. The evaluation criteria included measuring endometrial thickness on the day of embryo transfer and calculating pregnancy and implantation rates to compare outcomes between groups. Results: The study found no statistically significant differences in endometrial thickness (superovulated: 4.48 ± 1.36 mm, synchronized: 5.15 ± 1.58 mm), pregnancy rates (superovulated: 30.8%, synchronized: 41.7%), and implantation rates (superovulated: 14.3%, synchronized: 21.9%) between the groups (p > 0.05). Conclusion: The observations indicate that superovulated recipients are as effective as synchronized recipients for embryo transfer in cynomolgus monkeys. This suggests that superovulated recipients can serve as viable options, offering an efficient and practical approach to facilitate the generation of gene-edited models in this species.

Subcutaneous leiomyosarcoma in a cynomolgus macaque (Macaca fascicularis).

Leiomyosarcoma, a malignant tumour originating from smooth muscle cells, has rarely been documented in non-human primates. In this case study, a 7-year-old female cynomolgus macaque (Macaca fascicularis) presented with a rapidly growing mass overlying the left elbow joint. Radiographs indicated the presence of a soft tissue neoplasm without any associated bone involvement. The mass was surgically resected. Histological and immunohistochemical analyses revealed spindle-shaped cells with eosinophilic cytoplasm that resembled smooth muscle cells, exhibiting positive immunoreactions for vimentin, desmin and smooth muscle actin and a negative reaction for pan-cytokeratin. This is the first reported case of subcutaneous leiomyosarcoma in a cynomolgus macaque and provides important insights into the incidence and characteristics of this condition in this species.

Application of ultrasonographic human estimated foetal weight formulas to cynomolgus monkeys (Macaca fascicularis) at 129-132 days of gestation: A comparative study of estimated and actual birthweight.

BACKGROUND: Cynomolgus monkeys (Macaca fascicularis) are essential in biomedical research, including reproductive studies. However, the application of human estimated foetal weight (EFW) formulas using ultrasonography (USG) in these non-human primates is not well established. OBJECTIVES: This study aims to evaluate the applicability of human EFW formulas for estimating foetal weight in cynomolgus monkeys at approximately 130 days of gestation. METHODS: Our study involved nine pregnant cynomolgus monkeys. We measured foetal parameters, including biparietal diameter, head circumference, abdominal circumference and femur length using USG. The EFW was calculated using 11 human EFW formulas. The actual birthweight (ABW) was recorded following Cesarean section, the day after the EFW calculation. For comparing EFW and ABW, we employed statistical methods such as mean absolute percentage error (APE) and Bland-Altman analysis. RESULTS: The ABW ranged between 200.36 and 291.33 g. Among the 11 formulas, the Combs formula showed the lowest APE (4.3%) and highest correlation with ABW (p < 0.001). Notably, EFW and ABW differences for the Combs formula were ≤5% in 66.7% and ≤10% in 100% of cases. The Bland-Altman analysis supported these results, showing that all cases fell within the limits of agreement. CONCLUSIONS: The Combs formula is applicable for estimating the weight of cynomolgus monkey fetuses with USG at approximately 130 days of gestation. Our observations suggest that the Combs formula can be applied in the prenatal care and biomedical research of this species.

COMP-Ang1 accelerates chondrocyte maturation by decreasing HO-1 expression.

Endochondral ossification is essential for new bone formation and remodeling during the distraction stage. Endochondral ossification is attributed to chondrocyte maturation, which is induced by various factors, such as the cellular environment, gene transcription, and growth factor expression. Cartilage oligomeric matrix protein (COMP)-angiopoietin 1 (Ang1) is more soluble, stable, and potent than endogenous Ang1, and COMP-Ang1 treatment has osteogenic and angiogenic effects in an in vivo model of bone fracture healing. Although the osteogenic effects of COMP-Ang1 have been demonstrated, the precise mechanism by which COMP-Ang1 induces chondrocyte maturation and triggers endochondral ossification is not understood. Here, we investigated the possible mechanism by which COMP-Ang1 induces chondrocyte maturation. First, using a WST assay, we found that COMP-Ang1 is nontoxic in rat chondrocytes. Then, we isolated total RNA from COMP-Ang1-treated rat chondrocytes, and analyzed the decrease in chondrogenic gene expression and the increase in osteogenic gene expression using real-time RT-PCR. Gene and protein expression of heme oxygenase-1 (HO-1), which maintains chondrocytes in an immature stage, decreased in a dose-dependent manner upon COMP-Ang1 treatment. To clarify the relationship between HO-1 and COMP-Ang1 in chondrocyte maturation, we used cobalt protoporphyrin IX (CoPP IX), an HO-1 inducer, and tin protoporphyrin IX (SnPP-IX), an HO-1 inhibitor. Treatment with various combinations of CoPP IX, SnPP IX, and COMP-Ang1 confirmed that COMP-Ang1 accelerates chondrocyte maturation by reducing HO-1. In conclusion, our results suggest that COMP-Ang1 accelerates chondrocyte maturation by interacting with HO-1.

[6]-shogaol attenuates neuronal apoptosis in hydrogen peroxide-treated astrocytes through the up-regulation of neurotrophic factors.

Neuronal apoptosis induced by oxidative stress is a prominent feature of neurodegenerative disorders. [6]-shogaol, a bio-active compound in ginger, possesses potent anti-inflammatory actions and has recently emerged as a potential therapeutic agent for neurodegenerative disorders. However, the effects of [6]-shogaol on astroglial apoptosis following exogenously induced oxidative stress has not yet been investigated. Here, we show that the anti-apoptotic activity of [6]-shogaol in astrocytes following exposure to hydrogen peroxide (H2 O2 ) involves a marked up-regulation of neurotrophic factors such as nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor. Astrocytes co-treated with [6]-shogaol and H2 O2 for 1 h showed decrease in reactive oxygen species production compared with those only treated with H2 O2 . Moreover, [6]-shogaol counteracted the reduced expression of ERK1/2 in H2 O2 -treated astrocytes and protected these cells from oxidative stress and apoptosis by attenuating the impairment of mitochondrial function proteins such as Bcl-2 and Bcl-xL. Additionally, [6]-shogaol inhibits the expression of the apoptotic proteins Bax and caspase-3 in H2 O2 -treated astrocytes. This data suggest that following oxidative stress, [6]-shogaol protects astrocytes from oxidative damage through the up-regulating levels of neurotrophic factors. These findings provide further support for the use of [6]-shogaol as a therapeutic agent in neurodegenerative disorders.

Chemoprevention of a flavonoid fraction from Rhus verniciflua Stokes on aflatoxin B1-induced hepatic damage in mice.

Since aflatoxin B(1) (AFB(1))-mediated hepatic damage is related to the production of AFB(1)-8,9-epoxide and reactive oxygen species, bioactive compounds having antioxidant potentials are suggested to be capable of reducing AFB(1)-induced toxicity. We previously purified a mixture of flavonoids that we named RCMF (Rhus verniciflua Stokes chloroform-methanol fraction), from a traditional Korean food additive and herbal medicine. RCMF exhibited various biological effects, including antioxidant and antitumor activities. In this study, we examined whether RCMF protects against AFB(1)-induced liver injury using in vitro and in vivo systems. Pretreatment of HepG2 cells with RCMF significantly reduced AFB(1)-stimulated production of ROS and malondialdehyde (MDA) to the control levels. RCMF also prevented the reduction in HepG2 cell viability caused by AFB(1). Oral administration of RCMF to mice significantly suppressed an AFB(1)-induced increase in serum levels of alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase. It also prevented MDA formation and blocked decreases in glutathione levels and superoxide dismutase activities in the livers of AFB(1)-treated mice. In addition, RCMF supplementation prevented an AFB(1) -induced decrease in serum titers of IgA and IgG1. Collectively, these results suggest that RCMF attenuates AFB(1)-mediated damage to the liver, and that this effect is at least partially related to the restoration of antioxidant defense systems and an increase in AFB(1)-GSH conjugate formation.