Isolation, Structure Elucidition, and Immunosuppressive Activity of Diterpenoids from Ligularia fischeri.

Six new (1-3 and 6-8) and seven known diterpenoids were isolated from the whole plant of Ligularia fischeri. Compound 1 is a new 15,16-dinorerythroxylane-type diterpenoid possessing a C18 skeleton, and 2 is the first example of a 6/6/6/6/5/5-fused hexacyclic ent-kaurane diterpenoid with 19,20-olide and 11,16-epoxy moieties. The structures of the new compounds were elucidated by spectroscopic analysis and chemical methods. The absolute configurations of 1 and 7 were determined by single-crystal X-ray diffraction. Compounds 1-13 were evaluated for their immunosuppressive activity, and 4, 7, and 13 showed moderate inhibitory activities against human B lymphoblast HMy2.CIR cells with IC50 values of 56.3 ± 2.2, 13.3 ± 0.8, and 31.4 ± 0.9 µM, respectively.

Therapeutic effect of mitochondrial transplantation on burn injury.

As mitochondrial damage or dysfunction is commonly observed following burn injuries, we investigated whether mitochondrial transplantation (MT) can result in therapeutic benefits in the treatment of burns. Human immortalized epidermal cells (HaCaT) and Kunming mice were used to establish a heat-injured cell model and a deep partial-thickness skin burn animal model, respectively. The cell model was established by exposing HaCaT cells to 45 or 50 °C for 10 min, after which cell proliferation was assayed using fluorescent double-staining and colony formation assays, cell migration was assessed using colloidal gold migration and scratch assays, and cell cycle progression and apoptosis were measured by flow cytometry. Histopathological staining, immunohistochemistry, nick-end labeling analysis, and enzyme-linked immunosorbent assays were used to evaluate the effects of MT on inflammation, tissue recovery, apoptosis, and scar growth in a mouse model. The therapeutic effects were observed in the heat-injured HaCaT cell model. MT promoted cell viability, colony formation, proliferation, and migration; decreased G1 phase; promoted cell division; and decreased apoptosis. Wound-healing promotion, anti-inflammation (decreased mast cell aggregation, down-regulated of TNF-α, IL-1ß, IL-6, and up-regulated IL-10), acceleration of proliferation recovery (up-regulated CD34 and VEGF), apoptosis reduction, and scar formation reduction (decreased collagen I/III ratio and TGF-ß1) were observed in the MT mouse model. The MT mode of action was, however, not investigated in this study. In conclusion, our data indicate that MT exerts a therapeutic effect on burn injuries both in vitro and in vivo.

Baicalin attenuates acute skin damage induced by ultraviolet B via inhibiting pyroptosis.

As the outermost layer of the human body, the skin suffers from various external factors especially light damage, among which ultraviolet B (UVB) irradiation is common and possesses a relatively high biological damage capacity. Pyroptosis is a newly discovered type of programmed cell death, which can induce cell rupture and induce local inflammatory response. However, the molecular mechanisms of pyroptosis in photodamaged skin is poorly understood. Baicalin, a flavonoid extracted from the desiccated root of Scutellaria baicalensis Georgi (Huang Qin). Despite its antioxidant abilities, whether baicalin protects skin by attenuating UVB-induced pyroptosis remains unclear, which was the aim of this study. The UVB-induced acute skin damage model was established by using human immortalized keratinocytes (HaCaT cells) and Kunming (KM) strain mice. The protective dose selection for baicalin is 50 µM in vitro and 100 mg/kg in vivo. In in vitro study, UVB irradiation significantly decreased cell viability, increased cell death and oxidative stress in HaCaT cells, while pretreatment with baicalin improved these phenomena. Furthermore, the baicalin pretreatment notably suppressed nuclear factor kappa B (NF-κB) translocation, the NLRP3 inflammasome activation and gasdermin D (GSDMD) maturation, thus effectively attenuating UVB-induced pyroptosis. In in vivo study, the baicalin pretreatment mitigated epidermal hyperplasia, collagen fiber fragmentation, oxidative stress and pyroptosis in UVB-irradiated mouse skin. In a nutshell, this study suggests that baicalin could be a potential protective agent to attenuate acute skin damage induced by UVB irradiation through decreasing oxidative stress and suppressing NF-κB/NLRP3/GSDMD-involved pyroptosis.

Pyroptosis as a double-edged sword: The pathogenic and therapeutic roles in inflammatory diseases and cancers.

Pyroptosis is a programmed cell death mode discovered in recent years. It is caused by inflammasomes and the perforation of Gasdermin family proteins, and results in the release of inflammatory factors and triggering of an inflammatory cascade response. The pathways of pyroptosis include the caspase-1-dependent canonical pathway, the caspase-4/5/11-dependent non-canonical pathway, other caspase-dependent pathways and caspase-independent pathways. Its morphological features are different from other programmed cell death modes (apoptosis, autophagy, etc.). Pyroptosis can be observed microscopically that abundant pores are formed in the cell membrane, resulting in cell swelling and rupture, and eventually leading to the outflow of cellular contents. In addition to causing tissue damage and dysfunction through inflammation, pyroptosis can also become a potential cancer treatment strategy by reducing drug resistance in cancer cells. However, many details are still unclear on the molecular mechanisms of its role in pathogenicity and therapeutics, and therefore lots of work needs to be done. This article reviews the morphological characteristics, pathogenic and therapeutic mechanisms of pyroptosis and its related research progress in inflammatory diseases and cancers. It helps to further understand the mechanism of pyroptosis and provide new ideas for the research and prevention of inflammatory diseases and cancers.

Lycium barbarum polysaccharide inhibits blue-light-induced skin oxidative damage with the involvement of mitophagy.

Although blue light can damage the skin to a certain extent, the pathogenesis of its damage remains still unclear. The available evidence suggests that oxidative stress may be the main cause of its damage. Lycium barbarum polysaccharide (LBP) has antioxidative effects in a variety of cells. In this paper, we investigated the protective role of LBP and its mechanism of action related to mitophagy in blue-light-damaged skin cells. The findings indicated that in HaCaT cells and mouse skin, LBP pretreatment was effective in reducing blue-light-induced apoptosis and ameliorating the elevated level of cellular autophagy/mitophagy caused by excessive blue light exposure. The markers reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were used to assess oxidative stress. LBP could effectively inhibit blue-light-induced oxidative stress. It was also found that blue light exposure caused mitochondrial dysfunction in HaCaT cells, including increased intracellular calcium ion levels and decreased mitochondrial membrane potential. LBP pretreatment significantly relieved mitochondrial dysfunction in HaCaT cells. These findings imply that LBP pretreatment protects skin cells from damage induced by blue light irradiation and that mitophagy may be a significant factor in skin photodamage.

NADPH oxidase is involved in H2O2-induced differentiation of human promyelocytic leukaemia HL-60 cells.

The expression and activity of NADPH oxidase increase when HL-60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150-500 µM H2O2 inducing differentiation of HL-60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 µM H2O2. However, H2O2 stimulated the generation of intracellular superoxide (O2•-), which decreased in the presence of the two inhibitors. DPI also inhibited H2O2-induced ERK (extracellular-signal-regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL-60 cells induced by H2O2. This shows that H2O2 can activate NADPH oxidase, leading to O2•- production, followed by ERK activation and ultimately resulting in the differentiation of HL-60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.

Lycium Barbarum polysaccharide protects HaCaT cells from PM2.5-induced apoptosis via inhibiting oxidative stress, ER stress and autophagy.

Objectives: Lycium barbarum polysaccharide (LBP) is a natural polysaccharide extracted from Lycium barbarum that has anti-inflammatory, anti-apoptotic and anti-aging effects, and plays a role in the prevention and treatment of various diseases. In this study, we investigated the therapeutic effect of LBP on particulate matter 2.5 (PM2.5)-induced skin damage.Methods: Cell viability was analyzed by MTT and LDH assays. Apoptosis was analyzed by Annexin V-FITC/PI staining. Oxidative stress/damage were assessed by intracellular ROS levels, MDA content and SOD activity. The intracellular protein expression was analyzed by Western blot. Mitochondrial damage was assayed by mitochondrial membrane potential with JC-1 probe. LC3-GFP adenovirus was transfected into HaCaT cells to analyze intracellular autophagosome levels.Results: In PM2.5-treated HaCaT cells, LBP pretreatment reduced PM2.5-induced cytotoxicity, ameliorated cell morphology and reduced cell apoptosis. LBP also inhibited the expression levels of GRP78 and CHOP, reduced the conversion of LC3I to LC3II, inhibited Bax protein and activated Bcl-2 protein. Furthermore, LBP inhibited PM2.5-induced mitochondrial autophagy (mitophagy) and mitochondrial damage. PM2.5-induced autophagy was regulated by endoplasmic reticulum (ER) stress.Conclusion: LBP protects skin cells from PM2.5-induced cytotoxicity by regulating the oxidative stress-ER stress-autophagy-apoptosis signaling axis, revealing that LBP has a great potential for the skin protection.

Carbonation Resistance and Pore Structure of Mixed-Fiber-Reinforced Concrete Containing Fine Aggregates of Iron Ore Tailings.

The disposal of industrial by-product tailings has become an important issue in solving environmental pollution. In this study, 15%, 30%, 50%, and 70% iron tailings were used to replace the natural sand in concrete, and 1.5% steel fiber and 0-0.75% PVA fibers were added to the iron tailings concrete. The effects of the iron tailings replacement rate and the fiber content on the mechanical properties, carbonization depth, and concrete porosity were studied in a carbonization environment. The results demonstrated that the compressive and splitting tensile strengths of concrete first increased and subsequently decreased with an increase in the iron tailings replacement rate, while the carbonation depth and porosity initially decreased and subsequently increased. When the replacement rate of iron tailings was 30%, the compressive strength and split tensile strength were increased by 7.6% and 17.7%, respectively, and the porosity was reduced by 8.9%. The compressive strength, carbonation depth and porosity of single-doped steel-fiber concrete were superior to those of ordinary iron tailings concrete. However, compared with single-doped steel fiber, the performance of steel-PVA fiber was further improved. Based on the mechanical properties, the carbonation depth test results of the three aforementioned types of concrete, the mathematical expression of the uniaxial compression stress-strain curve of iron tailings concrete, and the prediction equation of the carbonation depth of mixed-fiber iron tailings concrete were proposed. This study provides a reference for the application and popularization of fiber-reinforced iron tailings concrete in carbonization environments.

Hydrogen sulfide protects retina from blue light-induced photodamage and degeneration via inhibiting ROS-mediated ER stress-CHOP apoptosis signal.

Background: Hydrogen sulfide (H2S) is a small reducing gas molecule with various biological functions such as anti-oxidative, anti-apoptotic and anti-inflammatory activities. In this study, we investigated the therapeutic effects of exogenous H2S in the experimental models of retinal photodamage in vivo and in vitro.Methods: Rats with open eyelids were pretreated with H2S (80~120 µmol/kg) for 10 days and then continuously exposed to blue light (435~445nm, 11.2W/m2) for 8 h to establish in vivo experimental model. ARPE-19 cells were pretreated with H2S and then exposed to blue light to establish in vitro experimental model.Results: In vivo experiments, H2S significantly ameliorated blue light-induced retinal oxidative stress, apoptosis and degeneration. Moreover, H2S inhibited the activation of blue light-induced endoplasmic reticulum (ER) stress CHOP apoptotic signaling. In vitro experiments, H2S improved blue light-induced oxidative stress and oxidative damage. H2S inhibited ROS-mediated activation of ER stress CHOP apoptotic signaling. H2S alleviated blue light-induced apoptosis and increases cell viability. The ER stress inhibitor 4-PBA alleviated blue light-induced apoptosis and increases cell viability.Conclusion: Taken together, these results indicate that H2S can inhibit ROS-mediated ER stress-CHOP apoptosis signal, thereby alleviating blue light-triggered retinal apoptosis and degeneration.

Blue light induces skin apoptosis and degeneration through activation of the endoplasmic reticulum stress-autophagy apoptosis axis: Protective role of hydrogen sulfide.

Research on the phototoxicity of blue light (BL) to the skin is increasing. Although blue light can induce oxidative stress, inflammation, and inhibition of proliferation in skin cells, the mechanism by which blue light damages the skin is not yet clear. Endoplasmic reticulum (ER) stress and autophagy are two mechanisms by which cells resist external interference factors and maintain cell homeostasis and normal function, and both can affect cell apoptosis. Interestingly, we have found that blue light (435 nm ~ 445 nm, 8000 lx, 6-24 h)-induced oxidative stress triggers the ER stress-CHOP (C/EBP homologous protein) signal and affects the protein levels of B-cell lymphoma-2 (Bcl-2) and Bcl2-associated X (Bax), thereby promoting apoptosis. In addition, blue light activates autophagy in skin cells, which intensifies cell death. When ER stress is inhibited, autophagy is subsequently inhibited, suggesting that blue light-induced autophagy is influenced by ER stress. These evidences suggest that blue light induces activation of reactive oxygen species (ROS)-ER stress-autophagy-apoptosis axis signaling, which further induces skin injury and apoptosis. This is the first report on the relationships among oxidative stress, ER stress, autophagy, and apoptosis in blue light-induced skin injury. Furthermore, we have studied the effect of hydrogen sulfide (H2S) on blue light-induced skin damage, and found that exogenous H2S can protect skin from blue light-induced damage by regulating the ROS-ER stress-autophagy-apoptosis axis. Our data shows that when we are exposed to blue light, such as sunbathing and jaundice treatment, H2S may be developed as a protective agent.

Structural integrity is essential for the protective effect of mitochondrial transplantation against UV-induced cell death.

Mitochondrial transplantation (MT) is a new technology developed in recent years, which injects healthy mitochondria directly into damaged tissues or blood vessels to play a therapeutic role. This technology has been studied in many animal models of various diseases including myocardial ischemia, cerebral stroke, liver and lung injury, and even has been successfully used in the treatment of childhood heart disease. MT can quickly improve tissue function within a few minutes after injection. The speed with which MT improves tissue function is frequently questioned, for it is hard to understand how the whole mitochondrion transports to the damaged sites, enters cells and functions within such a short period of time. Are there small molecules of mitochondrial component responsible for the function of MT? To test this hypothesis, we established an ultra-violet (UV)-irradiated HeLa cell model. The results of colony formation, sulforhodamine B (SRB), and Hoechst 33342/PI double staining assay strongly indicated that MT exhibited a significant protective effect against UV irradiation damage. The UV irradiation-induced cell cycle arresting at S phase, apoptosis, mitochondrial membrane potential (MMP) decreasing, and the related apoptosis signaling factors p-IKKα, p-p65, I-κB and the activation of caspase3 were all reversed by MT treatments to some extent. The mechanisms of MT were evaluated through comparing the effect of thermal inactivation, ultrasonic crushing, and repeated freezing and thawing treatments on MT function. These results denied the above hypothesis that mitochondrial component may be responsible for MT, excluded the function of ATP, mtDNA and other small molecules, and indicated that the mitochondria structural integrity is essential. We also evaluated the effect of Ca2+ concentrations (1 and 1.8 mM) on MT, and the results showed no effect was found in this UV-irradiated HeLa cell model. Our data support a potent anti-UV irradiation effect of MT, and that structural integrity of the mitochondria is critical for its function.

Chitooligosaccharides protect rat cortical neurons against copper induced damage by attenuating intracellular level of reactive oxygen species.

A large number of evidence has suggests that dyshomeostasis of the redox-active biometals such as copper and other metal ions can lead to oxidative stress in neurons, which plays a key role in the pathology of neurodegenerative disorders. Chitooligosaccharides (COSs) are biodegradation product of chitosan and demonstrated diverse biological activities, Here we first report that protective effects of COSs (M.W. 1500, DD. 90%) against Cu(II) induced neurotoxicity in primary cultured rat cortical neurons. The toxicity of Cu(II) to cortical neurons was obviously attenuated in a concentration-dependent manner by COSs pretreated. The data derived from lactate dehydrogenase (LDH) release and the Hoechst 33342 assay support the results from MTT assay. After DCFH assay, COSs were found to depress Cu(II) induced elevation in intracellular reactive oxygen species (ROS), These findings suggest that COSs protect against Cu(II) induced neurotoxicity in primary cortical neurons by interfering with an increase in intracellular reactive oxygen species (ROS).

Hydrogen Sulfide Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Apoptosis and Affects Autophagy.

Age-related macular degeneration (AMD) is a major cause of visual impairment and blindness among the elderly. AMD is characterized by retinal pigment epithelial (RPE) cell dysfunction. However, the pathogenesis of AMD is still unclear, and there is currently no effective treatment. Accumulated evidence indicates that oxidative stress and autophagy play a crucial role in the development of AMD. H2S is an antioxidant that can directly remove intracellular superoxide anions and hydrogen peroxide. The purpose of this study is to investigate the antioxidative effect of H2S in RPE cells and its role in autophagy. The results show that exogenous H2S (NaHS) pretreatment effectively reduces H2O2-induced oxidative stress, oxidative damage, apoptosis, and inflammation in ARPE-19 cells. NaHS pretreatment also decreased autophagy levels raised by H2O2, increased cell viability, and ameliorated cell morphological damage. Interestingly, the suppression of autophagy by its inhibitor 3-MA showed an increase of cell viability, amelioration of morphology, and a decrease of apoptosis. In summary, oxidative stress causes ARPE-19 cell injury by inducing cell autophagy. However exogenous H2S is shown to attenuate ARPE-19 cell injury, decrease apoptosis, and reduce the occurrence of autophagy-mediated by oxidative stress. These findings suggest that autophagy might play a crucial role in the development of AMD, and exogenous H2S has a potential value in the treatment of AMD.

An acute lytic cell death induced by xanthohumol obstructed ROS detecting in HL-60 cells.

Serum is an important component in cell culture medium. It also possesses potent antioxidant properties. Therefore, the conventional protocols for detecting reactive oxygen species (ROS) in cultured cells with fluorescent probes include washing and suspending cells with serum-free buffers, such as PBS. This transient serum deprivation is essential for the ROS detecting. Unfortunately, it may also cause unexpected results, which push us to choose more optimal experiment conditions. In the present study, we found an acute lytic cell death induced by xanthohumol (XN), which obstructed ROS detecting in human leukemia cell line HL-60 cells. XN induced ROS burst, caused cell swelling, membrane permeability increase, LDH release, and ultimately an acute lytic cell death and cell rupture. These effects could be alleviated by the antioxidant N-Acetyl-L-cysteine (NAC). Apoptosis, pyroptosis or necroptosis were not observed in this process. Results also indicated that 2% serum addition had already completely scavenged ROS induced by 10 µM XN. Taken together, it is strongly suggested to detecting ROS in a serum-free medium when studying where and how ROS generated in cells. The concentration at the ROS maximum point (10 µM XN in this study) can be selected as the optimal concentration.

Three new triterpenoids from Potentilla multicaulis.

Three new triterpenoids, 19-hydroxy-2,3-secours-12-ene-2,3,28-trioic acid 3- methyl ester (1), 19-hydroxy-1-oxo-2-nor-2,3-secours-12-ene-3,28-dioic acid (2), and (3beta,18alpha,19alpha)-3,28-dihydroxy-20,28-epoxyursan-24-oic acid (3), were isolated from the roots of Potentilla multicaulis. Their structures were elucidated on the basis of spectroscopic methods (IR, HR-ESI-MS, and 1D- and 2D-NMR). Compound 2b exhibited moderate cytotoxic activity against human promyelocytic leukemia (HL-60) cells.

Specific anticancer activity of a new bisabolane sesquiterpene against human leukemia cells inducing differentiation in vitro.

A bisabolane sesquiterpene, rel-(1S,4R,5S,6R)-4,5-diacetoxy-6- [(R)-5-hydroxy-1,5-dimethylhex-3-enyl]- 3-methylcyclohex-2-enyl (Z)-2-methylbut-2 -enoate, which was newly isolated from the roots of Leontopodium longifolium, presented specific anticancer activity against human leukemia HL-60 cells, but did not inhibit proliferation of human hepatoma SMMC-7721 cells and human normal hepatocytes L02 cells. Nitroblue tetrazolium (NBT) reduction, phagocytosis of latex beads, and cell electrophoresis all demonstrated that this bisabolane sesquiterpene presented its anticancer activity against human leukemia HL-60 cells in vitro via inducing cell differentiation. Our results may have implications for treatment of human leukemia with the sesquiterpene.

Diterpenoids from Salvia miltiorrhiza and Their Immune-Modulating Activity.

Danshen, the dried root of Salvia miltiorrhiza (Lamiaceae), is one of the most popular traditional herbal medicines commonly used in China. Recently, danshen has been used as a health-promoting functional tea to prevent diseases by strengthening the human immunity in China. To search for secondary metabolites with immune-modulating activity, a phytochemical investigation was carried out on the roots of S. miltiorrhiza, which led to the isolation of 6 new diterpenoids (1-4, 16, and 20) along with 20 known diterpenoids. The structures and absolute configurations of these new compounds were elucidated on the basis of spectroscopic analysis, X-ray diffraction analysis, calculated optical rotation, and calculated electronic circular dichroism spectra. Among these isolates, compounds 3, 17, 19, and 23 promoted the proliferation of HMy2.CIR, exhibiting a protective effect on lymphocytes at the concentration from 2.50 to 40 µM, whereas compounds 2, 7, 8, 10, 14, 18, 22, and 25 inhibited the cell proliferation in a concentration-dependent manner.

Determination of Hydroxylated Polybrominated Diphenyl Ethers in Chinese Aquatic Products by LC-MS/MS.

In this reported work, a sensitive and reliable method for detecting 11 hydroxylated polybrominated diphenyl ethers (OH-PBDEs) was established by a combination of pressurized liquid extraction (PLE) and liquid chromatography tandem mass spectrometry (LC/MS/MS). The aquatic products (mainly crayfish and grass carp) from 12 cities in China's HuBei province were examined for the presence of the target OH-PBDEs. The analytical process involved first extracting the OH-PBDEs from the crayfish and grass carp using a PLE, followed by purification using gel permeation chromatography. To eliminate the interference from matrix, isotopic dilution was used in the quantitative analysis. Compared with the existing methods, OH-PBDEs were determined without a need for derivatizing, and it was more efficient and quicker. The condition of extraction and cleanup were also optimized. Experimental results showed that the proposed method exhibited a low detection limit of 0.04-0.2 µg/kg, with a wide linearity range 1-400 ng/L and a good linear correlation coefficient (r2 > 0.990) using this method. These results indicated the steadiness of the established method has the advantages of high sensitivity and facile sample preparation without matrix interference.

New bisabolane sesquiterpenes and coumarin from Leontopodium longifolium.

From the roots of Leontopotium longifolium, three new bisabolane sesquiterpenes, rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-1,5-dimethylhexa-3,5-dienyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (1), rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (2), rel-(1R,2S,4R,5S)-4-acetoxy-2-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-5-methylcyclohexyl (Z)-2-methylbut-2-enoate (3), and a new coumarin, 2,3-dihydro-5-hydroxy-2-(1-methylethenyl)-7H-pyrano[2,3-g][1,4]benzodioxin-7-one (4) together with nine known compounds have been isolated. The structures of these compounds were established by spectroscopic methods. Compounds 1 and 2 exhibited moderate cytotoxic activities against human promyelocytic leukemia (HL-60) cells.

Oxidative stress is involved in inhibition of copper on histone acetylation in cells.

Our previous study demonstrates that copper induces histone hypoacetylation by inhibiting histone acetyltransferase (HAT) activity. However, it lacks direct evidences whether copper-inhibited histone acetylation right contributes to the toxicity of copper. Exposure of human leukemia cells (HL-60) to Cu2+ resulted in cell proliferation arrest and a concentration- and time-dependent decrease of histone acetylation. At the same time, Cu2+-induced significant increase of H2O2 and O2.- generation via a concentration- and time-dependent manner too. The histone acetylation was efficiently suppressed by exogenous H2O2, and enhanced by superoxide dismutase (the scavenger of O2.-), catalase (the scavenger of H2O2) or the combination of both, indicating that Cu2+ at least partially inhibited histone acetylation through triggering oxidative stress. Further studies found that sodium butyrate, the inhibitor of histone deacetylase (HDAC), which had no obvious effect on oxidative stress but increased histone acetylation at the concentration of 50 microM, attenuated Cu2+-inhibited cell proliferation, indicating that histone acetylation inhibition is simultaneously involved in the cytotoxicity of Cu2+. Considering the important role of histone acetylation in gene transcription and regulation of cell fate, the present study may open a new door to further understand the mechanism of Cu2+-induced toxicity.