Bone Marrow ; drug effects ; Humans ; Reactive Oxygen Species ; pharmacology

OBJECTIVE: To determine whether salvianolic acid B (Sal B) exerts protective effects on diabetic peripheral neuropathy by attenuating apoptosis and pyroptosis. METHODS: RSC96 cells were primarily cultured with DMEM (5.6 mmol/L glucose), hyperglycemia (HG, 125 mmol/L glucose) and Sal B (0.1, 1, and 10 µ mol/L). Cells proliferation was measured by 3-(4, 5-cimethylthiazol-2-yl)-2, 5-dilphenyltetrazolium bromide assay. Reactive oxygen species (ROS) generation and apoptosis rate were detected by flow cytometry analysis. Western blot was performed to analyze the expressions of poly ADP-ribose polymerase (PARP), cleaved-caspase 3, cleaved-caspase 9, Bcl-2, Bax, NLRP3, ASC, and interleukin (IL)-1β. RESULTS: Treatment with HG at a concentration of 125 mmol/L attenuated cellular proliferation, while Sal B alleviated this injury (P<0.05). In addition, Sal B inhibited HG-induced ROS production and apoptosis rate (P<0.05). Furthermore, treatment with Sal B down-regulated HG-induced PARP, cleaved-caspase 3, cleaved-caspase 9, Bax, NLRP3, ASC, and IL-1β expression, but mitigated HG-mediated down-regulation of Bcl-2 expression (P<0.05). CONCLUSION Sal B may protect RSC96 cells against HG-induced cellular injury via the inhibition of apoptosis and pyroptosis activated by ROS.
Apoptosis ; Benzofurans/pharmacology* ; Oxidative Stress ; Pyroptosis ; Reactive Oxygen Species/metabolism*

OBJECTIVETo investigate the effect of deltamethrin (DM) on production of reactive oxygen species (ROS) of rat pheochromocytoma (PC12) cells and its mechanism.

METHODSPC12 cells were treated with various dose of DM (0, 10 or 100 micromol/L) for 1, 6 or 12 h respectively. Furthermore, PC12 cells were treated with various dose of DM (0, 10 or 100 micromol/L) for 24 or 48 h, respectively. PC12 cells were pre-incubated with 10 mmol/L N-acetyl-L-cysteine (NAC) for 2 h, or with 500 micromol/L DL-Buthionine-[S, R]-Sulfoximine (BSO) for 16 h, or with 40 micromol/L tertiary butylhydroquinone (tBHQ) for 16 h, prior to exposure to DM and then with 10 micromol/L DM for 6 h. After treatment, ROS production in PC12 cells were measured by a molecular probe, 2', 7'-dichlorofluorescein diacetate (DCFH-DA).

RESULTSDM induced a dose-time dependent increase in ROS production (indicated by DCF fluorescence intensity). 10 micromol/L DM treatment for 6 h enhanced DCF fluorescence intensity that reached approximately 2.24 times of values of control group. Furthermore, a pretreatment with NAC, BSO or tBHQ significantly reduced the DM-enhanced DCF fluorescence intensity that reached approximately 22%, 62% or 38% of values of DM treatment, respectively (P < 0.05), indicating that all these pretreatments attenuate ROS production.

CONCLUSIONThe in vitro studies demonstrate that DM could enhance ROS production, and may be the influential factor for the decreased mercapto level and antioxidative function.

OBJECTIVETo investigate the effect of luteolin on cell growth and apoptosis of HepG2 cells in vitro.

METHODSCultured HepG2,HL60,A549 and LO2 cells were treated with luteolin for different doses (0 μg/ml,2.5 μg/ml,10 μg/ml and 20 μg/ml) and varied times (0 h,24 h,48 h and 72 h). Cell viability was measured with MTT assay and IC50 was calculated. The reactive oxygen species (ROS) levels in HepG2 cells treated with luteolin for 6 h and 12 h were measured with flow cytometry. Cell apoptosis of HepG2 cells treated with luteolin for 24h was examined with flow cytometry and Annexin V-FITC/PI. Expression levels of apoptosis pathway proteins (p53,ASPP2 and iASPP) in HepG2 cells were detected with western blot and the dose and time-effect was analyzed.

RESULTSLuteolin effectively inhibited tumor cell proliferation in a dose-and time-dependent manner,and the inhibition rates of 20 μg/ml Luteolin for 72 h were 39.34%,62.90%,57.57% and 62.90% to LO2,HepG2, HL60 and A549 cells,respectively. The intracellular ROS level was decreased in HepG2 cells by 13.88% and 41.11% after being treated with luteolin for 6 h and 12 h,respectively. The apoptosis rate of HepG2 cells treated with luteolin for 24 h was 14.43%,and western blot showed that luteolin reduced the expression level of iASPP by 77.07% and up-regulated the expression of p53 by 179.37% and ASPP2 by 725.02% in HepG2 cells treated with luteolin for 12 h.

CONCLUSIONLuteolin has ant-proliferative and pro-apoptotic activity on hepatoma HepG2 cells, which is associated with the altered expression of pro-apoptotic factors and decreased ROS level in HepG2 cells.

OBJECTIVESTo observe ultrastructural changes of spermatozoa after incubate with reactive oxygen species (ROS).

METHODSSpermatozoa of normal physiological functions selected from semen samples by Percoll gradient centrifugation technique were regarded as normal sperm models in present study. Ultrastructural changes of spermatozoa observed by transmission electron microscope after model spermatozoa were incubated with ROS generated by hypoxanthine and xanthine oxidase under aerobic environment.

RESULTSAfter model spermatozoa were incubated with ROS, impairment of various extent in membrane and acrosome of spermatozoa and abnormality in mitochondria of spermatozoa were found.

CONCLUSIONSExcessive ROS may cause ultrastructural change in membrane, acrosome and mitochondria of spermatozoa and impair function of spermatozoa.

Catalpol is an iridoid glycoside extracted from the root of Rehmannia glutinosa. It has been reported to have antioxidant stress effects. Adenosine 5' monophosphate-activated protein kinase( AMPK) plays an important role in inhibiting oxidative stress. This study was designed to investigate the protective effects of catalpol on TNF-α-exposed human aorta epithelial cells( HAECs) via inhibit oxidative stress,and the relationship between catalpol and AMPK was detected by RNA interference technique. Levels of superoxide dismutase( SOD),malonaldehyde( MDA),glutathione( GSH) and lactate dehydrogenase( LDH) were measured with a colorimetric assay kit. The level of ROS was measured with FACS calibur. Western blot was employed to detect the protein expression of AMPK,phosphorylated-AMPK and NOX4. Finally,RNA interference technique was used to investigate the role of AMPK in catalpol-induced protective effects. TNF-α treatment decreased the expression of phosphorylated-AMPK protein level,however,catalpol could reverse the decreased phosphorylated-AMPK level. Catalpol could inhibit NOX4 protein expression and decrease ROS overproduction. After using AMPK siRNA that effects of catalpol on ROS overproduction and NOX4 protein expression inhibition were attenuated. The above results suggest that catalpol inhibits oxidative stress in TNF-α-exposed HAECs by activating AMPK.
Humans ; Iridoid Glucosides ; pharmacology ; Iridoids ; Oxidative Stress ; Reactive Oxygen Species ; Tumor Necrosis Factor-alpha

Mitophagy is a process whereby cells selectively remove mitochondria through the mechanism of autophagy, which plays an important role in maintaining cellular homeostasis. In order to explore the effect of mitophagy genes on the antioxidant activities of Saccharomyces cerevisiae, mutants with deletion or overexpression of mitophagy genes ATG8, ATG11 and ATG32 were constructed respectively. The results indicated that overexpression of ATG8 and ATG11 genes significantly reduced the intracellular reactive oxygen species (ROS) content upon H₂O₂ stress for 6 h, which were 61.23% and 46.35% of the initial state, respectively. Notable, overexpression of ATG8 and ATG11 genes significantly increased the mitochondrial membrane potential (MMP) and ATP content, which were helpful to improve the antioxidant activities of the strains. On the other hand, deletion of ATG8, ATG11 and ATG32 caused mitochondrial damage and significantly decreased cell vitality, and caused the imbalance of intracellular ROS. The intracellular ROS content significantly increased to 174.27%, 128.68%, 200.92% of the initial state, respectively, upon H₂O₂ stress for 6 h. The results showed that ATG8, ATG11 and ATG32 might be potential targets for regulating the antioxidant properties of yeast, providing a new clue for further research.
Mitophagy/genetics* ; Saccharomyces cerevisiae/genetics* ; Antioxidants ; Hydrogen Peroxide/pharmacology* ; Reactive Oxygen Species

Mitochondrial reactive oxygen species (mROS) that are overproduced by mitochondrial dysfunction are linked to pathological conditions including sensory abnormalities. Here, we explored whether mROS overproduction induces itch through transient receptor potential canonical 3 (TRPC3), which is sensitive to ROS. Intradermal injection of antimycin A (AA), a selective inhibitor of mitochondrial electron transport chain complex III for mROS overproduction, produced robust scratching behavior in naïve mice, which was suppressed by MitoTEMPO, a mitochondria-selective ROS scavenger, and Pyr10, a TRPC3-specific blocker, but not by blockers of TRPA1 or TRPV1. AA activated subsets of trigeminal ganglion neurons and also induced inward currents, which were blocked by MitoTEMPO and Pyr10. Besides, dry skin-induced chronic scratching was relieved by MitoTEMPO and Pyr10, and also by resveratrol, an antioxidant. Taken together, our results suggest that mROS elicit itch through TRPC3, which may underlie chronic itch, representing a potential therapeutic target for chronic itch.
Animals ; Antioxidants/pharmacology* ; Mice ; Mitochondria ; Pruritus/chemically induced* ; Reactive Oxygen Species/metabolism* ; TRPA1 Cation Channel

This study aimed to investigate the effect of serum containing ginseng and Moutan Cortex on human umbilical vein endothelial cells(HUVEC) injured with hydrogen peroxide(H_2O_2). HUVEC injured with H_2O_2 were divided into 6 groups, namely blank group, model group, ginsenoside(TGG) group, total glucosides of Moutan Cortex(TGM) group, paeonol(P) group and TGG+TGM+P group. After 24 hours of co-culture with H_2O_2, the activities of succinate dehydrogenase(SDH) and Ca~(2+)-Mg~(2+)-ATP were detected by microenzyme labeling. The apoptosis rate, intracellular Ca~(2+) concentration, reactive oxygen species(ROS) and mitochondrial membrane potential(JC-1) were detected by flow cytometry. The expressions of mitochondrial apoptosis pathway-related proteins Bax, Bcl-2, cytochrome C, caspase-3 and caspase-9 were detected by Western blot. The results showed that H_2O_2 could significantly damage HUVEC, decrease the activity of SDH and Ca~(2+)-Mg~(2+)-ATP(P<0.01), while could increase the apoptosis+necrosis rate, JC-1 decline rate, ROS increase rate and Ca~(2+) concentration increase rate(P<0.01). Serum containing ginseng and Moutan Cortex could increase the activities of SDH and Ca~(2+)-Mg~(2+)-ATP to different degrees, decrease the apoptosis+necrosis rate, JC-1 decline rate, ROS increase rate and Ca~(2+) concentration increase rate(P<0.05 or P<0.01), and down-regulate the protein expressions of Bax, caspase-3, caspase-9, cytochrome C, and up-regulate the protein expression of Bcl-2. The results showed that serum containing ginseng and Moutan Cortex has a protective effect on vascular endothelial cell injury induced by ROS, and its mechanism may be related to the improvement of mitochondrial function and the inhibition of the activation of mitochondrial apoptosis pathway.
Apoptosis ; Drugs, Chinese Herbal/pharmacology* ; Human Umbilical Vein Endothelial Cells ; Humans ; Paeonia ; Panax ; Reactive Oxygen Species

Objective To observe the effect of cryptotanshinone on the ferroptosis of human liver cancer HepG2 cells. Methods The viability of the HepG2 cells cultured
Ferroptosis ; Hep G2 Cells ; Humans ; Liver Neoplasms ; Phenanthrenes/pharmacology* ; Reactive Oxygen Species