Objective: To investigate the effects and mechanism of interleukin-4-modified gold nanoparticle (IL-4-AuNP) on the wound healing of full-thickness skin defects in diabetic mice. Methods: Experimental research methods were adopted. Gold nanoparticle (AuNP) and IL-4-AuNP were synthesized by improving the methods described in published literature. The morphology of those two particles were photographed by transmission electron microscopy, and their particle sizes were calculated. The surface potential and hydration particle size of the two particles were detected by nanoparticle potentiometer and particle size analyzer, respectively. The clearance rate of IL-4-AuNP to hydrogen peroxide and superoxide anion was measured by hydrogen peroxide and superoxide anion kits, respectively. Mouse fibroblast line 3T3 cells were used and divided into the following groups by the random number table (the same below): blank control group, hydrogen peroxide alone group treated with hydrogen peroxide only, hydrogen peroxide+IL-4-AuNP group treated with IL-4-AuNP for 0.5 h and then treated with hydrogen peroxide. After 24 h of culture, the reactive oxygen species (ROS) levels of cells were detected by immunofluorescence method; cell count kit 8 was used to detect relative cell survival rate. The macrophage Raw264.7 mouse cells were then used and divided into blank control group and IL-4-AuNP group that treated with IL-4-AuNP. After 24 h of culture, the expression of arginase 1 (Arg-1) in cells was observed by immunofluorescence method. Twelve male BALB/c mice (mouse age, sex, and strain, the same below) aged 8 to 10 weeks were divided into IL-4-AuNP group and blank control group, treated accordingly. On the 16^th day of treatment, whole blood samples were collected from mice for analysis of white blood cell count (WBC), red blood cell count (RBC), hemoglobin level, or platelet count and the level of aspartate aminotransferase (AST), alanine transaminase (ALT), urea, or creatinine. The inflammation, bleeding, or necrosis in the heart, liver, spleen, lung, and kidney tissue of mice were detected by hematoxylin-eosin (HE). Another 36 mice were selected to make diabetic model, and the full-thickness skin defect wounds were made on the back of these mice. The wounds were divided into blank control group, AuNP alone group, and IL-4-AuNP group, with 12 mice in each group, and treated accordingly. On the 0 (immediately), 4^th, 9^th, and 15^th day of treatment, the wound condition was observed and the wound area was calculated. On the 9^th day of treatment, HE staining was used to detect the length of neonatal epithelium and the thickness of granulation tissue in the wound. On the 15^th day of treatment, immunofluorescence method was used to detect ROS level and the number of Arg-1 positive cells in the wound tissue. The number of samples was 6 in all cases. Data were statistically analyzed with independent sample t test, corrected t test, Tukey test, or Dunnett T3 test. Results: The size of prepared AuNP and IL-4-AuNP were uniform. The particle size, surface potential, and hydration particle size of AuNP and IL-4-AuNP were (13.0±2.1) and (13.9±2.5) nm, (-45.8±3.2) and (-20.3±2.2) mV, (14±3) and (16±4) nm, respectively. For IL-4-AuNP, the clearance rate to hydrogen peroxide and superoxide anion were (69±4)% and (52±5)%, respectively. After 24 h of culture, the ROS level of 3T3 in hydrogen peroxide alone group was significantly higher than that in blank control group (q=26.12, P<0.05); the ROS level of hydrogen peroxide+IL-4-AuNP group was significantly lower than that in hydrogen peroxide alone group (q=25.12, P<0.05) and close to that in blank control group (P>0.05). After 24 h of culture, the relative survival rate of 3T3 cells in hydrogen peroxide+IL-4-AuNP group was significantly higher than that in hydrogen peroxide alone group (t=51.44, P<0.05). After 24 h of culture, Arg-1 expression of Raw264.7 cells in IL-4-AuNP group was significantly higher than that in blank control group (t'=8.83, P<0.05).On the 16^th day of treatment, there were no significant statistically differences in WBC, RBC, hemoglobin level, or platelet count and the level of AST, ALT, urea, or creatinine of mice between blank control group and IL-4-AuNP group (P>0.05). No obvious inflammation, bleeding or necrosis was observed in the heart, liver, spleen, lung, and kidney of important organs in IL-4-AuNP group, and no significant changes were observed compared with blank control group. On the 0 and 4^th day of treatment, the wound area of diabetic mice in blank control group, AuNP alone group, and IL-4-AuNP group had no significant difference (P>0.05). On the 9^th day of treatment, the wound areas both in AuNP alone group and IL-4-AuNP group were significantly smaller than that in blank control group (with q values of 9.45 and 14.87, respectively, P<0.05), the wound area in IL-4-AuNP group was significantly smaller than that in AuNP alone group (q=5.42, P<0.05). On the 15^th day of treatment, the wound areas both in AuNP alone group and IL-4-AuNP group were significantly smaller than that in blank control group (with q values of 4.84 and 20.64, respectively, P<0.05), the wound area in IL-4-AuNP group was significantly smaller than that in AuNP alone group (q=15.80, P<0.05); moreover, inflammations such as redness and swelling were significantly reduced in IL-4-AuNP group compared with the other two groups. On the 9^th day of treatment, compared with blank control group and AuNP alone group, the length of neonatal epithelium in the wound of diabetic mice in IL-4-AuNP group was significantly longer (all P<0.05), and the thickness of the granulation tissue in the wound was significantly increased (with q values of 11.33 and 9.65, respectively, all P<0.05). On the 15^th day of treatment, compared with blank control group, ROS levels in wound tissue of diabetic mice in AuNP alone group and IL-4-AuNP group were significantly decreased (P<0.05). On the 15^th day of treatment, the number of Arg-1 positive cells in the wounds of diabetic mice in IL-4-AuNP group was significantly more than that in blank control group and AuNP alone group, respectively (all P<0.05). Conclusions: IL-4-AuNP is safe in vivo, and can improve the oxidative microenvironment by removing ROS and induce macrophage polarization towards M2 phenotype, thus promote efficient diabetic wound healing and regeneration of full-thickness skin defects in diabetic mice.
Mice ; Male ; Animals ; Interleukin-4 ; Gold/pharmacology* ; Diabetes Mellitus, Experimental ; Creatinine ; Hydrogen Peroxide ; Reactive Oxygen Species ; Superoxides ; Metal Nanoparticles ; Soft Tissue Injuries ; Antibodies ; Inflammation ; Necrosis ; Hemoglobins

Objective To study the effect and mechanism of blueberry on regulating the mitochondrial inner membrane protein mitofilin/Mic60 in an in vitro model of metabolic dysfunction-associated liver disease (MAFLD). Methods L02 human hepatocytes were induced by free fatty acids (FFA) to establish MAFLD cell model. A normal group, a model group, an 80 μg/mL blueberry treatment group, a Mic60 short hairpin RNA (Mic60 shRNA) transfection group, and Mic60 knockdown combined with an 80 μg/mL blueberry treatment group were established. The intracellular lipid deposition was observed by oil red O staining, and the effect of different concentrations of blueberry pulp on the survival rate of L02 cells treated with FFA was measured by MTT assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents were measured by visible spectrophotometry. The expression of reactive oxygen species (ROS) in hepatocytes was observed by fluorescence microscopy, and the mRNA and protein expression of Mic60 were detected by real-time quantitative PCR and Western blot analysis, respectively. Results After 24 hours of FFA stimulation, a large number of red lipid droplets in the cytoplasm of L02 cells was observed, and the survival rate of L02 cells treated with 80 μg/mL blueberry was higher. The results of ALT, AST, TG, TC, MDA and the fluorescence intensity of ROS in blueberry treated group were lower than those in model group, while the levels of SOD, GSH, Mic60 mRNA and protein in blueberry treated group were higher than those in model group. Conclusion Blueberry promotes the expression of Mic60, increases the levels of SOD and GSH in hepatocytes, and reduces the production of ROS, thus alleviating the injury of MAFLD hepatocytes and regulating the disorder of lipid metabolism.
Humans ; Blueberry Plants/chemistry* ; Hepatocytes/metabolism* ; Liver/metabolism* ; Liver Diseases/metabolism* ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/metabolism* ; Superoxides/metabolism* ; Mitochondrial Membranes/metabolism* ; Mitochondrial Proteins/metabolism* ; Plant Extracts/pharmacology*

Seed vigor is a key factor affecting seed quality. The mechanical drying process exerts a significant influence on rice seed vigor. The initial moisture content (IMC) and drying temperature are considered the main factors affecting rice seed vigor through mechanical drying. This study aimed to determine the optimum drying temperature for rice seeds according to the IMC, and elucidate the mechanisms mediating the effects of drying temperature and IMC on seed vigor. Rice seeds with three different IMCs (20%, 25%, and 30%) were dried to the target moisture content (14%) at four different drying temperatures. The results showed that the drying temperature and IMC had significant effects on the drying performance and vigor of the rice seeds. The upper limits of drying temperature for rice seeds with 20%, 25%, and 30% IMCs were 45, 42, and 38 °C, respectively. The drying rate and seed temperature increased significantly with increasing drying temperature. The drying temperature, drying rate, and seed temperature showed extremely significant negative correlations with germination energy (GE), germination rate, germination index (GI), and vigor index (VI). A high IMC and drying temperature probably induced a massive accumulation of hydrogen peroxide (H₂O₂) and superoxide anions in the seeds, enhanced superoxide dismutase (SOD) and catalase (CAT) activity, and increased the abscisic acid (ABA) content. In the early stage of seed germination, the IMC and drying temperature regulated seed germination through the metabolism of H₂O₂, gibberellin acid (GA), ABA, and α-amylase. These results indicate that the metabolism of reactive oxygen species (ROS), antioxidant enzymes, GA, ABA, and α-amylase might be involved in the mediation of the effects of drying temperature on seed vigor. The results of this study provide a theoretical basis and technical guidance for the mechanical drying of rice seeds.
Abscisic Acid/metabolism* ; Antioxidants/pharmacology* ; Catalase/metabolism* ; Gene Expression Regulation, Plant/drug effects* ; Germination ; Gibberellins/metabolism* ; Hydrogen Peroxide/chemistry* ; Malondialdehyde/chemistry* ; Oryza/metabolism* ; Oxygen/chemistry* ; Plant Proteins/genetics* ; Reactive Oxygen Species ; Seeds/metabolism* ; Superoxide Dismutase/metabolism* ; Superoxides/chemistry* ; Temperature ; Weather ; alpha-Amylases/metabolism*

BACKGROUND: Sixty percent of infants with severe neonatal hypoxic-ischemic encephalopathy die, while most survivors have permanent disabilities. Treatment for neonatal hypoxic-ischemic encephalopathy is limited to therapeutic hypothermia, but it does not offer complete protection. Here, we investigated whether hypoxia-inducible factor (HIF) promotes cell survival and suggested neuroprotective strategies.PURPOSE: HIF-1α deficient mice have increased brain injury after neonatal hypoxia-ischemia (HI), and the role of HIF-2α in HI is not well characterized. Copper-zinc superoxide dismutase (SOD)1 overexpression is not beneficial in neonatal HI. The expression of HIF-1α and HIF-2α was measured in SOD1 overexpressing mice and compared to wild-type littermates to see if alteration in expression explains this lack of benefit.METHODS: On postnatal day 9, C57Bl/6 mice were subjected to HI, and protein expression was measured by western blotting in the ipsilateral cortex of wild-type and SOD1 overexpressing mice to quantify HIF-1α and HIF-2α. Spectrin expression was also measured to characterize the mechanism of cell death.RESULTS: HIF-1α protein expression did not significantly change after HI injury in the SOD1 overexpressing or wild-type mouse cortex. However, HIF-2α protein expression increased 30 minutes after HI injury in the wild-type and SOD1 overexpressing mouse cortex and decreased to baseline value at 24 hours after HI injury. Spectrin 145/150 expression did not significantly change after HI injury in the SOD1 overexpressing or wild-type mouse cortex. However, spectrin 120 expression increased in both wild-type and SOD1 overexpressing mouse at 4 hours after HI, which decreased by 24 hours, indicating a greater role of apoptotic cell death.CONCLUSION: HIF-1α and HIF-2α may promote cell survival in neonatal HI in a cell-specific and regional fashion. Our findings suggest that early HIF-2α upregulation precedes apoptotic cell death and limits necrotic cell death. However, the influence of SOD was not clarified; it remains an intriguing factor in neonatal HI.
Animals ; Blotting, Western ; Brain Injuries ; Cell Death ; Cell Survival ; Humans ; Hypothermia, Induced ; Hypoxia-Ischemia, Brain ; Infant ; Mice ; Spectrin ; Superoxide Dismutase ; Superoxides ; Survivors ; Up-Regulation

BACKGROUND: Although Eriobotrya japonica leaves have been studied as a raw material for various cosmetic products, little is known about the anti-oxidant, anti-inflammatory, and anti-melanogenic activities of Eriobotrya japonica leaf ethanol extract (EJEE). METHODS: This study was conducted to evaluate the anti-oxidant, anti-inflammatory, and anti-melanogenic activities of EJEE using different in vitro models. In addition, we investigated the potential irritation of EJEE to skin and eye using animal alternative tests. RESULTS: The total content of polyphenols, one of the active constituents of EJEE, was analyzed by high-performance liquid chromatography and found to contain 88.68 mg tannic acid equivalent/g. EJEE showed a concentration-dependent 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity, and a superoxide dismutase-like activity. The anti-inflammatory effect of 0.5% (w/v) EJEE was demonstrated by a reduction in lipopolysaccharide-induced nitric oxide and tumor necrosis factor-alpha levels in RAW 264.7 cells. EJEE also significantly inhibited melanogenesis in melanocyte stimulating hormone-induced B16F1 cells. EJEE did not show any irritation in skin and eye in animal alternative test. CONCLUSIONS: These results indicate that the EJEE possesses anti-oxidant, anti-inflammatory, and anti-melanogenic activities, while it did not induce toxicity or irritation in neither skin nor eye. Therefore, EJEE can be used as a cosmetic ingredient for skin improvement.
Animals ; Chromatography, Liquid ; Eriobotrya ; Ethanol ; In Vitro Techniques ; Melanocytes ; Nitric Oxide ; Polyphenols ; RAW 264.7 Cells ; Skin ; Superoxides ; Tannins ; Tumor Necrosis Factor-alpha

Many of the fungicides and antibiotics currently available against plant pathogens are of limited use due to the emergence of resistant strains. In this study, we examined the effects of diphenyleneiodonium chloride (DPIC), an inhibitor of the superoxide producing enzyme NADPH oxidase, against fungal and bacterial plant pathogens. We found that DPIC inhibits fungal spore germination and bacterial cell proliferation. In addition, we demonstrated the potent antibacterial activity of DPIC using rice heads infected with the bacterial pathogen Burkholderia glumae which causes bacterial panicle blight (BPB). We found that treatment with DPIC reduced BPB when applied during the initial flowering stage of the rice heads. These results suggest that DPIC could serve as a new and useful antimicrobial agent in agriculture.
Agriculture ; Anti-Bacterial Agents ; Burkholderia ; Cell Proliferation ; Flowers ; Germination ; Head ; NADPH Oxidase ; Plants ; Spores, Fungal ; Superoxides

OBJECTIVE: Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H₂O₂ stimuli. METHODS: Modified microfluidics and imaging techniques were used to determine O₂ ^•- levels and construct an O₂ ^•- reaction network. To elucidate the consequences of increased O₂ ^•- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca²⁺ levels, mitochondrial Ca²⁺ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. RESULTS AND CONCLUSIONS Results from a modified microchip demonstrated that 1 mmol/L H₂O₂ induced a rapid increase in cellular O₂ ^•- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca²⁺ and mitochondrial Ca²⁺ by confocal laser scanning microscopy and confirmed that Ca²⁺ stores in the endoplasmic reticulum were the primary source of H₂O₂-induced cytosolic Ca²⁺ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca²⁺ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.
Apoptosis ; Calcium/metabolism* ; Calcium Signaling ; Caspases/metabolism* ; Cell Line, Tumor ; Cell Lineage ; Cytosol/metabolism* ; Glutathione/metabolism* ; Humans ; Hydrogen Peroxide/chemistry* ; Mitochondria/metabolism* ; Oxidation-Reduction ; Oxidative Stress ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Superoxides/chemistry*

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.
Acetylcysteine ; Ascorbic Acid ; Humans ; Mitochondria ; Parasites ; Reactive Oxygen Species ; Retinaldehyde ; Superoxides ; Toxoplasma ; Toxoplasmosis

BACKGROUND: Helicobacter pylori increases production of reactive oxygen species (ROS), which activates inflammatory and carcinogenesis-related signaling pathways in gastric epithelial cells. Therefore, reducing ROS, by upregulating antioxidant enzyme, such as superoxide dismutase (SOD), may be a novel strategy to prevent H. pylori-associated gastric diseases. Astaxanthin is an antioxidant carotenoid that prevents oxidative stress-induced cell injury. The present study was aimed to determine whether H. pylori decreases SOD activity by changing the levels of SOD1/SOD2 and whether astaxanthin prevents changes in SOD levels and activity in H. pylori-infected gastric epithelial AGS cells. METHODS: AGS cells were pre-treated with astaxanthin for 3 hours prior to H. pylori infection and cultured for 1 hour in the presence of H. pylori. SOD levels and activity were assessed by Western blot analysis and a commercial assay kit, respectively. Mitochondrial ROS was determined using MitoSOX fluorescence. RESULTS: H. pylori decreased SOD activity and the SOD2 level, but increased mitochondrial ROS in AGS cells. The SOD1 level was not changed by H. pylori infection. Astaxanthin prevented H. pylori-induced decreases in the SOD2 level and SOD activity and reduced mitochondrial ROS in AGS cells. CONCLUSIONS: Consumption of astaxanthin-rich food may prevent the development of H. pylori-associated gastric disorders by suppressing mitochondrial oxidative stress.
Blotting, Western ; Epithelial Cells ; Fluorescence ; Helicobacter pylori ; Helicobacter ; Oxidative Stress ; Reactive Oxygen Species ; Stomach Diseases ; Superoxide Dismutase ; Superoxides

BACKGROUND: Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. METHODS: RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. RESULTS: The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. CONCLUSION: This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.
Acid Phosphatase ; Carbonic Anhydrase II ; Cathepsin K ; Chloride Channels ; Cytoplasm ; Gene Expression ; In Vitro Techniques ; Matrix Metalloproteinase 9 ; Membrane Potential, Mitochondrial ; Metabolism ; Osteoclasts ; Phosphotransferases ; RANK Ligand ; Reactive Oxygen Species ; Receptor Activator of Nuclear Factor-kappa B ; Superoxides ; T-Lymphocytes