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

Xenogenic organ transplantation has been considered the most promising strategy in providing possible substitutes with the physiological function of the failing organs as well as solving the problem of insufficient donor sources. However, the xenograft, suffered from immune rejection and ischemia-reperfusion injury (IRI), causes massive reactive oxygen species (ROS) expression and the subsequent cell apoptosis, leading to the xenograft failure. Our previous study found a positive role of PPAR-γ in anti-inflammation through its immunomodulation effects, which inspires us to apply PPAR-γ agonist rosiglitazone (RSG) to address survival issue of xenograft with the potential to eliminate the excessive ROS. In this study, xenogenic bioroot was constructed by wrapping the dental follicle cells (DFC) with porcine extracellular matrix (pECM). The hydrogen peroxide (H₂O₂)-induced DFC was pretreated with RSG to observe its protection on the damaged biological function. Immunoflourescence staining and transmission electron microscope were used to detect the intracellular ROS level. SD rat orthotopic transplantation model and superoxide dismutase 1 (SOD1) knockout mice subcutaneous transplantation model were applied to explore the regenerative outcome of the xenograft. It showed that RSG pretreatment significantly reduced the adverse effects of H₂O₂ on DFC with decreased intracellular ROS expression and alleviated mitochondrial damage. In vivo results confirmed RSG administration substantially enhanced the host's antioxidant capacity with reduced osteoclasts formation and increased periodontal ligament-like tissue regeneration efficiency, maximumly maintaining the xenograft function. We considered that RSG preconditioning could preserve the biological properties of the transplanted stem cells under oxidative stress (OS) microenvironment and promote organ regeneration by attenuating the inflammatory reaction and OS injury.
Mice ; Humans ; Rats ; Animals ; Swine ; PPAR gamma/pharmacology* ; Reactive Oxygen Species/pharmacology* ; Heterografts ; Hydrogen Peroxide/pharmacology* ; Rats, Sprague-Dawley ; Rosiglitazone/pharmacology* ; Oxidative Stress

Six new ent-abietane diterpenoids, abientaphlogatones A-F (1-6), along with two undescribed ent-abietane diterpenoid glucosides, abientaphlogasides A-B (7-8) and four known analogs were isolated from the aerial parts ofPhlogacanthus curviflorus (P. curviflorus). The structures of these compounds were determined using high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), one-dimensional and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, electronic circular dichroism (ECD) spectra, and quantum chemical calculations. Notably, compounds 5 and 6 represented the first reported instances of ent-norabietane diterpenoids from the genus Phlogacanthus. In the β-hematin formation inhibition assay, compounds 2, 4, 7-10, and 12 displayed antimalarial activity, with IC₅₀ values of 12.97-65.01 μmol·L^-1. Furthermore, compounds 4, 5, 8, and 10 demonstrated neuroprotective activity in PC12 cell injury models induced by H₂O₂ and MPP⁺.
Abietanes/pharmacology* ; Antimalarials ; Hydrogen Peroxide ; Biological Assay ; Plant Components, Aerial

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

Objective To investigate the effect of H₂O₂-induced oxidative stress on autophagy and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs). Methods hBMSCs were isolated and cultured. The cells were divided into control group, 3-MA group, H₂O₂ group, H₂O₂ combined with 3-MA group. DCFH-DA staining was used to analyze the level of reactive oxygen species (ROS). hBMSCs were treated with 0, 50, 100, 200, 400 μmol/L H₂O₂, and then the cell viability was detected by CCK-8 assay. The level of autophagy was detected by monodansylcadaverine (MDC) staining and LysoTracker Red staining. The cell apoptosis was detected by flow cytometry. Western blotting was used to detect the expression of beclin 1, mTOR, phosphorylated mTOR (p-mTOR), cleaved caspase-3(c-caspase-3) and caspase-3 proteins. Results Compared with the control group and 3-MA group, ROS level and autophagosomes were increased and the proliferation and apoptosis were decreased in H₂O₂ group. The protein expression of beclin 1, mTOR, c-caspase-3 was up-regulated, while the p-mTOR was down-regulated. Compared with the 3-MA group, the H₂O₂ combined with 3-MA group also had an increased ROS level and autophagosomes, but not with significantly increased apoptosis rate; The protein expression of beclin 1, mTOR, c-caspase-3 was up-regulated, and the p-mTOR was down-regulated. Conclusion H₂O₂ can induce hMSCs to trigger oxidative stress response. It enhances the autophagy and inhibits the proliferation and apoptosis of hBMSCs.
Humans ; Beclin-1/metabolism* ; Caspase 3/metabolism* ; Reactive Oxygen Species/metabolism* ; Hydrogen Peroxide/pharmacology* ; Apoptosis ; TOR Serine-Threonine Kinases/metabolism* ; Oxidative Stress ; Autophagy ; Mesenchymal Stem Cells/metabolism* ; Cell Proliferation

OBJECTIVES: To explore the physicochemical characteristics and biocompatibility of calcium peroxide (CPO)-loaded polycaprolactone (PCL) microparticle. METHODS: The CPO/PCL particles were prepared. The morphology and elemental distribution of CPO, PCL and CPO/PCL particles were observed with scanning electron microscopy and energy dispersive spectroscopy, respectively. Rat adipose mesenchymal stem cells were isolated and treated with different concentrations (0.10%, 0.25%, 0.50%, 1.00%) of CPO or CPO/PCL particles. The mesenchymal stem cells were cultured in normal media or osteogenic differentiation media under the hypoxia/normoxia conditions, and the amount of released O₂ and H₂O₂ after CPO/PCL treatment were detected. The gene expressions of alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), osteopontin (OPN) and osteocalcin (OCN) were detected by realtime RT-PCR. SD rats were subcutaneously injected with 1.00% CPO/PCL particles and the pathological changes and infiltration of immune cells were observed with HE staining and immunohistochemistry at day 7 and day 14 after injection. RESULTS: Scanning electron microscope showed that CPO particles had a polygonal structure, PCL particles were in a small spherical plastic particle state, and CPO/PCL particles had a block-like crystal structure. Energy dispersive spectroscopy revealed that PCL particles showed no calcium mapping, while CPO/PCL particles showed obvious and uniform calcium mapping. The concentrations of O₂ and H₂O₂ released by CPO/PCL particles were lower than those of CPO group, and the oxygen release time was longer. The expressions of Alp, Runx2, Ocn and Opn increased with the higher content of CPO/PCL particles under hypoxia in osteogenic differentiation culture and normal culture, and the induction was more obvious under osteogenic differentiation conditions (all P<0.05). HE staining results showed that the muscle tissue fibers around the injection site were scattered and disorderly distributed, with varying sizes and thicknesses at day 7 after particle injection. Significant vascular congestion, widened gaps, mild interstitial congestion, local edema, inflammatory cell infiltration, and large area vacuolization were observed in some tissues of rats. At day 14 after microparticle injection, the muscle tissue around the injection site and the tissue fibers at the microparticle implantation site were arranged neatly, and the gap size was not thickened, the vascular congestion, local inflammatory cell infiltration, and vacuolization were significantly improved compared with those at day 7. The immunohistochemical staining results showed that the expressions of CD3 and CD68 positive cells significantly increased in the surrounding muscle tissue, and were densely distributed in a large area at day 7 after particle injection. At day 14 of microparticle injection, the numbers of CD3 and CD68 positive cells in peripheral muscle tissue and tissue at the site of particle implantation were lower than those at day 7 (all P<0.01). CONCLUSIONS CPO/PCL particles have good oxygen release activity, low damage to tissue, and excellent biocompatibility.
Rats ; Animals ; Osteogenesis ; Core Binding Factor Alpha 1 Subunit ; Rats, Sprague-Dawley ; Hydrogen Peroxide/pharmacology* ; Cell Differentiation ; Oxygen ; Hypoxia ; Cells, Cultured

Deep venous thrombosis (DVT) poses a major challenge to public health worldwide. Endothelial cell injury evokes inflammatory and oxidative responses that contribute to thrombus formation. Tea polyphenol (TP) in the form of epigallocatechin-3-gallate (EGCG) has anti-inflammatory and oxidative effect that may ameliorate DVT. However, the precise mechanism remains incompletely understood. The current study was designed to investigate the anti-DVT mechanism of EGCG in combination with warfarin (an oral anticoagulant). Rabbits were randomly divided into five groups. A DVT model of rats was established through ligation of the inferior vena cava (IVC) and left common iliac vein, and the animals were orally administered with EGCG, warfarin, or vehicle for seven days. In vitro studies included pretreatment of human umbilical vein endothelial cells (HUVECs) with different concentrations of EGCG for 2 h before exposure to hydrogen peroxide. Thrombus weight and length were examined. Histopathological changes were observed by hematoxylin-eosin staining. Blood samples were collected for detecting coagulation function, including thrombin and prothrombin times, activated partial thromboplastin time, and fibrinogen levels. Protein expression in thrombosed IVCs and HUVECs was evaluated by Western blot, immunohistochemical analysis, and/or immunofluorescence staining. RT-qPCR was used to determine the levels of AGTR-1 and VEGF mRNA in IVCs and HUVECs. The viability of HUVECs was examined by CCK-8 assay. Flow cytometry was performed to detect cell apoptosis and ROS generation was assessed by 2',7'-dichlorofluorescein diacetate reagent. In vitro and invivo studies showed that EGCG combined with warfarin significantly reduced thrombus weight and length, and apoptosis in HUVECs. Our findings indicated that the combination of EGCG and warfarin protects HUVECs from oxidative stress and prevents apoptosis. However, HIF-1α silencing weakened these effects, which indicated that HIF-1α may participate in DVT. Furthermore, HIF-1α silencing significantly up-regulated cell apoptosis and ROS generation, and enhanced VEGF expression and the activation of the PI3K/AKT and ERK1/2 signaling pathways. In conclusion, our results indicate that EGCG combined with warfarin modifies HIF-1α and VEGF to prevent DVT in rabbits through anti-inflammation via the PI3K/AKT and ERK1/2 signaling pathways.
Animals ; Anticoagulants/pharmacology* ; Catechin/analogs & derivatives* ; Eosine Yellowish-(YS)/pharmacology* ; Fibrinogen/pharmacology* ; Hematoxylin/pharmacology* ; Human Umbilical Vein Endothelial Cells ; Humans ; Hydrogen Peroxide/pharmacology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; MAP Kinase Signaling System ; Phosphatidylinositol 3-Kinases/metabolism* ; Polyphenols/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; RNA, Messenger ; Rabbits ; Rats ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Sincalide/pharmacology* ; Tea ; Thrombin/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Venous Thrombosis/pathology* ; Warfarin/pharmacology*

Five undescribed sesquiterpenoids (1-5), and nine known sesquiterpenoids (6-14) were obtained from the fruits of Litsea lancilimba Merr. by LC-MS/MS molecular networking strategies. Litsemene A (1) possessed a unique 8-member ring through unexpected cyclization of the methyl group on C-10 of guaiane. Their structures were elucidated by spectroscopic techniques including IR, UV, NMR, HR-ESI-MS, and their absolute configurations were assigned by ECD calculations. All isolated sesquiterpenoids were analyzed by bioinformatics and evaluated for their neuroprotective effects against H₂O₂-induced injury in human neuroblastoma SH-SY5Y cells.
Chromatography, Liquid ; Humans ; Hydrogen Peroxide/toxicity* ; Litsea ; Molecular Structure ; Neuroblastoma/drug therapy* ; Neuroprotective Agents/pharmacology* ; Sesquiterpenes/chemistry* ; Tandem Mass Spectrometry

Hallmarks of the pathophysiology of glaucoma are oxidative stress and apoptotic death of retinal ganglion cells (RGCs). Ginkgo biloba extract (EGb) with multi-target, multi-pathway functions has been reported to exert positive pharmacological effects on oxidative stress and damaged RGCs. However, the ingredients and anti-apoptotic targets of EGb in the treatment of open-angle glaucoma (OAG) have not been fully elucidated. Therefore, in-depth analysis is necessary for further research. Ginkgo biloba-related and anti-apoptotic targets were identified and then combined to obtain the intersection, representing the potential anti-apoptotic targets of Ginkgo biloba. In addition, compound-anti-apoptotic target and OAG-target protein-protein interaction network were merged to obtain five core genes and compound-OAG-anti-apoptotic target protein-protein interaction network. Consequently, the active compounds and anti-apoptotic targets of Ginkgo biloba in the treatment of OAG were identified, namely luteolin, β-sitosterol, kaempferol, stigmasterol, quercetin, and p53, Bax, Bcl-2, Caspase-3 and Caspase-9, respectively. For the anti-apoptotic targets of Ginkgo biloba in the treatment of OAG, Gene Ontology (GO) and pathway analysis were executed to confirm the gene functions of Ginkgo biloba in antagonizing apoptosis of RGCs. The pathway enrichment was mainly involved in transcriptional activation of p53 responsive genes, activation of caspases and apoptotic processes. Finally, we confirmed the results of the network analysis by H₂O₂ treated RGC-5 cells in vitro. The results demonstrated that EGb protection can effectively diminish H₂O₂-induced apoptosis by inhibiting p53 acetylation, reducing the ratio of Bax/Bcl-2 and suppressing the expression of specific cleavage of Caspase-9 and Caspase-3.
Ginkgo biloba ; Glaucoma, Open-Angle ; Humans ; Hydrogen Peroxide ; Network Pharmacology ; Plant Extracts ; Retinal Ganglion Cells

Captopril can have nephrotoxic effects, which are largely attributed to accumulated renin and "escaped" angiotensin II (Ang II). Here we test whether angiotensin converting enzyme-1 (ACE1) inhibition damages kidneys via alteration of renal afferent arteriolar responses to Ang II and inflammatory signaling. C57Bl/6 mice were given vehicle or captopril (60 mg/kg per day) for four weeks. Hypertension was obtained by minipump supplying Ang II (400 ng/kg per min) during the second 2 weeks. We assessed kidney histology by periodic acid-Schiff (PAS) and Masson staining, glomerular filtration rate (GFR) by FITC-labeled inulin clearance, and responses to Ang II assessed in afferent arterioles in vitro. Moreover, arteriolar H₂O₂ and catalase, plasma renin were assayed by commercial kits, and mRNAs of renin receptor, transforming growth factor-β (TGF-β) and cyclooxygenase-2 (COX-2) in the renal cortex, mRNAs of angiotensin receptor-1 (AT₁R) and AT₂R in the preglomerular arterioles were detected by RT-qPCR. The results showed that, compared to vehicle, mice given captopril showed lowered blood pressure, reduced GFR, increased plasma renin, renal interstitial fibrosis and tubular epithelial vacuolar degeneration, increased expression of mRNAs of renal TGF-β and COX-2, decreased production of H₂O₂ and increased catalase activity in preglomerular arterioles and enhanced afferent arteriolar Ang II contractions. The latter were blunted by incubation with H₂O₂. The mRNAs of renal microvascular AT₁R and AT₂R remained unaffected by captopril. Ang II-infused mice showed increased blood pressure and reduced afferent arteriolar Ang II responses. Administration of captopril to the Ang II-infused mice normalized blood pressure, but not arteriolar Ang II responses. We conclude that inhibition of ACE1 enhances renal microvascular reactivity to Ang II and may enhance important inflammatory pathways.
Angiotensin II/pharmacology* ; Animals ; Arterioles/metabolism* ; Captopril/pharmacology* ; Hydrogen Peroxide/pharmacology* ; Kidney ; Mice