OBJECTIVE: To explore the mechanism by which ω-3 polyunsaturated fatty acids (hereinafter referred to as "ω-3") exert antioxidant stress protection and promote osteogenic differentiation in MC3T3-E1 cells, and to reveal the relationship between ω-3 and the key antioxidant stress pathway involving nuclear factor E2-related factor 2 (Nrf2) and NAD (P) H quinone oxidoreductase 1 (NQO1) in MC3T3-E1 cells. METHODS: The optimal concentration of H ₂O ₂ (used to establish the oxidative stress model of MC3T3-E1 cells in vitro) and the optimal intervention concentrations of ω-3 were screened by cell counting kit 8. MC3T3-E1 cells were divided into blank control group, oxidative stress group (H ₂O ₂), low-dose ω-3 group (H ₂O ₂+low-dose ω-3), and high-dose ω-3 group (H ₂O ₂+high-dose ω-3). After osteoblastic differentiation for 7 or 14 days, the intracellular reactive oxygen species (ROS) level was measured by fluorescence staining and flow cytometry, and the mitochondrial morphological changes were observed by biological transmission electron microscope; the expression levels of Nrf2, NQO1, heme oxygenase 1 (HO-1), Mitofusin 1 (Mfn1), and Mfn2 were detected by Western blot to evaluate the cells' antioxidant stress capacity; the expression levels of Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were detected by immunofluorescence staining and Western blot; osteogenic potential of MC3T3-E1 cells was evaluated by alkaline phosphatase (ALP) staining and alizarin red staining. RESULTS: Compared with the oxidative stress group, the content of ROS in the low and high dose ω-3 groups significantly decreased, and the protein expressions of Nrf2, NQO1, and HO-1 significantly increased ( P<0.05). At the same time, the mitochondrial morphology of MC3T3-E1 cells improved, and the expressions of mitochondrial morphology-related proteins Mfn1 and Mfn2 significantly increased ( P<0.05). ALP staining and alizarin red staining showed that the low-dose and high-dose ω-3 groups showed stronger osteogenic ability, and the expressions of osteogenesis-related proteins RUNX2 and OCN significantly increased ( P<0.05). And the above results showed a dose-dependence in the two ω-3 treatment groups ( P<0.05). CONCLUSION ω-3 can enhance the antioxidant capacity of MC3T3-E1 cells under oxidative stress conditions and upregulate their osteogenic activity, possibly through the Nrf2/NQO1 signaling pathway.
Oxidative Stress/drug effects* ; NF-E2-Related Factor 2/metabolism* ; NAD(P)H Dehydrogenase (Quinone)/metabolism* ; Animals ; Mice ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Fatty Acids, Omega-3/pharmacology* ; Signal Transduction/drug effects* ; Osteoblasts/drug effects* ; Reactive Oxygen Species/metabolism* ; Cell Line ; Hydrogen Peroxide/pharmacology* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Antioxidants/pharmacology* ; Heme Oxygenase-1/metabolism*

OBJECTIVE: To analyze the influences of dihydromyricetin on the proliferation and apoptosis of chondrocytes in osteoarthritis induced by hydrogen peroxide (H₂O₂) through reactive oxygen species (ROS)/p38 mitogen activated protein kinase (p38-MAPK) pathway. METHODS: Five C57BL/6J mice were euthanized by cervical dislocation after anesthesia. Chondrocytes were extracted and cultured.After passage, the chondrocytes were divided into control group, H2O2 group (0.8 μmol·L^-1 H₂O₂), dihydromyricetin low concentration group (0.8 μmol·L^-1 H₂O₂+20 μmol·L^-1 dihydromyricetin), dihydromyricetin high concentration group (0.8 μmol·L^-1 H₂O₂+80 μmol·L^-1 dihydromyricetin), and ROS inhibitor N-acetylcysteine (NAC) group (0.8 μmol·L^-1 H₂O₂+5 mmol·L^-1 NAC). The activity of chondrocytes was measured by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate of chondrocytes was measured by Hoechst 33342 method. The level of ROS in chondrocytes was measured by 2, 7-dichlorofluorescein diacetate (DCFH-DA) fluorescence probe.The level of Type II collagen α1 (Col2α1) mRNA was measured by qRT-PCR.And the expression of Col2α1, p-p38-MAPK/p38-MAPK, B cell lymphoma gene-2 (Bcl-2) and Bcl-2 associated X protein (Bax) proteins was detected by Western blot. RESULTS: The chondrocytes showed swirling fibrous mass, and the expression of COL2α was positive. Compared with the control group, the chondrocyte viability, apoptosis rate, ROS fluorescence intensity, p-p38-MAPK/p38-MAPK, and the expression of Bax protein in H2O22 group increased, the level of Col2α1 mRNA, and the expression of Col2α1 and Bcl-2 proteins decreased (P<0.05). Compared with H₂O₂ group, the chondrocyte viability, apoptosis rate, ROS fluorescence intensity, p-p38-MAPK/p38-MAPK, and the expression of Bax protein in dihydromyricetin low concentration group, dihydromyricetin high concentration group, and NAC group decreased, the level of Col2α1 mRNA, and the expression of Col2α1 and Bcl-2 proteins increased (P<0.05). CONCLUSION Dihydromyricetin may inhibit chondrocyte apoptosis, inflammatory reaction and oxidative stress by inhibiting ROS/p38-MAPK pathway. Dihydromyricetin may be a potential drug for treating osteoarthritis.
Animals ; Chondrocytes/metabolism* ; Apoptosis/drug effects* ; Hydrogen Peroxide/toxicity* ; Osteoarthritis/physiopathology* ; Mice, Inbred C57BL ; Reactive Oxygen Species/metabolism* ; Mice ; Flavonols/pharmacology* ; p38 Mitogen-Activated Protein Kinases/genetics* ; Cell Proliferation/drug effects* ; Male ; Signal Transduction/drug effects* ; MAP Kinase Signaling System/drug effects* ; Cells, Cultured

The caries-preventive effects of arginine have been attributed to its impact on biofilm composition and pH. Recent in vitro studies suggest that arginine also affects the production of biofilm matrix components that contribute to virulence, but this mechanism has not been investigated clinically. This randomized, placebo-controlled, triple-blind, split-mouth in situ trial assessed arginine's impact on the microbial composition, matrix architecture, and microscale pH of biofilms from caries-active patients (N = 10). We also examined whether individual differences in the pH response to arginine were related to biofilm composition and matrix structure. Biofilms were grown for four days on carriers attached to intraoral splints. Three times daily, the biofilms were treated extraorally with sucrose (5 min), followed by arginine or placebo (30 min), in a split-mouth design. After growth, the microscale biofilm pH response to sucrose was monitored by pH ratiometry. Microbial biofilm composition and carbohydrate matrix architecture were analyzed by 16S rRNA gene sequencing and fluorescence lectin-binding analysis, respectively. Arginine treatment significantly mitigated sucrose-induced pH drops, reduced total carbohydrate matrix production, and altered the spatial distribution of fucose- and galactose-containing carbohydrates. Both arginine- and placebo-treated biofilms were dominated by streptococci and Veillonella spp. Paired analyses showed a significant reduction in mitis/oralis group streptococci and a non-significant increase in several arginine metabolizers in arginine-treated biofilms. Individual pH responses were not significantly associated with the abundance of specific bacterial taxa or carbohydrate matrix components. In conclusion, arginine reduced the virulence of biofilms from caries-active patients through multiple mechanisms, including suppressing matrix carbohydrate production.
Biofilms/drug effects* ; Humans ; Arginine/pharmacology* ; Hydrogen-Ion Concentration ; Dental Caries/prevention & control* ; Male ; Female ; Adult ; Double-Blind Method ; Sucrose/pharmacology*

OBJECTIVE: To explore the protective effects and underlying mechanisms of H ₂S against lipid peroxidation-mediated carbonyl stress in the uranium-treated NRK-52E cells. METHODS: Cell viability was evaluated using CCK-8 assay. Apoptosis was measured using flow cytometry. Reagent kits were used to detect carbonyl stress markers malondialdehyde, 4-hydroxynonenal, thiobarbituric acid reactive substances, and protein carbonylation. Aldehyde-protein adduct formation and alcohol dehydrogenase, aldehyde dehydrogenase 2, aldo-keto reductase, nuclear factor E2-related factor 2 (Nrf2), and cystathionine β-synthase (CBS) expression were determined using western blotting or real-time PCR. Sulforaphane (SFP) was used to activate Nrf2. RNA interference was used to inhibit CBS expression. RESULTS: GYY4137 (an H ₂S donor) pretreatment significantly reversed the uranium-induced increase in carbonyl stress markers and aldehyde-protein adducts. GYY4137 effectively restored the uranium-decreased Nrf2 expression, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2, accompanied by a reversal of the uranium-decreased expression of CBS and aldehyde-metabolizing enzymes. The application of CBS siRNA efficiently abrogated the SFP-enhanced effects on the expression of CBS, Nrf2 activation, nuclear translocation, and ratio of nuclear to cytoplasmic Nrf2 and concomitantly reversed the SFP-enhanced effects of the uranium-induced mRNA expression of aldehyde-metabolizing enzymes. Simultaneously, CBS siRNA reversed the SFP-mediated alleviation of the uranium-induced increase in reactive aldehyde levels, apoptosis rates, and uranium-induced cell viability. CONCLUSION H ₂S induces Nrf2 activation and nuclear translocation, which modulates the expression of aldehyde-metabolizing enzymes and the CBS/H ₂S axis. Simultaneously, the Nrf2-controlled CBS/H ₂S axis may at least partially promote Nrf2 activation and nuclear translocation. These events form a cycle-regulating mode through which H ₂S attenuates the carbonyl stress-mediated NRK-52E cytotoxicity triggered by uranium.
NF-E2-Related Factor 2/genetics* ; Animals ; Hydrogen Sulfide/pharmacology* ; Rats ; Signal Transduction/drug effects* ; Lipid Peroxidation/drug effects* ; Cell Line ; Uranium/toxicity* ; Antioxidant Response Elements ; Kidney/metabolism* ; Oxidative Stress/drug effects* ; Cell Survival/drug effects* ; Apoptosis/drug effects*

OBJECTIVES: Investigating the protective effect of naringenin (NAR) on the osteogenic potential of human periodontal ligament stem cells (hPDLSCs) under oxidative stress and its related mechanisms. METHODS: The oxidative damage model of hPDLSCs was established using hydrogen peroxide (H₂O₂) andthe hPDLSCs were treated with different concentrations of NAR and 0.5 μmol/L forkhead box protein O-1 (FOXO1) inhibitor AS1842856. After that, the cell counting kit-8 (CCK8) was used to determine the optimal concentrations of H₂O₂ and NAR. The alkaline phosphatase (ALP) staining and real time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR) were employed to assess the expression of ALP, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) in hPDLSCs of each group. The enzyme-linked immunosorbent assay (ELISA) and 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining were utilized to evaluate the expression of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in hPDLSCs. Meanwhile, qRT-PCR and western blot were used to detect the expression levels of FOXO1 and β-catenin, both are pathway related genes and proteins. RESULTS: H₂O₂ exposure led to an increase in oxidative damage in hPDLSCs, characterized by a rise in intracellular ROS levels and increased expression of MDA and LDH (P<0.05). At the same time, the osteogenic differentiation ability of hPDLSCs decreased, as evidenced by lighter ALP staining and reduced expression levels of osteogenic differentiation-related genes ALP, RUNX2 and OCN (P<0.05). Co-treatment with NAR alleviated the oxidative damage in hPDLSCs, enhanced their antioxidant capacity, and restored their osteogenic ability. The FOXO1 inhibitor AS1842856 downregulated the expression of β-catenin (P<0.05) and significantly diminished both the antioxidant effect of NAR and its ability to restore osteogenesis (P<0.05). CONCLUSIONS NAR can enhance the antioxidant capacity of hPDLSCs by activating the FOXO1/β-catenin signaling pathway within hPDLSCs, thereby mitigating oxidative stress damage and alleviating the loss of osteogenic capacity.
Humans ; Oxidative Stress/drug effects* ; Periodontal Ligament/cytology* ; Hydrogen Peroxide ; Forkhead Box Protein O1/metabolism* ; Stem Cells/cytology* ; Flavanones/pharmacology* ; beta Catenin/metabolism* ; Osteogenesis/drug effects* ; Signal Transduction ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Alkaline Phosphatase/metabolism* ; Osteocalcin/metabolism* ; Cells, Cultured ; Cell Differentiation/drug effects*

OBJECTIVES: This study aims to investigate the impact of glutathione S-transferase (GST) on the environmental adaptability of Streptococcus mutans (S. mutans). METHODS: A GST knockout strain ΔgsT was constructed. Transcriptomic sequencing was performed to analyze the gene expression differences between the wild-type S. mutans UA159 and its GST knockout strain ΔgsT. Comprehensive functional assessments, including acid tolerance assays, hydrogen peroxide challenge assays, nutrient limitation growth assays, and fluorescence in situ hybridization, were conducted to evaluate the acid tolerance, antioxidant stress resistance, growth kinetics, and interspecies competitive ability of ΔgsT within plaque biofilms. RESULTS: Compared with the wild-type S. mutans, 198 genes in ΔgsT were significantly differentially expressed and enriched in pathways related to metabolism, stress response, and energy homeostasis. The survival rate of ΔgsT in acid tolerance assays was markedly reduced (P<0.01). After 15 min of hydrogen peroxide challenge, the survival rate of ΔgsT decreased to 38.12% (wild type, 71.75%). Under nutrient-limiting conditions, ΔgsT exhibited a significantly lower final OD₆₀₀ value than the wild-type strain (P<0.05). In the biofilm competition assays, the proportion of S. mutans ΔgsT in the mixed biofilm (8.50%) was significantly lower than that of the wild type (16.89%) (P<0.05). CONCLUSIONS GST enhances the acid resistance, oxidative stress tolerance, and nutrient adaptation of S. mutans by regulating metabolism-related and stress response-related genes.
Streptococcus mutans/enzymology* ; Biofilms ; Glutathione Transferase/physiology* ; Adaptation, Physiological ; Hydrogen Peroxide/pharmacology* ; Gene Expression Regulation, Bacterial ; Oxidative Stress ; Metabolic Reprogramming

Reactive oxygen species (ROS) are major contributors to radiation therapy-induced side effects in cancer patients. A fusion antioxidant enzyme comprising glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and a transmembrane peptide has been shown to effectively mitigate ROS-induced damage. To enhance its targeting capability, the fusion protein was further modified by incorporating a matrix metalloproteinase-2/9 substrate peptide (X) and the transmembrane peptide R9, yielding the antioxidant enzyme GST-SOD1-X-R9 (GS1XR). This modification reduced its transmembrane ability in tumor cells, thereby selectively protecting normal cells from oxidative stress. However, the use of non-human GST poses potential immunogenicity risks. In this study, we employed seamless cloning technology to construct an expression vector containing the human GST gene to replace the non-human GST gene, and then expressed and purified novel fusion antioxidant enzymes GS1R and GS1XR. The protective effects of newly constructed GS1R and GS1XR against hydrogen peroxide (H₂O₂)-induced oxidative damage in L-02 cells were then evaluated using GS1 as a control. Enzymatic activity assays revealed that the specific activity of GST in GS1XR remained unchanged compared to the unmodified protein, while SOD activity was enhanced. Exposure to 200 μmol/L H₂O₂ transiently activated the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway; however, this activation diminished after 24 h, reducing cell viability to 48.4%. Both GS1R and GS1XR effectively scavenged intracellular ROS, directly counteracting oxidative stress and promoting Nrf2 nuclear translocation, thereby activating antioxidant pathways and restoring cell viability to normal levels. The two enzymes showed comparable efficacy. In contrast, GS1, lacking transmembrane capability, was restricted to scavenging extracellular ROS and provided only limited protection. In conclusion, both novel fusion antioxidant enzymes demonstrated significant potential in safeguarding normal cells from ROS-mediated oxidative damage. The findings provide a foundation for further investigation in related field.
Humans ; Oxidative Stress/drug effects* ; Hydrogen Peroxide ; Antioxidants/metabolism* ; Glutathione Transferase/metabolism* ; Recombinant Fusion Proteins/pharmacology* ; Superoxide Dismutase-1 ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/biosynthesis*

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

OBJECTIVE: To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells). METHODS: HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na₂S₂O₄. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting. RESULTS: Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01). CONCLUSIONS Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.
Mice ; Animals ; Oxygen/metabolism* ; Beclin-1/pharmacology* ; Glucose/metabolism* ; Actins ; Sincalide ; Autophagy/physiology* ; Hydrogen/pharmacology* ; Reperfusion Injury ; Apoptosis