Reactive Oxygen Species Intensity Research Articles

Selenium protects against Pb-induced renal oxidative injury in weaning rats and human renal tubular epithelial cells through activating NRF2

BackgroundLead (Pb) poisoning posing a crucial health risk, especially among children, causing devastating damage not only to brain development, but also to kidney function. Thus, an urgent need persists to identify highly effective, safe, and low-toxicity drugs for the treatment of Pb poisoning. The present study focused on exploring the protective effects of Se on Pb-induced nephrotoxicity in weaning rats and human renal tubular epithelial cells, and investigated the possible mechanisms. MethodsForty weaning rats were randomly divided into four groups in vivo: control, Pb-exposed, Pb+Se and Se. Serum creatinine (Cr), urea nitrogen (BUN) and hematoxylin and eosin (H&E) staining were performed to evaluate renal function. The activities of antioxidant enzymes in the kidney tissue were determined. In vitro experiments were performed using human renal tubular epithelial cells (HK-2 cells). The cytotoxicity of Pb and Se was detected by 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Inverted fluorescence microscope was used to investigate cell morphological changes and the fluorescence intensity of reactive oxygen species (ROS). The oxidative stress parameters were measured by a multi-detection reader. Nuclear factor-erythroid-2-related factor (NRF2) signaling pathways were measured by Western blot and reverse transcription polymerase chain reaction (RT-PCR) in HK-2 cells. ResultsWe found that Se alleviated Pb-induced kidney injury by relieving oxidative stress and reducing the inflammatory index. Se significantly increased the activity of the antioxidant enzymes glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), whereas it decreased the excessive release of malondialdehyde (MDA) in the kidneys of weaning rats and HK-2 cells. Additionally, Se enhanced the antioxidant defense systems via activating the NRF2 transcription factor, thereby promoting the to downstream expression of heme oxygenase 1. Furthermore, genes encoding glutamate-cysteine ligase synthetase catalytic (GCLC), glutamate-cysteine ligase synthetase modifier (GCLM) and NADPH quinone oxidoreductase 1 (NQO1), downstream targets of NRF2, formed a positive feedback loop with NRF2 during oxidative stress responses. The MTT assay results revealed a significant decrease in cell viability with Se treatment, and the cytoprotective role of Se was blocked upon knockdown of NRF2 by small interfering RNA (siRNA). MDA activity results also showed that NRF2 knockdown inhibited the NRF2-dependent transcriptional activity of Se. ConclusionsOur findings demonstrate that Se ameliorated Pb-induced nephrotoxicity by reducing oxidative stress both in vivo and in vitro. The molecular mechanism underlying Se’s action in Pb-induced kidney injury is related to the activation of the NRF2 transcription factor and the activity of antioxidant enzymes, ultimately suppressing ROS accumulation.

Plum blossom low molecular weight polypeptide protects HaCaT cells against UVB-induced oxidative damage in vitro and underlying mechanism.

Increasing amounts of ultraviolet radiation occur as ozone depletion causes the earth's ozone layer to be destroyed, making antioxidant efficacy a research hotspot. Previous studies on plum blossom have mostly focused on Volatile Oils, Flavonoids, Phenylpropanoids, and other compounds, whereas few studies have focused on low molecular weight polypeptide (LMWP) of plum blossom. This research provides a reference for the deep processing and utilization of plum blossom. (a) Plum blossom low molecular weight polypeptides protect HaCaT cells against UVB-induced oxidative damage in vitro and the underlying mechanism. (b) Improve the theoretical basis for the intense processing and utilization of plum blossom. The safe concentration of LMWP and the survival rate of HaCaT cells were determined using the CCK-8 experiment. The fluorescence intensity of reactive oxygen species (ROS) was identified using the dichlorofluorescin diacetate (DCFH-DA) method; Superoxide dismutase (SOD) and malondialdehyde (MDA) concentrations were measured in ruptured cells; Western blot analysis was used to examine the expression levels of three proteins: nuclear factor E2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and benzoquinone oxidoreductase 1 (NQO-1). It was noted that a certain concentration of LMWP could promote cell proliferation. In oxidatively damaged HaCaT cells, SOD levels and survival rates were markedly reduced, but ROS and MDA levels were elevated. However, after treatment with LMWP, the survival rate of the cells and SOD levels were markedly increased, and the levels of ROS and MDA were markedly decreased. As shown by Western blotting, the model group exhibited lower levels of Nrf2, HO-1, and NQO-1 expression than the control group, whereas LMWP-treated cells had significantly higher levels of Nrf2, HO-1, and NQO-1 expression than their model-treated counterparts. LMMP can effectively protect HaCaT cells against oxidative damage in vitro induced by UVB, and the underlying mechanism is linked to the activation of the transcription factor Nrf2.

Inhibition of Transverse Aortic Constriction Induced Myocardial Hypertrophy by Knocking out the Target of the Early Growth Response-1 to Inhibit Apoptosis and Autophagy

Myocardial hypertrophy, a significant contributor to the development of heart failure, continues to be prevalent. Early growth response-1 (EGR-1) is closely linked to the development of diverse myocardial conditions. The target of EGR1 (TOE1) is a critical factor in myocardial hypertrophy, but its regulatory function remains unclear. Myocardial cell injury was induced by angiotensin II. TOE1 knockout mice and cells were generated to investigate its impact on myocardial hypertrophy. TUNEL staining was employed to assess cell apoptosis. Furthermore, western blotting and qRT-PCR were performed to measure the expression of target genes. The results revealed that knockout of TOE1 effectively inhibited myocardial hypertrophy and injury caused by transverse aortic constriction. In vivo experiments demonstrated that TOE1 knockout improved myocardial function and suppressed inflammatory factors, oxidative stress, apoptosis, and autophagy levels. In vitro, TOE1 knockout suppressed cell apoptosis, mitochondrial damage, and the intensity of reactive oxygen species. Additionally, it inhibited the expression of apoptosis- and autophagy-related genes. These findings introduce a promising avenue for preventing and treating myocardial hypertrophy.

Bithionol Restores Sensitivity of Multidrug-Resistant Gram-Negative Bacteria to Colistin with Antimicrobial and Anti-biofilm Effects.

Being among the few last-resort antibiotics, colistin (COL) has been used to treat severe infectious diseases, such as those caused by multidrug-resistant Gram-negative bacteria (MDR GNB). However, the appearance of colistin-resistant (COL-R) GNB has been frequently reported. Therefore, novel antimicrobial strategies need to be urgently sought to address this resistance challenge. In the present study, antimicrobial drug screening conducted revealed that bithionol (BT), approved by the Food and Drug Administration and used as an anthelminthic drug for paragonimiasis, exhibited a synergistic antibacterial effect with COL. Clinically isolated COL-R GNB were used as candidates to evaluate the synergistic antibacterial activity. The results revealed that BT could significantly reverse the sensitivity of COL-R GNB to COL. Furthermore, the combined application of BT and COL can reduce bacterial biofilm formation and have a scavenging effect on the mature biofilm in vitro. The damage caused to the bacterial cell membrane integrity by the BT/COL combination was observed under a fluorescence microscope. The fluorescence intensity of reactive oxygen species also increased in the experimental group. The BT/COL combination also exhibited a synergistic antibacterial effect in vivo. Importantly, BT was confirmed to be safe at the highest concentrations that exerted synergistic effects on all tested strains. In conclusion, our findings demonstrated that BT exerted synergistic antimicrobial and anti-biofilm effects when combined with COL against MDR organisms, especially COL-R GNB, in vitro and in vivo. The findings thus provide a reference for the clinical response to the serious challenge of MDR GNB and the exploitation of the potential antibacterial activities of existing clinical non-antibacterial drugs.

Anti-Inflammatory and Antioxidant Properties of Squalene in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio).

Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO4-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO4 treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO4-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.

Erythropoietin exerts neuroprotective effect against hypoxic ischemic brain injury in neonatal mice by activating AMPK pathway and upregulating UCP2

Purpose: To investigate the neuroprotective effect of erythropoietin (EPO) against hypoxic-ischemic brain injury in neonatal mice, and the underlying mechanism(s) of action.
 Methods: Sixty neonatal mice were assigned to 3 groups: sham, model and EPO intervention groups, each with 20 mice. Western blot assay was used to determine changes in the protein expressions of AMPK, UCP2 and KLF2. Fluorescence intensity of reactive oxygen species (ROS) was evaluated with flow cytometry.
 Results: The protein expressions of AMPK and KLF2 in the cerebral tissues of EPO intervention mice were significantly up-regulated, relative to model mice (p < 0.05), while UCP2 protein level was also significantly higher in EPO intervention mice than in model mice (p < 0.05). Mean fluorescence intensity of ROS was significantly higher in the cerebral cortex of model mice than in sham mice, but it was down-regulated in the cerebral cortex of mice in EPO intervention group, relative to the model group (p < 0.05).
 Conclusion: EPO exerts neuroprotective effect by activating AMPK pathway, up-regulating protein expression of UCP2, inhibiting production of mitochondrial ROS, and reducing oxidative stress in brain tissue. Thus, EPO has potentials for use in clinical practice as a neuroprotective agent.

Theaflavin-3,3'-Digallate Protects Cartilage from Degradation by Modulating Inflammation and Antioxidant Pathways.

Background Osteoarthritis (OA) is a common degenerative joint disease that may be closely linked to inflammation and oxidative stress destroying the balance of cartilage matrix. Theaflavin-3,3′-digallate (TFDG), a natural substance derived from black tea, has been reported to restrict the activity of inflammatory cytokines and effectively eliminate reactive oxygen species (ROS) in various diseases. However, it is not clear whether TFDG can improve OA. Methods Chondrocytes were treated with or without IL-1β and 20 μM and 40 μM TFDG. The effect of TFDG on the proliferation of chondrocytes was detected by CCK8. RT-qPCR was used to detect the gene expression of inflammatory factors, extracellular matrix synthesis, and degradation genes. Western blot and immunofluorescence assays were used to detect the protein expression. The fluorescence intensity of reactive oxygen species labeled by DCFH-DA was detected by flow cytometry. We established an OA rat model by performing destabilized medial meniscus (DMM) surgery to observe whether TFDG can protect chondrocytes under arthritis in vivo. Results TFDG was found to inhibit proinflammatory factors (IL-6, TNF-α, iNOS, and PGE) and matrix-degrading enzymes (MMP13, MMP3, and ADAMTS5) expression and protected extracellular matrix components of chondrocytes (ACAN, COL2, and SOX9). TFDG accelerated the scavenging of ROS caused by IL-1β according to the Nrf2 signaling pathway activation. At the same time, TFDG suppressed the PI3K/AKT/NF-κB and MAPK signaling pathways to delay the inflammatory process. The cartilage of DMM rats receiving TFDG showed lower Osteoarthritis Research Society International (OARSI) scores and expressed higher levels of COL2 and Nrf2 compared with those of rats in the DMM group. Conclusion TFDG could protect cartilage from degradation and alleviate osteoarthritis in rats, which suggests that TFDG has potential as a drug candidate for OA therapy.

Effects of Peroxiredoxin 6 and Its Mutants on the Isoproterenol Induced Myocardial Injury in H9C2 Cells and Rats.

Background Peroxiredoxin 6 (PRDX6) is an important antioxidant enzyme, with a potential application value in the treatment of diseases caused by oxidative damage. Methods PRDX6 and a mutant (mPRDX6) were heterologously expressed by using an E.coli expression system and purified by Ni-affinity chromatography. Isoproterenol (ISO) was used to induce a myocardial cell injury model and an animal myocardial injury model. After the treatment with PRDX6 and mPRDX6, the proliferation activity of H9C2 cells was detected by Cell Counting Kit-8 (CCK8) method; the apoptosis was evaluated by flow cytometry, and the histological changes of myocardial cells were observed by hematoxylin and eosin (H&E) staining, the levels of catalase (CAT), glutathione peroxidase (GPX), malondialdehyde (MDA), and superoxide dismutase (SOD) in ISO-treated H9C2 cells as well as in the heart tissue and serum of rats treated with ISO were detected, and the expression levels of Bax, Bcl-2 and peroxisome proliferators-activated receptors-γ (PPAR-γ) proteins were detected by Western blot. Results PRDX6 and mPRDX6 were successfully expressed and purified. The results of efficacy study showed that the mutant mPRDX6, in which the phospholipaseA2 (PLA2) activity of PRDX6 was deleted by site directed mutation, had a better protective effect against the myocardial injury than PRDX6. CCK8 results showed that compared with that in ISO group, the proliferation activity of H9C2 cells was significantly enhanced (P < 0.01), the apoptosis rate was significantly decreased (P < 0.01), and the fluorescence intensity of reactive oxygen species (ROS) was significantly decreased (P < 0.01) in mPRDX6 group. The results of H&E staining showed that the myocardial injury was alleviated to a certain extent in mPRDX6 group. Compared with those in ISO group, the activities of CAT, GPX, and SOD in H9C2 cells and the heart tissue and serum of rats were significantly increased (P < 0.05), while the contents of MDA were significantly decreased (P < 0.05). Western blot analysis showed that the expression level of Bcl-2 in H9C2 cells was significantly decreased (P < 0.01), and that of Bax and PPAR-γ was significantly increased (P < 0.05). Conclusion mPRDX6 has a protective effect against the myocardial injury induced by ISO, and the mechanism may be related to its antioxidation. This study may provide a theoretical basis for the research and development of drugs used for the treatment of myocardial injury.

Effects of exosomes from human adipose-derived mesenchymal stem cells on pulmonary vascular endothelial cells injury in septic mice and its mechanism

Effects of exosomes from human adipose-derived mesenchymal stem cells on pulmonary vascular endothelial cells injury in septic mice and its mechanism

Sevoflurane diminishes neurogenesis and promotes ferroptosis in embryonic prefrontal cortex via inhibiting nuclear factor-erythroid 2-related factor 2 expression.

Prenatal sevoflurane exposure may pose neurotoxicity to embryonic brain development and lead to cognitive dysfunction in offspring, but the underlying mechanism is still unclear. We aimed to investigate whether sevoflurane could cause neurogenesis abnormality and ferroptosis in embryonic prefrontal cortex (PFC) and to identify the role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the sevoflurane-related neurotoxicity. We used the rodents and primary neural stem cells to examine whether sevoflurane impacted proliferation, differentiation, ferroptosis and apoptosis in the neural stem cells of embryonic PFC. In addition, the expression of Nrf2 and the intensity of reactive oxygen species (ROS) were also assessed to explore the underlying molecular mechanism. Our results showed that sevoflurane exposure in third trimester could lead to neurogenesis inhibition and ferroptosis in-vivo embryonic PFC, with little influence on apoptosis. Moreover, a significant decrease in the expression of Nrf2 as well as an increase in ROS accumulation were also found in neural stem cells after sevoflurane anesthesia. We conclude that Nrf2-related neurogenesis inhibition and ferroptosis are a central mechanism contributing to sevoflurane-induced neurotoxicity in embryonic brain. The results of the present study are the first to demonstrate that ferroptosis and the expression of Nrf2 are involved in sevoflurane-related neurotoxicity in embryonic brain, which provides new vision for consideration in anesthesia-associated neurological abnormalities.

Mitochondrial-bound hexokinase 1 can affect the glucolipid metabolism and reactive oxygen species production in goose fatty liver

To investigate the functions of hexokinase 1 (HK1) in the formation of goose fatty liver, a total of 40 healthy 63-day-old male Landes geese were selected and randomly assigned to a control group and an overfeeding treatment. In addition, the overexpression or RNA interference assay of HK1, and transcriptome analysis after HK1 overexpression were performed in the goose primary hepatocytes. Data showed that the mRNA expression of hepatic HK1 was upregulated in overfed treatment compared to control on the 19 days of overfeeding. The expression of HK1 was increased in 50 mM glucose treatment in hepatocytes. Moreover, overexpression of HK1 tended to increase the relative lipid accumulation level and had weakened fluorescence intensity of reactive oxygen species (ROS), while knockdown of HK1 resulted in a tendency of relative lipid content decrease and had enhanced fluorescence intensity of ROS in cells in comparison to the control. The verification of transcriptome analysis indicated that the expression of ceruloplasmin (CP), acyl-CoA dehydrogenase medium-chain (ACADM), phosphoglucomutase 2 (PGM2), and phospholipase A2 group IVA (PLA2G4A) was significantly induced by HK1 overexpression, while that of enoyl-CoA hydratase, short chain 1 (ECHS1), cytochrome P450 family 2 subfamily C member 19 (CYP2C19), carnitine palmitoyltransferase 1 A (CPT1A), and oxidative stress-induced growth inhibitor 1 (OSGIN1) was inhibited. In summary, HK1 could promote fat deposition by affecting the process of glucolipid metabolism in the formation of goose fatty liver. Additionally, HK1 might decrease the ROS level in hepatocytes by regulating the expression of redox-related genes. HIGHLIGHTS The HK1 gene may promote the goose fatty liver production. The HK1 gene may be used as a research target to decrease the reactive oxygen species level in fatty liver.

CONTEMPORARY APPROACHES TO PROGNOSTICATION AND MANAGEMENT OF PELVIC RADIATION INJURIES IN GYNECOLOGICAL CANCER PATIENTS.

Rapid development of radiotherapeutic techniques and implementation of radiation therapy (RT) nanotechnologies in practice, taking into account principles of radiobiology, ensures that the planned dose will bedelivered to the target volume with minimal irradiation of healthy tissues while maintaining the guaranteed RTquality. Therefore, further advance of RT involves not only implementation of the new technologies in radiationpractice, but also the intensive developments in fields of radiation medicine and clinical radiobiology. search for optimal models of the high-energy (HDR - high dose rate) brachytherapy (BT) using the 192Irsource in comparison with effects of the reference gamma radiation from 60Co, thereby, to increase the effectivenessof chemoradiation therapy (CRT) of gynecological cancer patients (GCPs) with minimal radiation loads on criticalorgans and tissues in the tumor environment. The radiobiological study was aimed to determine the feasibility ofusing the transmembrane potential (TMP) and intensity of reactive oxygen species (ROS) production in peripheralblood lymphocytes (PBL) as predictors of radiosensitivity of non-malignant cells from the tumor environment or itsbed in order to minimize the RT complications in GCPs. Patients (n = 115) with cancer stages II-III, T2-3N0-1M0 were managed with comprehensiveconservative treatment. Three groups of patients were selected depending on the applied HDR BT method against abackground of the administered chemosensitizing agents. Blood samples of GCPs (n = 24) before the RT initiationand of apparently healthy individuals (AHIs, i.e. the control group, n = 18) were taken for the radiobiologicalresearch. Review of the direct results of 60Co or 192Ir sources use in HDR BT and of the follow-up data showed theincreased tumor positive response in the main study groups after CRT course by respectively 16.6 % and 20.1 % incomparison with 60Со HDR BT administration. Concerning local reactions it was noted that grade II radiation reactions were almost absent in the main groups. According to the results of radiobiological studies, it was establishedthat TMP level in PBL of GCPs was 1.36 times higher than in AHIs. Thus, the emerging of late radiation injuries depended on the accuracy of of individual computer planning and correct reproduction of the planned RT course, timely correction of treatment programs, use of a complexof rational medical prophylaxis, severity of tumor process and concomitant disorders, as well as on the used type ofHDR radiation sources (192Ir and 60Co). Changes in TMP values and intensity of ROS production in PBL of GCPs in comparison with AHIs, and the high values of these parameters in PBL of individual patients are a rationale to specifythem as additional indicators characterizing the possibility of radiation complications before the RT planning.

Effective-component compatibility of Bufei Yishen formula protects COPD rats against PM2.5-induced oxidative stress via miR-155/FOXO3a pathway

Ambient particulate matter <2.5 µm (PM2.5) has been identified as a critical risk factor in chronic obstructive pulmonary disease (COPD) exacerbation, but therapies for this condition are limited. Effective-component compatibility of Bufei Yishen formula (ECC-BYF) exhibits beneficial efficacy on COPD rats. However, its effect on PM2.5-aggravated COPD rats are considered to be uncertain. In this study, we used an established PM2.5-aggravated COPD rat model in vivo to evaluate the protective effect of ECC-BYF, and focused on its antioxidative role in PM2.5-stimulated bronchial epithelial cells via regulating microRNA (miR)−155/ forkhead box class O3a (FOXO3a) pathway. As expected, PM2.5-aggravated COPD rats showed a reduction of lung function, persistent lung inflammation, and remodeling of lung tissue. In comparison, ECC-BYF administration significantly enhanced lung function, alleviated alveolar destruction, inflammatory cell infiltration, mucus hypersecretion, and collagen deposition, along with diminishing inflammatory cytokine production and oxidative stress. Furthermore, ECC-BYF pretreatment markedly decreased the fluorescence intensity of reactive oxygen species (ROS) in PM2.5-induced human bronchial epithelial (Beas-2B) cells and primary mouse tracheal epithelial cells (MTECs), as well as reversing the imbalance between oxidants and antioxidants in Beas-2B. Meanwhile, ECC-BYF elevated FOXO3a while inhibiting miR-155 expression dose -dependently. In vitro transfection of miR-155 mimic into Beas-2B significantly decreased FOXO3a protein expression, accompanied by the reduced superoxide dismutase 2 (SOD2) and catalase (CAT) expressions, thus eliminating the protective effect of ECC-BYF on PM2.5-evoked oxidative stress. Nonethless, FOXO3a overexpression could partially restore the antioxidative effect of ECC-BYF. In conclusion, ECC-BYF can protect pre-existing COPD against PM2.5 contamination by exerting a profound antioxidative influence via regulating miR-155/FOXO3a signaling.

Simulated physiological oocyte maturation (SPOM) improves developmental competence of in vitro produced goat embryos

The effect of simulated physiological oocyte maturation on the developmental competence, reactive oxygen species production and apoptosis rate of in vitro produced goat embryos were studied in the present experiment. Oocytes and spermatozoa were recovered from ovaries and epididymis, respectively, procured from a local small animal abattoir. The oocytes aspirated from the ovaries were allocated into two groups, control (subjected to routine in vitro maturation, fertilization and culture) and simulated physiological oocyte maturation (SPOM) group (subjected to prematuration, followed by routine in vitro maturation, fertilization and culture). The SPOM group showed a significantly (p < 0.05) higher maturation and blastocyst rates (90.60 ± 0.46% and 29.09 ± 2.59%, respectively) as compared to the control group (85.29 ± 0.98% and 24.09 ± 1.08%). The intensity of reactive oxygen species of the embryos in the control group (14.98 ± 0.83 pixels/embryo) was significantly (p < 0.05) higher than the SPOM group (9.60 ± 0.76 pixels/embryo). The apoptosis rate was also significantly (p < 0.05) higher in the embryos of the control group (9.18 ± 1.07%) as compared to the SPOM group (5.71 ± 0.90%). In conclusion, the simulated physiological oocyte maturation system significantly increases the developmental competence of the oocytes and decreases the intensity of reactive oxygen species and embryonic apoptosis in abattoir derived goat embryos.

Modifications of proteins of membrane-cytoskeleton complex and production of reactive oxygen species in erythrocytes cryopreserved with polyethylene glycol

Protein modifications in the membrane-cytoskeleton complex (MCC) of human erythrocytes, as well as changes in the intensity of reactive oxygen species (ROS) production upon cell cryopreservation with polyethylene glycol (PEG) were investigated. The protein profile of ghosts of erythrocytes frozen with PEG has common features with both the control and cells frozen without cryoprotectant. PEG makes it possible to restrict the structural rearrangements of the main MCC proteins under the effect of extreme factors and to restrain the amount of high molecular weight polypeptide complexes induced by the protein-cross-linking reagent diamide at the control level, in contrast to cells frozen without a cryoprotectant. However, changes related to the protein peroxiredoxin 2 in ghosts of erythrocytes cryopreserved with PEG are also attributed to cells frozen without a cryoprotectant that may be associated with the activation of oxidative processes. This is evidenced by a 10-fold increase in ROS formation in erythrocytes frozen under PEG protection. Thus, upon cryopreservation of erythrocytes with PEG, certain disorders in MCC proteins may be associated with increased formation of ROS, which may contribute to the disorganization of the structural components of MCC and disrupt the stability of cryopreserved cells under physiological conditions.

Number Concentration of Ultrafine Bubble Being Effective in Promoting Barley Seed Germination

The test method for evaluating the promotion effect of Ultrafine Bubble (UFB) on barley seed germination was published as an ISO standard in July 2019. There should be a minimum number concentration of UFB that can provoke early seed germination, but it is still unclear. Hence, we conducted a germination examination by applying UFB water containing different number concentrations. The results showed that the promotion of germination is observed statistically with UFB water whose number concentration is in the range of 10 to the 8th power/ml. The difference in the number concentration of UFB was also supported by ESR analysis. After the application of ultrasonic sound of 43 kHz for 30 s, UFB water containing UFB of 6.03 x 108 particles/mL showed the larger signal intensity of reactive oxygen species which was composed more of hydroxyl radicals than that of 1.26 x 108 particles/mL. This fact indicated the difference in the number concentration of UFB without any effect of foreign matter included in UFB water.

Effect of metformin on mitochondrial pathway of apoptosis and oxidative stress in cell model of nonalcoholic fatty liver disease

Objective: To investigate the effects of metformin on mitochondrial pathway of apoptosis and oxidative stress in cell model of nonalcoholic fatty liver disease. Methods: An in vitro cell model of nonalcoholic fatty liver disease was established using 0.6 mmol/L oleic acid to induce lipid accumulation in HepG2 cells. HepG2 cells were divided into control (Con) group, oleic acid (OA) group, and metformin-low (1mmol/L) and high (10mmol/L) dose group. Oil Red O stain was used to detect intracellular lipid droplet distribution. The levels of alanine aminotransferase and aspartate aminotransferase in the culture supernatant were detected by assay kits. DCFH-DA method was used to detect the reactive oxygen species of HepG2 cells. Double staining flow cytometry was used to detect the apoptosis rate of HepG2 cells. Western blot was used to detect caspase-3, B-lymphocyte lymphoma-related protein, B-cell lymphoma 2, and cytochrome c protein. One-way analysis of variance was used to compare the data between groups. Results: Oleic acid-induced HepG2 cells were significantly increased with lipid droplets. Low and high-dose metformin had reduced intracellular lipid droplets accumulation. The effect of metformin in the high-dose group was more significant than that in the low-dose group. Aspartate aminotransferase and alanine aminotransferase in HepG2 cells of OA group were significantly increased, which were (43.41 ± 7.11) U/L and (29.56 ± 4.11) U/L, respectively. The intracellular aspartate aminotransferase and alanine aminotransferase were decreased significantly after the treatment with low and high-dose metformin, which were (32.44 ± 4.08)U/L, (19.31 ± 3.03) U/L, (26.00 ± 3.11) U/L and (15.11 ± 4.11) U/L, respectively and the differences were statistically significant (P < 0.05). DCFH-DA test results showed that the fluorescence intensity of reactive oxygen species in the oleic acid group was 41.21% ± 4.23%, while the fluorescence intensity of reactive oxygen species in the low and high-dose metformin groups were reduced to 27.44% ± 3.91%, and 17.55% ± 5.11%, respectively and the differences between the groups were statistically significant (P < 0.05). The results of flow cytometry analysis showed that the cell apoptosis rate of the OA group was significantly higher than that of the Con group (12.12% ± 0.72% vs. 3.04% ± 0.57%, P < 0.05).The apoptosis rate of HepG2 cells was significantly reduced after metformin treatment at low and high doses (8.71% ± 0.71%, 5.71% ± 0.61%, P < 0.05). Western blot results showed that compared with the Con group, the expressions of B-lymphocyte lymphoma-related protein, cytochrome c, and caspase-3 were increased in the OA group, while the B-cell lymphoma 2 were decreased (P < 0.05). The expression of B-lymphocyte lymphoma-related protein, cytochrome c, and caspase-3 protein in HepG2 cells was decreased after treatment with low and high-dose metformin, while B-cell lymphoma 2 was increased (P < 0.05). Conclusion: Metformin can effectively alleviate steatosis and improve the HepG2 function in cell model of nonalcoholic fatty liver disease. The mechanism of metformin may be related to the reduction of oxidative stress injury, the regulation of protein expression related to mitochondrial apoptosis pathway and the inhibition of cell apoptosis.

The impact of uric acid treatment on mitochlondria morphology and function in a steatosis model of L-02 cells

Background and objectives: Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic diseases worldwide. Oxidative stress (OS) is a major contributor toward NAFLD development, while mitochondria play a central role in OS. Our previous study has shown that uric acid (UA), as a dual function metabolite, could alleviate OS. This study examined the impact of UA on mitochondria morphology and function in a model of steatosis using L-02 cells to explore the pathogenesis of NAFLD. Methods: The L‑02 hepatocyte cell line was used to develop a steatosis cell model via 0.3 mM oleic acid (OA) over 24 h, subsequently treated with uric acid (UA) dose of 5, 10, and 20 mg/dL for 24, 48, and 72 h, respectively. The fluorescence intensity of reactive oxygen species (ROS), apoptosis rate, and activity of Succinate dehydrogenase(SDH), cytochrome oxidas(CCO), and adenosine triphosphate(ATP) synthase in electron transport chain (ETC), as well as the content of ATP and 8-OH-dG were examined; ultrastructure was observed under an electron microscope. Results: Treatment with UA at a concentration of 5 and 10 mg/dL decreased the rate of ROS production, apoptosis, and 8-OH-dG concentration, while supporting ATP recovery, and SDH activity in the steatosis model cell. It also promoted lipid droplet metabolism; however, the recovery of mitochondria morphology was not obvious. Conclusions: Treatment with UA dose of 5 and 10 mg/dL could protect mitochondria from OS damage. Future research requires a more stable and effective model. keywords : electron transport chain, oxidative stress, nonalcoholic fatty liver disease

Effects of a Combined Mitochondria-Targeted Treatment on the State of Mitochondria and Synaptic Membranes from the Brains of Diabetic Rats

On samples of the mitochondria and synaptic membranes isolated from rat brains using differential centrifugation, we tried to evaluate the neuroprotective efficacy of a combination of mitochondriaspecific antioxidants, acetyl-L-carnitine (ALC) and α-lipoic acid (LA), with nicotinamide (NAm), against diabetes-induced disorders in the CNS. Three groups of adult male Wistar rats were examined; these were control intact rats (group C), animals with experimental streptozotocin (STZ)-induced diabetes (group D; 6 weeks after STZ injections), and diabetic rats treated during the two final weeks of the above period by a combination of ALC, LA, and NAm (separate daily injections; doses 100, 50, and 100 mg/kg body mass, respectively; group D+T). At the day of preparation of the organelle samples, the mean blood glucose levels in groups C, D, and D+T were 4.8, 20.3, and 15.4 mM, respectively. The intensity of reactive oxygen species (ROS) production in the brain mitochondria from rats of group D measured by fluorescent analyses using 2’,7’-dichlorofluorescein diacetate was, on average, 37.2% greater than that in group C. Co-treatment provided a significant decrease in the above index in group D+T (27.8% in comparison with group D). Diabetes led to dramatic intensification of the CYP2E1 protein level in the liver of group D animals (242% vs. group C). In group D+T, this index was 33.1% lower than that in group D.

Met-enkephalin inhibits ROS production through Wnt/β-catenin signaling in the ZF4 cells of zebrafish

Opioid neuropeptides are developed early in the course of a long evolutionary process. As the endogenous messengers of immune system, opioid neuropeptides participate in regulating immune response. In this study, the mechanism that Met-enkephalin (M-ENK) inhibits ROS production through Wnt/β-catenin signaling was investigated in the ZF4 cells of zebrafish. ZF4 cells were exposed to 0, 10, 20, 40, 80, and 160 μM Met-enkephalin (M-ENK) for 24 h, and the cell viability was detected with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The cell viability was significantly increased by 10, 20, 40, 80, and 160 μM M-ENK. After ZF4 cells were exposed to 0, 20, 40, and 80 μM M-ENK for 24 h, the mRNA expression of Wnt10b, β-catenin, and CCAAT/enhancer binding protein α (C/EBPα) was significantly increased by 40 and 80 μM M-ENK. However, the mRNA and protein expression of GSK-3β was significantly decreased by 40 and 80 μM M-ENK. The protein expression of β-catenin was significantly induced by 40 and 80 μM M-ENK, while the protein expression of p-β-catenin was significantly decreased by 20, 40, and 80 μM M-ENK. In addition, the mRNA expression of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The levels of H2O2, ·OH, and O2·- were significantly decreased, but the activity of CAT, SOD, and GSH-PX was significantly increased by 40 and 80 μM M-ENK. The fluorescence intensity of reactive oxygen species (ROS) was decreased, and that of mitochondrial membrane potential (MMP) was increased with the increase of M-ENK concentration in ZF4 cells. The results showed that M-ENK could induce Wnt/β-catenin signaling, which further inhibited ROS production through the induction of C/EBPα, MMP, and the activities of antioxidant enzymes.