H2O2-induced Oxidative Stress Research Articles

Structural characterization of three acidic polysaccharides from Opuntia dillenii Haw. fruits and their protective effect against hydrogen peroxide-induced oxidative stress in Huh-7 cells

Three novel acidic polysaccharide fractions (OFPP-1, OFPP-2, OFPP-3) with different molecular weights (803.7, 555.1 and 414.5 kDa) were isolated from the peeled Opuntia dillenii Haw. fruits by alkali-extraction, graded alcohol precipitation and column chromatography. Structural analysis indicated that OFPPs were pectic polysaccharides consisting of rhamnose, arabinose and galactose residues. The backbone of OFPP-1 consisted of a repeating unit →6-α-D-GalpA-(1→2)-α-L-Rhap-(1→ with T-α-D-GalpA-(1→6)-α-D-GalpA-(1→4)-α-D-Glcp-(1→, T-β-D-Xylp-(1→6)-α-D-GalpA-(1→4)-α-D-Glcp-(1→ or T-α-D-GalpA-(1→3)-α-L-Araf-(1→ as the side chains. The backbone of OFPP-2 consisted of a disaccharide repeating unit →2)-α-L-Rhap-(1→4)-β-D-GalpA-(1→ with T-β-L-Araf-(1→ as the branches substituted at the O-4 position of →2,4)-α-L-Rhap-(1→. Whereas the backbone of OFPP-3 was →2,4)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-β-L-Araf-(1→ or →2,4)-α-L-Rhap-(1→2)-α-L-Rhap-(1→4)-β-D-GalpA-(1→, which was branched at the O-4 position of →2,4)-α-L-Rhap-(1→. Moreover, these 3 polysaccharide fractions could protect Huh-7 cells against H2O2-induced oxidative stress to different extents by decreasing the MDA content and increasing the SOD, CAT, GSH-Px activities and the GSH level in the Huh-7 cells. These results suggest that OFPPs have the potential to be used as natural antioxidants.

5(S)-5-carboxystrictosidine from the root of Mappianthus iodoides ameliorates H2O2-induced apoptosis in H9c2 cardiomyocytes via PI3K/AKT and ERK pathways

5(S)-5-carboxystrictosidine (5-CS) is a compound found in the root of Mappianthus iodoides, a traditional Chinese medicine used for the treatment of coronary artery disease. The aim of the present study was to investigate the protective effect of 5-CS against oxidative stress-induced apoptosis in H9c2 cardiomyocytes and the underlying mechanisms. 5-CS pretreatment significantly protected against H2O2-induced cell death, LDH leakage and malondialdehyde (MDA) production which are indicators for oxidative stress injury. 5-CS also enhanced the activity of SOD and CAT. In addition, 5-CS pretreatment significantly inhibited H2O2-induced apoptosis, as determined by flow cytometer, suppressed the activity of caspase-3 and caspase-9 and attenuated the activation of cleaved caspase-3 and caspase-9. 5-CS also increased Akt and ERK activation altered by H2O2 using Western blot analysis. The PI3K-specific inhibitor LY294002 abolished 5-CS-induced Akt activation. The ERK-specific inhibitor PD98059 abolished 5-CS-induced ERK activation. Both LY294002 and PD98059 attenuated the protective effect of 5-CS on H9c2 cardiomyocytes against H2O2-induced apoptosis and cell death. Taken together, these results demonstrate that 5-CS prevents H2O2-induced oxidative stress injury in H9c2 cells by enhancing the activity of the endogenous antioxidant enzymes, inhibiting apoptosis, and modulating PI3K/Akt and ERK signaling pathways.

Differential Performance of Xanthophylls in Combination with Phenol Classes against H2O2-Induced Oxidative Stress: An In Vitro Analysis Using Retinal Pigment Epithelial Cells.

Xanthophylls, vital for ocular defense against blue light and reactive oxygen species, are prone to oxidative degradation; however, they may be regenerated antioxidant-rich plant phenols. Despite certain in vitro evidence, clinical studies show inconsistent findings and this may be due to varying phenolic reduction potentials. Therefore, the current study aims to investigate the ocular protective effect of various plant phenols combined with xanthophyll. Human retinal pigment epithelial cells (ARPE-19) are subjected to oxidative stress induced by hydrogen peroxide (H2O2) after xanthophyll and phenol pretreatment. Assessments include xanthophyll uptake, total antioxidant capacity, cell viability, intracellular reactive oxygen species levels, apoptosis, phagocytosis, and vascular endothelial growth factor formation. The study finds that while the combination of lutein with phenols does not show significant protective effects compared to lutein-only, zeaxanthin combined with phenols exhibits enhanced protection compared to both the zeaxanthin-only and control groups. The research reveals the complex relationship between xanthophylls and phenols, suggesting that the advantageous effects of their combination might vary among different xanthophylls. Caution is necessary when applying molecular theories to ocular health, and this necessitates further research, serving as a basis for proposing clinical trials to evaluate the efficacy of specific xanthophyll and phenol combinations.

Aqueous sage leave extract attenuates inflammation and oxidant-induced genotoxicity in human peripheral blood mononuclear cells.

Traditional medicine has used sage (Salvia officinalis L.) preparations for centuries to prevent and treat various inflammatory and oxidative stress-induced conditions. The aim of this in vitro study was to determine the bioactive properties of a sage leave extract obtained with environmentally friendly aqueous extraction and lyophilisation in primary human peripheral blood cells. To that end we measured the total phenolic and flavonoid content (TPC and TFC, respectively) with gas chromatography-mass spectrometry (GC-MS). Non-cytotoxic concentrations determined with the trypan blue assay were used to assess the antioxidant (DPPH, ABTS, and PAB assay), antigenotoxic (CBMN assay), immunomodulatory (IL-1β and TNF-α), and neuroprotective effects (AChE inhibition). The extract contained high TPC (162 mg GAE/g of dry extract) and TFC (39.47 mg QE/g of dry extract) concentrations, while β-thujone content was unexpectedly low (below 0.9 %). Strong radical-scavenging activity combined with glutathione reductase activation led to a decrease in basal and H2O2-induced oxidative stress and DNA damage. A decrease in TNF-α and increase in IL-1β levels suggest complex immunomodulatory response that could contribute to antioxidant and, together with mild AChE inhibition, neuroprotective effects. Overall, this study has demonstrated that aqueous sage leave extract reduces the levels of thujone, 1,8-cineole, pinene, and terpene ketones that could be toxic in high concentrations, while maintaining high concentrations of biologically active protective compounds which have a potential to prevent and/or treat inflammatory and oxidative stress-related conditions.

Exosomes derived from adipose tissue-derived stem cells alleviated H2O2-induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway

Hypertrophic scar (HS) is characterized by excessive collagen deposition and myofibroblasts activation. Endothelial-to-mesenchymal transition (EndoMT) and oxidative stress were pivotal in skin fibrosis process. Exosomes derived from adipose tissue-derived stem cells (ADSC-Exo) have the potential to attenuate EndoMT and inhibit fibrosis. The study revealed reactive oxygen species (ROS) levels were increased during EndoMT occurrence of dermal vasculature of HS. The morphology of endothelial cells exposure to H2O2, serving as an in vitro model of oxidative stress damage, transitioned from a cobblestone-like appearance to a spindle-like shape. Additionally, the levels of endothelial markers decreased in H2O2-treated endothelial cell, while the expression of fibrotic markers increased. Furthermore, H2O2 facilitated the accumulation of ROS, inhibited cell proliferation, retarded its migration and suppressed tube formation in endothelial cell. However, ADSC-Exo counteracted the biological effects induced by H2O2. Subsequently, miRNAs sequencing analysis revealed the significance of mir-486-3p in endothelial cell exposed to H2O2 and ADSC-Exo. Mir-486-3p overexpression enhanced the acceleration of EndoMT, its inhibitors represented the attenuation of EndoMT. Meanwhile, the target regulatory relationship was observed between mir-486-3p and Sirt6, whereby Sirt6 exerted its anti-EndoMT effect through Smad2/3 signaling pathway. Besides, our research had successfully demonstrated the impact of ADSC-Exo and mir-486-3p on animal models. These findings of our study collectively elucidated that ADSC-Exo effectively alleviated H2O2-induced ROS and EndoMT by inhibiting the mir-486-3p/Sirt6/Smad axis.Graphical A schematic diagram summarizing the impact of ADSC-Exo on oxidative stress and endothelial-to-mesenchymal transition in endothelial cells was presented in this study. ADSC-Exo effectively alleviated the accumulation of ROS in endothelial cells induced by H2O2 and suppressed the pro-fibrotic function through modulation of the mir-486-3p/Sirt6/Smad signaling pathway. ADSC-Exo attenuated the up-regulation of mir-486-3p in endothelial cells exposure to H2O2, establishing a target relationship between mir-486-3p and Sirt6. Overexpression of Sirt6 inhibited the occurrence of endothelial-to-mesenchymal transition, thereby suppressing collagen deposition and myofibroblasts activity by the regulation of Smad2/3 phosphorylation. Consequently, this led to a reduction in hypertrophic scar formation.Graphical Highlights1. The occurrence and development of ROS and endothelial-to-mesenchymal transition promoted hypertrophic scar fibrosis;2. H2O2 induced oxidative stress and EndoMT of endothelial cells, whereas ADSC-Exo ameliorated the process;3. Mir-486-3p was crucial for H2O2-induced EndoMT and the improvement of ADSC-Exo;4. Mir-486-3p directly targeted Sirt6 to facilitate EndoMT by regulating Smad signaling pathway.

Biological Characteristics, Domesticated Cultivation Protocol, Antioxidant Activity, and Protective Effects against Cellular Oxidative Stress of an Underutilized Medicinal Mushroom: Fomitopsis palustris.

Brown-rot fungus is one of the important medicinal mushrooms, which include some species within the genus Fomitopsis. This study identified wild macrofungi collected from a broad-leaved tree in Liaoning Province as Fomitopsis palustris using both morphological and molecular methods. To elucidate the potential medicinal and economic value of F. palustris, we conducted single-factor and orthogonal tests to optimize its mycelium culture conditions. Subsequently, we completed liquid culture and domestic cultivation based on these findings. Furthermore, crude polysaccharides were extracted from the cultivated fruiting bodies of F. palustris and their antioxidant activity was evaluated using chemical methods and cell-based models. The results showed that the optimal culture conditions for F. palustris mycelium were glucose as the carbon source, yeast extract powder as the nitrogen source, pH 6.0, and a temperature of 35 °C. Moreover, temperature was found to have the most significant impact on mycelial growth. The liquid strains were fermented for 6 days and then inoculated into a cultivation substrate composed of broadleaf sawdust, resulting in mature fruiting bodies in approximately 60 days. The crude polysaccharides extracted from the cultivated fruiting bodies of F. palustris (FPPs) possess in vitro scavenging abilities against DPPH radicals and OH radicals, as well as a certain ferric-reducing antioxidant power. Additionally, FPPs effectively mitigated H2O2-induced oxidative stress in RAW264.7cells by enhancing the intracellular activity of antioxidant enzymes such as SOD and CAT, scavenging excess ROS, and reducing MDA levels. This study provides preliminarily evidence of the potential medicinal and economic value of F. palustris and offers initial data for the future development and utilization of this species.

Loading rutin on surfaces by the layer-by-layer assembly technique to improve the oxidation resistance and osteogenesis of titanium implants in osteoporotic rats

The purpose of this study was to construct a rutin-controlled release system on the surface of Ti substrates and investigate its effects on osteogenesis and osseointegration on the surface of implants. The base layer, polyethylenimine (PEI), was immobilised on a titanium substrate. Then, hyaluronic acid (HA)/chitosan (CS)-rutin (RT) multilayer films were assembled on the PEI using layer-by-layer (LBL) assembly technology. We used scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and contact angle measurements to examine all Ti samples. The drug release test of rutin was also carried out to detect the slow-release performance. The osteogenic abilities of the samples were evaluated by experiments on an osteoporosis rat model and MC3T3-E1 cells. The results (SEM, FTIR and contact angle measurements) all confirmed that the PEI substrate layer and HA/CS-RT multilayer film were effectively immobilised on titanium. The drug release test revealed that a rutin controlled release mechanism had been successfully established. Furthermore, the in vitro data revealed that osteoblasts on the coated titanium matrix had greater adhesion, proliferation, and differentiation capacity than the osteoblasts on the pure titanium surface. When MC3T3-E1 cells were exposed to H2O2-induced oxidative stress in vitro, cell-based tests revealed great tolerance and increased osteogenic potential on HA/CS-RT substrates. We also found that the HA/CS-RT coating significantly increased the new bone mass around the implant. The LBL-deposited HA/CS-RT multilayer coating on the titanium base surface established an excellent rutin-controlled release system, which significantly improved osseointegration and promoted osteogenesis under oxidative stress conditions, suggesting a new implant therapy strategy for patients with osteoporosis.

L-Serine Protects Murine Retinal Ganglion Cells from Oxidative Stress via Modulation of Mitochondrial Dysfunction

Purpose This study aimed to investigate the effects of l-serine on mitochondrial dysfunction in retinal ganglion cells after exposure to H2O2-induced oxidative stress. Methods Retinal ganglion cells obtained from C57BL6 mice (postnatal days 1–4) were purified and cultured. A cell viability assay was performed following exposure to H2O2-induced oxidative stress to assess the cytoprotective effects of l-serine on retinal ganglion cells. Flow cytometry with CellROX Deep Red and MitoSOX dyes was performed to analyze the cytoplasmic and mitochondrial reactive oxygen species levels, respectively. Staining with the fluorescent probe JC-1 was used to detect changes in the mitochondrial membrane potential. The oxygen consumption rate and Bioenergetic Health Index were used to evaluate mitochondrial respiration. Results H2O2 treatment was found to induce mitochondrial dysfunction in retinal ganglion cells. Pretreatment with l-serine prevented cytotoxicity and significantly increased the viability of retinal ganglion cells following exposure to H2O2-induced oxidative stress (p < .05). l-Serine alleviated reactive oxygen species production in retinal ganglion cells following exposure to H2O2-induced oxidative (p < .05). Further, it successfully mitigated H2O2-induced mitochondrial depolarization in retinal ganglion cells (p < .05) and significantly increased the oxygen consumption rate and Bioenergetic Health Index in retinal ganglion cells following exposure to H2O2-induced oxidative stress (p < .05). Conclusion Pretreatment with l-serine protected retinal ganglion cells from H2O2-induced oxidative stress by improving mitochondrial function. The findings of the present study suggest that l-serine is a potential candidate for treatment of reactive oxygen species-related ocular diseases such as mitochondrial optic neuropathies.

Disruption of NADPH homeostasis by total flavonoids from Adinandra nitida Merr. ex Li leaves triggers ROS-dependent p53 activation leading to apoptosis in non-small cell lung cancer cells

Ethnopharmacological relevanceAdinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. Aim of the studyTo explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. Materials and methodsHuman NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0–160 μg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 μg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-H2DCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under H2O2-induced oxidative stress (0–40 μM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0–1 μM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP+ pool measurement and NADPH rescue (0–10 μM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. ResultsTFAN significantly inhibited A549 cells (wild-type p53) but not NCI–H1299 cells (p53-deficient), NCI–H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to H2O2-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP+ change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. ConclusionThe disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.

Wine- and stir-frying processing of Cuscutae Semen enhance its ability to alleviate oxidative stress and apoptosis via the Keap 1-Nrf2/HO-1 and PI3K/AKT pathways in H2O2-challenged KGN human granulosa cell line

BackgroundCuscutae Semen (CS) has been prescribed in traditional Chinese medicine (TCM) for millennia as an aging inhibitor, an anti-inflammatory agent, a pain reliever, and an aphrodisiac. Its three main forms include crude Cuscutae Semen (CCS), wine-processed CS (WCS), and stir-frying-processed CS (SFCS). Premature ovarian insufficiency (POI) is a globally occurring medical condition. The present work sought a highly efficacious multi-target therapeutic approach against POI with minimal side effects. Finally, it analyzed the relative differences among CCS, WCS and SFCS in terms of their therapeutic efficacy and modes of action against H2O2-challenged KGN human granulosa cell line.MethodsIn this study, ultrahigh-performance liquid chromatography (UPLC)-Q-ExactiveTM Orbitrap-mass spectrometry (MS), oxidative stress indices, reactive oxygen species (ROS), Mitochondrial membrane potential (MMP), real-time PCR, Western blotting, and molecular docking were used to investigate the protective effect of CCS, WCS and SFCS on KGN cells oxidative stress and apoptosis mechanisms.ResultsThe results confirmed that pretreatment with CCS, WCS and SFCS reduced H2O2-induced oxidative damage, accompanied by declining ROS levels and malondialdehyde (MDA) accumulation in the KGN cells. CCS, WCS and SFCS upregulated the expression of antioxidative levels (GSH, GSH/GSSG ratio, SOD, T-AOC),mitochondrial membrane potential (MMP) and the relative mRNA(Nrf2, Keap1, NQO-1, HO-1, SOD-1, CAT). They inhibited apoptosis by upregulating Bcl-2, downregulating Bax, cleaved caspase-9, and cleaved caspase-3, and lowering the Bax/Bcl-2 ratio. They also exerted antioxidant efficacy by partially activating the PI3K/Akt and Keap1-Nrf2/HO-1 signaling pathways.ConclusionsThe results of the present work demonstrated the inhibitory efficacy of CCS, WCS and SFCS against H2O2-induced oxidative stress and apoptosis in KGN cells and showed that the associated mechanisms included Keap1-Nrf2/HO-1 activation, P-PI3K upregulation, and P-Akt-mediated PI3K-Akt pathway induction.

Kaempferol derivatives from Hippophae rhamnoides Linn. ameliorate H2O2-induced oxidative stress in SH-SY5Y cells by upregulating Nrf2.

As a medicinal and edible food, Hippophae rhamnoides Linn. subsp. sinensis Rousi is plenty of bioactive secondary metabolites including flavonoids and their derivatives, which have a certain protective effect on oxidative damage. The present study isolated three new kaempferol derivatives (compound 1-3), named Hippophandine A-C, from H. rhamnoides seed residue. The structures of Hippophandine A-C were explicated by HR-ESI-MS, NMR, and chemical analyses. Hippophandine A-C (1, 5, and 10 µM) were used to attenuate H2O2-induced cell death in SH-SY5Y and explore the mechanism in oxidative damage. The results elucidated that Hippophandine A-C reduced MDA, and increased the relative SOD, GSH, CAT contents, Nrf2 and HO-1 expression in H2O2-induced SH-SY5Y.

Human gingival mesenchymal stem cells-lyosecretome attenuates adverse effect of hydrogen peroxide-induced oxidative stress on osteoblast cells

ObjectiveTo determine total protein content, antioxidant activity, and protective ability of lyophilized human gingival mesenchymal stem cells (hGMSCs)-secretome in hydrogen peroxide (H2O2) induced oxidative stress model. MethodsHuman GMSCs were cultured to obtain a conditioned medium (secretome), then lyophilized to produce lyosecretome. Total protein was determined by bicinchoninic acid assay (BCA) and SDS-PAGE to improve protein measurements. Antioxidant concentration was measured by ABTS assay, while the protective ability of lyosecretome against oxidative stress was determined by the metabolic activity of osteoblast cells. The study group was divided into a control group (culture medium) and a lyosecretome treatment group (0.0; 0.157, 0.313, 0.625, 1.25, 2.5, 5, and 10 mg/mL + H2O2). ResultsLyosecretome had a protein concentration of 2086.00 ± 0.20 μg/ml, with a molecular weight of 174, 74, 61, 55, and 26 kDa, which are thought to facilitate cell migration, as well as bind cytokines and growth factors. Lyosecretome also provided the highest antioxidant activity of 93.51% at a concentration of 4.8 mg/ml, with an IC50 value of 2.08 mg/ml. The highest cell metabolic activity (79.53 ± 2.41%) was shown in the 1.25 mg/ml lyosecretome treatment group. All concentrations of hGMSC-lyosecretome attenuate the adverse effect of H2O2-induced oxidative stress. ConclusionLyosecretome obtained from hGMSCs can maintain metabolic activity in osteoblast cells as protection against H2O2 oxidative stress.

Knocking Down PIAS3 Reduces H2O2-induced Oxidative Stress Injury in HT22 Cells.

Oxidative stress is involved in the pathological processes of many neurodegenerative diseases. Protein modification by small ubiquitin-like modifiers (SUMOs) has been implicated in oxidative stress injury. By conjugating SUMOs to their selective protein substrates, SUMO ligases play critical roles in regulating functions of proteins involved in oxidative stress injury. In this study, we screened siRNAs to knockdown the SUMO ligase PIAS3 to assess its role in H2O2-induced injury in HT22 cells. H2O2 stimulation increased total protein SUMOylation, facilitated intracellular reactive oxygen species (ROS) release, increased cleaved caspase-3 levels, promoted p38 and JNK activation (phosphorylation), upregulated apoptosis, and decreased cell viability. The siRNA against PIAS3 329-347 (siPIAS3-329) markedly downregulated the protein expression of PIAS3 and reversed these effects, whereas siNC (negative control) had no effect. Our findings demonstrate that PIAS3-mediated SUMOylation facilitates oxidative stress injury and p38/JNK-mediated cell apoptosis and that PIAS3 is a potential target to protect against oxidative stress injury.

A protoplast-based transient gene expression assay for the identification of heat and oxidative stress-regulatory genes in perennial ryegrass

BackgroundWith the accumulating omics data, an efficient and time-saving transient assay to express target genes is desired. Mesophyll protoplasts, maintaining most stress-physiological responses and cellular activities as intact plants, offer an alternative transient assay to study target genes’ effects on heat and oxidative stress responses.ResultsIn this study, a perennial ryegrass (Loliumperenne L.) mesophyll protoplast-based assay was established to effectively over- or down-regulate target genes. The relative expression levels of the target genes could be quantified using RT-qPCR, and the effects of heat and H2O2-induced oxidative stress on protoplasts’ viability could be quantitatively measured. The practicality of the assay was demonstrated by identifying the potential thermos-sensor genes LpTT3.1/LpTT3.2 in ryegrass that over-expressing these genes significantly altered protoplasts’ viability rates after heat stress.ConclusionThis protoplast-based rapid stress regulatory gene identification assay was briefed as ‘PRIDA’ that will complement the stable genetic transformation studies to rapidly identify candidate stress-regulatory genes in perennial ryegrass and other grass species.

Genome-Wide Identification and Expression Profile Analysis of the Phenylalanine Ammonia-Lyase Gene Family in Hevea brasiliensis.

The majority of the world's natural rubber comes from the rubber tree (Hevea brasiliensis). As a key enzyme for synthesizing phenylpropanoid compounds, phenylalanine ammonia-lyase (PAL) has a critical role in plant satisfactory growth and environmental adaptation. To clarify the characteristics of rubber tree PAL family genes, a genome-wide characterization of rubber tree PALs was conducted in this study. Eight PAL genes (HbPAL1-HbPAL8), which spread over chromosomes 3, 7, 8, 10, 12, 13, 14, 16, and 18, were found to be present in the genome of H. brasiliensis. Phylogenetic analysis classified HbPALs into groups I and II, and the group I HbPALs (HbPAL1-HbPAL6) displayed similar conserved motif compositions and gene architectures. Tissue expression patterns of HbPALs quantified by quantitative real-time PCR (qPCR) proved that distinct HbPALs exhibited varying tissue expression patterns. The HbPAL promoters contained a plethora of cis-acting elements that responded to hormones and stress, and the qPCR analysis demonstrated that abiotic stressors like cold, drought, salt, and H2O2-induced oxidative stress, as well as hormones like salicylic acid, abscisic acid, ethylene, and methyl jasmonate, controlled the expression of HbPALs. The majority of HbPALs were also regulated by powdery mildew, anthracnose, and Corynespora leaf fall disease infection. In addition, HbPAL1, HbPAL4, and HbPAL7 were significantly up-regulated in the bark of tapping panel dryness rubber trees relative to that of healthy trees. Our results provide a thorough comprehension of the characteristics of HbPAL genes and set the groundwork for further investigation of the biological functions of HbPALs in rubber trees.

Global Transcriptome Analysis Reveals Corresponding Genes and Key Pathways Involved in Oxidative Stress in Mouse Small Intestinal Cells

Abstract Understanding the molecular mechanisms involved in the effects of oxidative stress in humans and animals is important to minimize the damage it causes, leading to various intestinal diseases. Our aim is to study the genes and pathways involved in oxidative stress in the gut using mouse small intestinal epithelial cells (MODE-K) as a model. The MODE-K cell line was divided into two different groups: one group was treated with hydrogen peroxide (H2O2) and the other group was not. To analyze the effects of H2O2 exposure, cell viability, apoptosis rate and reactive oxygen species (ROS) levels were determined. Next, transcriptome sequencing was performed, differentially expressed genes (DEGs) were identified and function annotation was performed, followed by a series of bioinformatics analyses. Real-time PCR was used to confirm the transcriptome data. Our results showed that H2O2-induced oxidative stress significantly increased ROS synthesis and promoted cell apoptosis in mouse small intestinal epithelial cells. During oxidative stress, 1207 DEGs (859 up-regulated, 348 down-regulated) were identified. According to GO analysis, DEGs are annotated into 51 different GO classifications including 22 biological processes, 15 cellular components and 14 molecular functions. In addition, using KEGG, PPI and correlation analysis, the two most significant subnetworks were identified. Ten correlated nodal DEGs of the first subnetwork correspond to MAPK, NF-kappa B and PI3K-AKT signaling pathways, and six correlated DEGs of the second subnetwork are associated with mitochondria. KDM6B was found to link these two subnetworks. The results suggest that oxidative stress affects epithelial growth, metabolism and apoptosis in a mouse model of intestinal cells through signaling pathways such as MAPK and PI3K/AKT/NF-kappa B, and mitochondria-related genes that are interconnected through the PTGS2-KDM6B-MT-ATP6 pathway.

Polydopamine-Coated Kaempferol-Loaded MOF Nanoparticles: A Novel Therapeutic Strategy for Postoperative Neurocognitive Disorder.

The primary objective of this study was to develop an innovative nanomedicine-based therapeutic strategy to alleviate Postoperative Neurocognitive Disorder (PND) in patients undergoing surgery. To achieve this goal, polydopamine-coated Kaempferol-loaded Metal-Organic Framework nanoparticles (pDA/KAE@ZIF-8) were synthesized and evaluated. The study involved encapsulating Kaempferol (KAE) within ZIF-8 nanoparticles, followed by coating with polydopamine (PDA) to enhance biocompatibility and targeted delivery. The characterization of these nanoparticles (NPs) was conducted using various techniques including Scanning Electron Microscopy, Fourier-Transform Infrared Spectroscopy, X-ray Diffraction, and Ultraviolet-Visible spectroscopy. The efficacy of pDA/KAE@ZIF-8 NPs was tested in both in vitro and in vivo models, specifically focusing on their ability to penetrate the blood-brain barrier and protect neuronal cells against oxidative stress. The study found that pDA/KAE@ZIF-8 NPs efficiently penetrated the blood-brain barrier and were significantly taken up by neuronal cells. These nanoparticles demonstrated remarkable Reactive Oxygen Species (ROS) scavenging capabilities and stability under physiological conditions. In vitro studies showed that pDA/KAE@ZIF-8 NPs provided protection to HT-22 neuronal cells against H2O2-induced oxidative stress, reduced the levels of pro-inflammatory cytokines, and decreased apoptosis rates. In a PND mouse model, the treatment with pDA/KAE@ZIF-8 NPs significantly improved cognitive functions, surpassing the effects of KAE alone. This improvement was substantiated through behavioral tests and a noted reduction in hippocampal inflammation. The findings from this study underscore the potential of pDA/KAE@ZIF-8 NPs as an effective nanotherapeutic agent for PND. This approach offers a novel direction in the postoperative care of elderly patients, with the potential to transform the therapeutic landscape for neurocognitive disorders following surgery. The application of nanotechnology in this context opens new avenues for more effective and targeted treatments, thereby improving the quality of life for patients suffering from PND.

A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two β-domains (β2β1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

Huaier Polysaccharide Alleviates Dextran Sulphate Sodium Salt-Induced Colitis by Inhibiting Inflammation and Oxidative Stress, Maintaining the Intestinal Barrier, and Modulating Gut Microbiota.

The incidence of ulcerative colitis (UC) is increasing annually, and UC has a serious impact on patients' lives. Polysaccharides have gained attention as potential drug candidates for treating ulcerative colitis (UC) in recent years. Huaier (Trametes robiniophila Murr) is a fungus that has been used clinically for more than 1000 years, and its bioactive polysaccharide components have been reported to possess immunomodulatory effects, antitumour potential, and renoprotective effects. In this study, we aimed to examine the protective effects and mechanisms of Huaier polysaccharide (HP) against UC. Based on the H2O2-induced oxidative stress model in HT-29 cells and the dextran sulphate sodium salt (DSS)-induced UC model, we demonstrated that Huaier polysaccharides significantly alleviated DSS-induced colitis (weight loss, elevated disease activity index (DAI) scores, and colonic shortening). In addition, HP inhibited oxidative stress and inflammation and alleviated DSS-induced intestinal barrier damage. It also significantly promoted the expression of the mucin Muc2. Furthermore, HP reduced the abundance of harmful bacteria Escherichia-Shigella and promoted the abundance of beneficial bacteria Muribaculaceae_unclassified, Anaerotruncus, and Ruminococcaceae_unclassified to regulate the intestinal flora disturbance caused by DSS. Nontargeted metabolomics revealed that HP intervention would modulate metabolism by promoting levels of 3-hydroxybutyric acid, phosphatidylcholine (PC), and phosphatidylethanolamine (PE). These results demonstrated that HP had the ability to mitigate DSS-induced UC by suppressing oxidative stress and inflammation, maintaining the intestinal barrier, and modulating the intestinal flora. These findings will expand our knowledge of how HP functions and offer a theoretical foundation for using HP as a potential prebiotic to prevent UC.

Use of Optical Redox Imaging to Quantify Alveolar Macrophage Redox State in Infants: Proof of Concept Experiments in a Murine Model and Human Tracheal Aspirates Samples.

Emerging data indicate that lung macrophages (LM) may provide a novel biomarker to classify disease endotypes in bronchopulmonary dysplasia (BPD), a form of infant chronic lung disease, and that augmentation of the LM phenotype may be a potential therapeutic target. To contribute to this area of research, we first used Optical Redox Imaging (ORI) to characterize the responses to H2O2-induced oxidative stress and caffeine treatment in an in vitro model of mouse alveolar macrophages (AM). H2O2 caused a dose-dependent decrease in NADH and an increase in FAD-containing flavoproteins (Fp) and the redox ratio Fp/(NADH + Fp). Caffeine treatment did not affect Fp but significantly decreased NADH with doses of ≥50 µM, and 1000 µM caffeine treatment significantly increased the redox ratio and decreased the baseline level of mitochondrial ROS (reactive oxygen species). However, regardless of whether AM were pretreated with caffeine or not, the mitochondrial ROS levels increased to similar levels after H2O2 challenge. We then investigated the feasibility of utilizing ORI to examine macrophage redox status in tracheal aspirate (TA) samples obtained from premature infants receiving invasive ventilation. We observed significant heterogeneity in NADH, Fp, Fp/(NADH + Fp), and mitochondrial ROS of the TA macrophages. We found a possible positive correlation between gestational age and NADH and a negative correlation between mean airway pressure and NADH that provides hypotheses for future testing. Our study demonstrates that ORI is a feasible technique to characterize macrophage redox state in infant TA samples and supports further use of this method to investigate lung macrophage-mediated disease endotypes in BPD.