Reduction Of Reactive Oxygen Species Research Articles

Gpnmb silencing protects against hyperoxia-induced acute lung injury by inhibition of mitochondrial-mediated apoptosis.

Background: Hyperoxia-induced acute lung injury (HALI) is a complication to ventilation in patients with respiratory failure, which can lead to acute inflammatory lung injury and chronic lung disease. The aim of this study was to integrate bioinformatics analysis to identify key genes associated with HALI and validate their role in H2O2-induced cell injury model.Methods: Integrated bioinformatics analysis was performed to screen vital genes involved in hyperoxia-induced lung injury (HLI). CCK-8 and flow cytometry assays were performed to assess cell viability and apoptosis. Western blotting was performed to assess protein expression.Results: In this study, glycoprotein non-metastatic melanoma protein B (Gpnmb) was identified as a key gene in HLI by integrated bioinformatics analysis of 4 Gene Expression Omnibus (GEO) datasets (GSE97804, GSE51039, GSE76301 and GSE87350). Knockdown of Gpnmb increased cell viability and decreased apoptosis in H2O2-treated MLE-12 cells, suggesting that Gpnmb was a proapoptotic gene during HALI. Western blotting results showed that knockdown of Gpnmb reduced the expression of Bcl-2 associated X (BAX) and cleaved-caspase 3, and increased the expression of Bcl-2 in H2O2 treated MLE-12 cells. Furthermore, Gpnmb knockdown could significantly reduce reactive oxygen species (ROS) generation and improve the mitochondrial membrane potential.Conclusion: The present study showed that knockdown of Gpnmb may protect against HLI by repressing mitochondrial-mediated apoptosis.

A photocrosslinkable and anti-inflammatory hydrogel of loxoprofen-conjugated chitosan methacrylate.

Polymer-drug conjugates are widely used for drug delivery. Herein, we report an injectable hydrogel for local delivery of nonsteroidal anti-inflammatory drugs (NSAIDs) using chitosan (CS) as a carrier polymer. Loxoprofen (LOX) was conjugated to the backbone of CS via carbodiimide chemistry to obtain the LOX-CS conjugate. This conjugation transformed the water-insoluble unmodified CS into the water-soluble LOX-CS conjugate. In particular, the LOX-CS conjugate did not precipitate at pH 7, allowing smooth subsequent chemical modification with methacrylic anhydride (MA) to synthesize LOX-CS methacrylate (LOX-CS-MA) with significantly higher methacrylation substitution. The LOX-CS-MA was capable of in situ gel formation under visible light irradiation in the presence of a benzoin-2,4,6-trimethylbenzoylphosphinate lithium (LAP) photoinitiator. Our results show that the LOX-CS-MA hydrogel exhibited good cytocompatibility and blood compatibility. It promoted M2 polarization, inhibited pro-inflammatory gene expression, and upregulated anti-inflammatory gene expression of macrophages. Furthermore, the LOX-CS-MA hydrogel significantly reduced reactive oxygen species (ROS) and nitric oxide (NO) produced by lipopolysaccharide (LPS)-stimulated macrophages. A subcutaneous implanted LOX-CS-MA hydrogel in a rat model revealed significantly reduced inflammatory cell density, decreased cell infiltration, and a much thinner fibrous capsule compared to the CS methacrylate (CS-MA) hydrogel, thus markedly alleviating the inflammatory response. This study highlights the feasibility of CS-drug conjugates in preparing CS-based methacrylate hydrogels for sustained drug release.

Antioxidative effects of molybdenum and its association with reduced prevalence of hyperuricemia in the adult population.

The relationship between molybdenum and kidney-related disease outcomes, including hyperuricemia, is not well investigated. This study aims to determine whether molybdenum and its antioxidative property are associated with systemic inflammation and kidney-related disease parameters including hyperuricemia. Urinary molybdenum's epidemiological relationship to hyperuricemia and kidney-disease related outcomes was evaluated in 15,370 adult participants in the National Health and Nutrition Examination Survey (NHANES) collected between 1999 and 2016. Individuals' urinary molybdenum levels were corrected to their urinary creatinine concentrations. The association between urinary molybdenum-to-creatinine ratio and kidney-disease related outcomes were assessed by multivariable linear and logistic regression analyses, adjusting for covariates including age, sex, ethnicity, diabetes mellitus, hypertension, body mass index, and estimated glomerular filtration rate. Antimony and tungsten were used as control trace metals. Experimentally, HK-2 cell was used to assess molybdenum's antioxidative properties. HK-2 cells were challenged with H2O2-induced oxidative stress. Oxidative stress was measured using a fluorescent microplate assay for reactive oxygen species (ROS) and antioxidation levels were assessed by measuring the expression of manganese superoxide dismutase. In the adult NHANES population, urinary molybdenum-to-creatinine ratio was significantly associated with decreased serum uric acid (β, -0.119; 95% CI, -0.148 to -0.090) concentrations, and decreased prevalence of hyperuricemia (OR, 0.73; 95% CI, 0.64-0.83) and gout (OR, 0.71; 95% CI, 0.52-0.94). Higher urinary molybdenum levels were associated with lower levels of systemic oxidative stress (gamma-glutamyltransferase levels; β, -0.052; 95% CI, -0.067 to -0.037) and inflammation (C-reactive protein levels; β, -0.184; 95% CI, -0.220 to -0.148). In HK-2 cells under H2O2-induced oxidative stress, molybdenum upregulated manganese superoxide dismutase expression and decreased oxidative stress. Urinary molybdenum levels are associated with decreased prevalence of hyperuricemia and gout in adult population. Molybdenum's antioxidative properties might have acted as an important mechanism for the reduction of systemic inflammation, ROS, and uric acid levels.

Construction of tissue-engineered vascular grafts with enhanced patency by integrating heparin, cell-adhesive peptide, and carbon monoxide nanogenerators into acellular blood vessels

Small-diameter tissue-engineered vascular grafts (sdTEVGs) have garnered significant attention as a potential treatment modality for vascular bypass grafting and replacement therapy. However, the intimal hyperplasia and thrombosis are two major complications that impair graft patency during transplantation. To address this issue, we fabricated the covalent-organic framework (COF)-based carbon monoxide (CO) nanogenerator-and co-immobilized with LXW-7 peptide and heparin to establish a multifunctional surface on TEVGs constructed from acellular blood vessels for preventing thrombosis and stenosis. The cell-adhesive peptide LXW-7 could capture endothelial-forming cells (EFCs) to promote endothelialization, while the antithrombotic molecule heparin prevented thrombus formation. The reactive oxygen species (ROS)-triggered CO release suppressed the adhesion and activation of macrophages, leading to the reduction of ROS and inflammatory factors. As a result, the endothelial-to-mesenchymal transition (EndMT) triggered by inflammation was restricted, facilitating the maintenance of the homeostasis of the neo-endothelium and preventing pathological remodeling in TEVGs. When transplanted in vivo, these vascular grafts exhibited negligible intimal hyperplasia and remained patent for 3 months. This achievement provided a novel approach for constructing antithrombotic and anti-hyperplastic TEVGs.

Direct in vitro action of estrone on uterine and white adipose tissue in obesity

The hypothesis whether estrone (E1) could exhibit a direct action at uterus and white adipose tissue (WAT), under obesity was tested. In uterine tissue of obese rats, E1 increased nitric oxide (NO) synthesis, and reduced reactive oxygen species (ROS) production. The anti-oxidative action of E1 was sustained under inflammatory stress or high glucose levels. ICI 182780 or G15 compounds were employed as ER or GPER antagonists respectively. The action of E1 on ROS release involved ER participation; instead GPER mediated the acute stimulation on NO production. The antioxidative effect depends on NO-ROS balance. NO synthase (NOS) blockage suppressed the reduction in ROS synthesis elicited by E1, effect mediated by cNOS and not by iNOS. On WAT explants, E1 reduced ROS and thiobarbituric acid reactive substances production, and diminished leptin release. In summary, the data provide evidence that, in uterus and WAT, E1 counteracts inflammatory and oxidative stress induced by obesity.

Hydrogen gas alleviates acute ethanol-induced hepatotoxicity in mice via modulating TLR4/9 innate immune signaling and pyroptosis

Alcoholic liver disease (ALD), which is induced by chronic heavy alcohol consumption, accompanies complicated pathological mechanisms, including oxidative stress, inflammation, cell death, epigenetic changes and acetaldehyde-mediated toxicity. Hydrogen (H2) is the lightest gas with multiple biological effects such as high selective anti-oxidation, anti-inflammation and anti-apoptosis. However, the dose effects and innate immune mechanisms of intraperitoneal injection of H2 on ALD are limited. Here, we used acute ethanol-induced hepatotoxicity mice models to estimate the actions of intraperitoneal injection of H2 on ALD. The effects of H2 on acute ethanol-induced liver damage were examined by hepatic oil red O staining, quantitative PCR (qPCR) for lipid metabolic genes, hepatic triglyceride (TG) and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Hepatic mitochondrial superoxide (MitoSOX), 3-nitrotyrosine (3-NT), malondialdehyde (MDA), and glutathione (GSH) levels were examined to evaluate oxidative stress. Immunoblot, and immunofluorescence staining were used to further confirm the innate immune molecular targets of H2. Our results showed that intraperitoneal injection of H2 improved acute ethanol-induced liver injury in mice in a dose dependent manner, as indicated by decreasing serum ALT and AST levels, hepatic TG levels, and increasing lipid export genes (Mttp and Apob) mRNA levels and reducing fatty acid uptake gene (CD36) mRNA levels. Mechanistically, H2 inhibited hepatic oxidative stress as indicated by reducing reactive oxygen species (ROS), 3-NT, and MDA levels in the liver, while increasing hepatic GSH levels; inhibited the overactived TLR4/9-NF-κB-TNF-α/IL-1β/IL-18 innate immune signaling; suppressed the canonical Caspase-1-GSDMD pyroptosis signaling, and the non-canonical pyroptosis signaling, such as Caspase-11-GSDMD, Caspase-8-GSDMD and Caspase-3-GSDME signaling. Therefore, our study highlights that intraperitoneal injection of H2 may represent a novel therapeutic and safe strategy for ALD via modulating oxidative stress, innate immunity and pyroptosis.

EVALUATING THE EFFECT OF PRODUCTS EXTRACTED FROM PANAX BIPINNATIFIDUS SEEM ON MULTIPLE MYELOMA CELLS

Panax bipinnatifidus Seem (PB) is a precious medicinal plant in Vietnam. Although known for its many biological activities, the impact of this herb on multiple myeloma has not been elucidated. Objective: The study was conducted to evaluate the effects of total saponin extract from PB on the viability, reactive oxygen species (ROS), and mitochondrial cardiolipin content of multiple myeloma cells. Methods: Human myeloma cell line (KMS-20) cells were divided into 2 groups: control group and experimental group. In the control group, cells were grown in medium containing DMSO 0.05%. In the experimental group, cells were grown in medium containing total saponin extract (PB-TS) at different concentration ranges. Taxol is an anti-cancer drug used as a positive control in the study. Cell viability, ROS, and cardiolipin content were determined using suitable kits. Results: IC50 values of PB-TS for KMS-20 cells were 4.43±0.78 (µg/ml), higher than the IC50 value of Taxol (2.88±0.51 ng/ml). Besides, the CM-H2DCFDA fluorescence density of the cell group treated with Taxol and PB-TS increased to 121.52 ± 5.16% and 134.27 ± 3.59% compared to the control group (p<0.05). Moreover, the NAO fluorescence density of the cell group treated with Taxol and PB-TS also decreased to 62.73±6.87% and 41.56± 8.52%, respectively (p<0.05 compared with the control). Conclusion: The results of the study showed that total saponin from PB exhibited strong toxicity to KMS-20 cells through stimulation of ROS production and reduction of mitochondrial cardiolipin content.

Role of silica nanoparticles in enhancing drought tolerance of cereal crops

Cereal crops are essential for providing essential nutrients and energy in the daily human diet. Additionally, they have a crucial role as a significant constituent of cattle feed, hence enhancing meat production. Drought, being an abiotic stressor, adversely affects the growth and yield of numerous crops on a global scale. This issue poses a significant and pressing obstacle to maintaining global cereal crop production and ensuring food security. Nanoparticles have become a valuable resource for improving cereal crop yield and productivity under ongoing rapid climate change and escalating drought conditions. Among these, silica nanoparticles (SiNPs) have demonstrated their potential for agricultural applications in regions with limited water availability. Drought stress has detrimental effects on cereal crops, impacting their growth, metabolic, and physiological processes, hampering water and nutrient absorption, disrupting cellular membranes, damaging the photosynthetic apparatus, and reducing antioxidant activities by altering gene expression. SiNPs help preserve cellular membranes, regulate water balance, and improve water and nutrient absorption, resulting in a substantial enhancement in plant growth under water-deficit conditions. SiNPs also protect the photosynthetic system and enhance its efficiency, facilitate the accumulation of phenolics, hormones, osmolytes, antioxidant activities, and gene expression, thus empowering plants with increased resistance to drought stress. Moreover, SiNPs decrease leaf water loss by promoting stomatal closure, primarily by fostering the accumulation of abscisic acid (ABA) and mitigating oxidative stress damage by activating the antioxidant defence system and reducing reactive oxygen species (ROS). However, a limited number of studies examine the role of SiNPs in cereal crops under drought stress conditions. In this review, we highlighted the promising potential of SiNPs to improve cereal crop resilience by changing morpho-histological traits, antioxidant properties, and gene expression to maintain food security in drought-prone areas. This study will aid researchers in using SiNPs as an environmentally benign way to improving drought resistance in cereal crops in order to fulfill global food supply needs.

Indian Almond (Terminalia catappa Linn.) Leaf Extract Extends Lifespan by Improving Lipid Metabolism and Antioxidant Activity Dependent on AMPK Signaling Pathway in Caenorhabditis elegans under High-Glucose-Diet Conditions.

This study aimed to evaluate the antioxidant and antiaging effects of Indian almond (Terminalia catappa Linn.) leaf extract (TCE) on high-glucose (GLU)-induced obese Caenorhabditis elegans. Since TCE contains high contents of flavonoids and phenolics, strong radical scavenging activity was confirmed in vitro. The stress-resistance effect of TCE was confirmed under thermal and oxidative stress conditions at nontoxic tested concentrations (6.25, 12.5, and 25 μg/mL). GLU at 2% caused lipid and reactive oxygen species (ROS) accumulation in C. elegans, and TCE inhibited lipid and ROS accumulation under both normal and 2% GLU conditions in a concentration-dependent manner. In addition, TCE proved to be effective in prolonging the lifespan of C. elegans under normal and 2% GLU conditions. The ROS reduction effect of TCE was abolished in mutants deficient in daf-16/FOXO and skn-1/Nrf-2. In addition, the lifespan-extending effect of TCE in these two mutants disappeared. The lifespan-extending effect was abolished even in atgl-1/ATGL-deficiency mutants. The TCE effect was reduced in aak-1/AMPK-deficient mutants and completely abolished under 2% GLU conditions. Therefore, the effect of prolonging lifespan by inhibiting lipid and ROS accumulation under the high GLU conditions of TCE is considered to be the result of atgl-1, daf-16, and skn-1 being downregulated by aak-1. These results suggest that the physiological potential of TCE contributes to antiaging under metabolic disorders.

Reducing Cholesterol Level in Live Macrophages Improves Delivery Performance by Enhancing Blood Shear Stress Adaptation.

In recent years, live-cell-based drug delivery systems have gained considerable attention. However, shear stress, which accompanies blood flow, may cause cell death and weaken the delivery performance. In this study, we found that reducing cholesterol in macrophage plasma membranes enhanced their tumor targeting ability by more than 2-fold. Our study demonstrates that the reduced cholesterol level deactivated the mammalian target of rapamycin (mTOR) and consequently promoted the nuclear translocation of transcription factor EB (TFEB), which in turn enhanced the expression of superoxide dismutase (SOD) to reduce reactive oxygen species (ROS) induced by shear stress. A proof-of-concept system using low cholesterol macrophages attached to MXene (e.g., l-RX) was fabricated. In a melanoma mouse model, l-RX and laser irradiation treatments eliminated tumors with no recurrences observed in mice. Therefore, cholesterol reduction is a simple and effective way to enhance the targeting performance of macrophage-based drug delivery systems.

Genome- and transcriptome-wide identification of trehalose-6-phosphate phosphatases (TPP) gene family and their expression patterns under abiotic stress and exogenous trehalose in soybean

BackgroundTrehalose-6-phosphate phosphatase (TPP) is an essential enzyme catalyzing trehalose synthesis, an important regulatory factor for plant development and stress response in higher plants. However, the TPP gene family in soybean has not been reported.ResultsA comprehensive analysis of the TPP gene family identified 18 GmTPPs classified into eight groups based on the phylogenetic relationships and the conservation of protein in six monocot and eudicot plants. The closely linked subfamilies had similar motifs and intron/exon numbers. Segmental duplication was the main driving force of soybean GmTPPs expansion. In addition, analysis of the cis-regulatory elements and promoter regions of GmTPPs revealed that GmTPPs regulated the response to several abiotic stresses. Moreover, RNA-seq and qRT-PCR analysis of the tissue-specific GmTPPs under different abiotic stresses revealed that most GmTPPs were associated with response to different stresses, including cold, drought, saline-alkali, and exogenous trehalose. Notably, exogenous trehalose treatment up-regulated the expression of most TPP genes under saline-alkali conditions while increasing the carbohydrate and trehalose levels and reducing reactive oxygen species (ROS) accumulation in soybean sprouts, especially in the saline-alkali tolerant genotype. Furthermore, the interaction network and miRNA target prediction revealed that GmTPPs interacted with abiotic stress response-related transcription factors.ConclusionsThe findings in this study lay a foundation for further functional studies on TPP-based breeding to improve soybean development and stress tolerance.

Exogenous Melatonin Promotes Cold Tolerance in Grape Seedlings: Physiological, Transcriptomic, and Functional Evidence.

Melatonin (MEL) is an antioxidant molecule that enhances plant tolerance to environmental stress. However, the mechanisms by which MEL regulates cold signaling pathways in grapes under cold stress remain elusive. Here, we investigated the physiological and transcriptomic changes in grape seedlings treated with exogenous MEL to determine their protective role under cold stress. Results showed that 150 μM MEL effectively attenuated cold-induced cell damage by reducing reactive oxygen species (ROS) and preserving the chloroplast structure and function. MEL also inhibited tannin degradation, which contributed to its protective effect. Exogenous MEL promoted the synthesis of endogenous MEL, abscisic acid, auxin, and cytokinin while inhibiting gibberellin. Transcriptomic profiling revealed 776 differentially expressed transcripts in MEL-treated samples compared to controls. Functional analysis of a candidate hub gene, VvHSFA6b, showed that its overexpression in grape calli enhances cold tolerance by activating jasmonic acid synthesis pathway genes, promoting JA accumulation, and inhibiting JAZ-repressed transcription factors.

Inhibition of Protein Disulfide Isomerase Attenuates Osteoclast Differentiation and Function via the Readjustment of Cellular Redox State in Postmenopausal Osteoporosis.

Due to the accumulation of reactive oxygen species (ROS) and heightened activity of osteoclasts, postmenopausal osteoporosis could cause severe pathological bone destruction. Protein disulfide isomerase (PDI), an endoplasmic prototypic thiol isomerase, plays a central role in affecting cellular redox state. To test whether suppression of PDI could inhibit osteoclastogenesis through cellular redox regulation, bioinformatics network analysis was performed on the causative genes, followed by biological validation on the osteoclastogenesis in vitro and ovariectomy (OVX) mice model in vivo. The analysis identified PDI as one of gene targets for postmenopausal osteoporosis, which was positively expressed during osteoclastogenesis. Therefore, PDI expression inhibitor and chaperone activity inhibitor were used to verify the effects of PDI inhibitors on osteoclastogenesis. Results demonstrated that PDI inhibitors could reduce osteoclast number and inhibit resorption function via suppression on osteoclast marker genes. The mechanisms behind the scenes were the PDI inhibitors-caused intracellular ROS reduction via enhancement of the antioxidant system. Micro-CT and histological results indicated PDI inhibitors could effectively alleviate or even prevent bone loss in OVX mice. In conclusion, our findings unveiled the suppressive effects of PDI inhibitors on osteoclastogenesis by reducing intracellular ROS, providing new therapeutic options for postmenopausal osteoporosis.

The Inhibition of Mitogen-Activated Protein Kinases (MAPKs) and NF-κB Underlies the Neuroprotective Capacity of a Cinnamon/Curcumin/Turmeric Spice Blend in Aβ-Exposed THP-1 Cells.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by an increased level of β-amyloid (Aβ) protein deposition in the brain, yet the exact etiology remains elusive. Nowadays, treatments only target symptoms, thus the search for novel strategies is constantly stimulated, and looking to natural substances from the plant kingdom. The aim of this study was to investigate the neuroprotective effects of a spice blend composed of cinnamon bark and two different turmeric root extracts (CCSB) in Aβ-exposed THP-1 cells as a model of neuroinflammation. In abiotic assays, CCSB demonstrated an antioxidant capacity up to three times stronger than Trolox in the ORAC assay, and it reduced reactive oxygen species (ROS) induced by the amyloid fragment in THP-1 cells by up to 39.7%. Moreover, CCSB lowered the Aβ stimulated secretion of the pro-inflammatory cytokines IL-1β and IL-6 by up to 24.9% and 43.4%, respectively, along with their gene expression by up to 25.2% and 43.1%, respectively. The mechanism involved the mitogen-activated protein kinases ERK, JNK and p38, whose phosphorylation was reduced by up to 51.5%, 73.7%, and 58.2%, respectively. In addition, phosphorylation of p65, one of the five components forming NF-κB, was reduced by up to 86.1%. Our results suggest that CCSB can counteract the neuroinflammatory stimulus induced by Aβ-exposure in THP-1 cells, and therefore can be considered a potential candidate for AD management.

The function role of HIGD1A in nonalcoholic steatohepatitis from chronic hepatitis B

Background Accompanied by the growing prevalence of nonalcoholic fatty liver disease (NAFLD), the coexistence of chronic hepatitis B (CHB) and NAFLD has increased. In the context of CHB, there is limited understanding of the factors that influence the development of NASH. Methods We enrolled CHB combined NAFLD patients who had liver biopsy and divided them to NASH vs. non-NASH groups. A whole transcriptome chip was used to examine the expression profiles of long noncoding RNAs (lncRNAs) and mRNA in biopsied liver tissues. The function analysis of HIGD1A were performed. We knocked down or overexpressed HIGD1A in HepG2.2.15 cells by transient transfection of siRNA-HIGD1A or pcDNA-HIGD1A. In vivo investigations were conducted using hepatitis B virus (HBV) transgenic mice. Results In 65 patients with CHB and NAFLD, 28 were patients with NASH, and 37 were those without NASH. After screening 582 differentially expressed mRNAs, GO analysis revealed differentially expressed mRNAs acting on nicotinamide adenine dinucleotide phosphate (NADPH), which influenced redox enzyme activity. KEGG analysis also shown that they were involved in the NAFLD signaling pathway. The function analysis revealed that HIGD1A was associated with the mitochondrion. Then, both in vivo and in vitro CHB model, HIGD1A was significantly higher in the NASH group than in the non-NASH group. HIGD1A knockdown impaired mitochondrial transmembrane potential and induced cell apoptosis in HepG2.2.15 cells added oleic acid and palmitate. On the contrary, hepatic HIGD1A overexpression ameliorated free fatty acids-induced apoptosis and oxidative stress. Furthermore, HIGD1A reduced reactive oxygen species (ROS) level by increasing glutathione (GSH) expression, but Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK)/Acetyl-CoA carboxylase (ACC) pathway was not involved. Conclusion Both in vivo and in vitro CHB model, an upward trend of HIGD1A was observed in the NASH-related inflammatory response. HIGDIA played a protective role in cells against oxidative stress. Our data suggested that HIGD1A may be a positive regulator of NASH within the CHB context.

Klotho activation of Nrf2 inhibits the ferroptosis signaling pathway to ameliorate sepsis-associated acute kidney injury.

Sepsis-associated acute kidney injury (SA-AKI) is a common complication of sepsis and greatly increases patient mortality. Recombinant human Klotho protein (Klotho) is a protective protein that can be secreted by the kidney. The aim of this study was to explore the protective effect of Klotho on SA-AKI and its molecular mechanism. In vivo, a mouse SA-AKI model was constructed by cecum ligation perforation (CLP). In vitro, a human renal tubular cell epithelial cell line (HK2) was induced with lipopolysaccharide (LPS) in the SA-AKI model. Determine renal injury markers, inflammatory factors, oxidative stress and molecular proteins related to the ferroptosis signaling pathway. Klotho reduced the release of renal injury markers and inflammatory cytokines, decreased oxidative stress, improved renal histopathological changes, ameliorated mitochondrial damage in mouse renal tubular epithelial cells, increased HK2 cell viability and reduced reactive oxygen species (ROS) accumulation. Exogenous supplementation with Klotho increased the Klotho content in circulating blood, renal tissue and HK2 cells. In the SA-AKI model, Klotho attenuated renal tissue injury, increased HK2 cell viability, decreased inflammatory factor expression and oxidative stress, restored tubular epithelial mitochondrial function, and increased its level in circulating blood, renal tissue and HK2 cells. Klotho probably exerts its protective effects by activating Nrf2 to inhibit the ferroptosis signaling pathway.

Collagen as a Nanonization Strategy for Enhancing Natural Antioxidants in Cancer Treatment

The purpose of this research was to determine whether nanoscale resveratrol (RSV), collagen added to RSV, is more effective than regular RSV. Collagen is a sturdy emulsifier made in the human body. RSV is an antioxidant which has anti-inflammatory properties, allowing it to reduce reactive oxygen species (ROS) levels, which can induce cancer when in excess. However, RSV is not absorbed easily by the human body since it is excreted easily, making it less effective. This research paper was focused on overcoming the low bioavailability of RSV through reducing particle size. The concept behind this is nanotechnology, using technology to study 1 to 100 nanometer structures. Smaller RSV particles have increased surface area, which dissolves faster in the bloodstream. The hypothesis is that RSV particles would decrease by 8 times the volume, while the ROS levels in 3T3L1 cells would decrease with nanoscale RSV by 50%. The particle size distribution of RSV was measured with a spectrophotometer, while the ROS levels in 3T3L1 cells were measured by ELISA. The collagen-RSV solution had a greater peak than the regular RSV solution, indicating the particle size of RSV decreased after adding collagen. Also, the nanoscale RSV could reduce ROS levels more than regular RSV. This evidence supports the hypothesis that nanoscale RSV is effective in reducing ROS levels, and is smaller than regular RSV.

Enhancement of Mitochondrial Membrane Potential and Pro-Survival Pathways with Azole Compound C7 Resulting in Expansion of Hematopoietic Stem and Progenitor Cells

Ex-vivo expansion of hematopoietic stem & progenitor cells (HSPC) could improve hematopoietic recovery and reduce post-transplant complications, especially for umbilical cord blood (UCB) grafts where limited total cell numbers result in delayed engraftment. Freshly-thawed UCB mononucleated cells (MNCs) or purified CD34 + cells were cultured in animal component free medium supplemented with C7 and a basal cytokine cocktail. The effects of the expansion protocol were measured with phenotypic, in-vitro, and in-vivo functional assays. A range of molecular techniques were also performed to elucidate the mechanisms of action of C7. In previous studies, expansion cultures initiated with UCB-MNCs and supplemented with C7 resulted in significant expansions of UCB HSPC over 11 days and these grafts demonstrated elevated and sustained multi-lineage engraftment of human cells in primary NSG mice (Bari et al., 2018). In UCB-CD34+ enriched cultures, the addition of C7 to a basal cytokine cocktail also boosted absolute CD45+CD34(BR)+CD38-CD45RA- progenitor expansion by 283±29.4 fold over 11 days which was 1.86±0.12 fold higher than control cultures (p<0.001; n=6). Furthermore, C7 induced significant expansion of primitive HSPC in UCB-MNC defined by phenotype CD45+CD34(BR)+CD38-CD45RA-CD90+CD49f+ by more than 633±8.5 fold, which is 7.5±0.16 fold higher compared to control cultures (p<0.01; n=3). The addition of C7 to CD34+ cells from mobilized peripheral blood (PB) and bone marrow (BM) also resulted in a significant expansion of HSPCs (p<0.05; n=3) (Fig 1A). When expanded UCB-CD34+ were engrafted in NSG mice, NSG PB analysis at week 3 and week 28 post-transplantation showed significantly higher (p<0.01) human CD45+ cell engraftment in C7 expanded graft (16.4%±2.6) than cytokine expanded graft (7.8%±0.9). NSG BM analysis at week 31 also exhibited similar trends for human CD45+ cell engraftment in C7 expanded grafts with multilineage reconstitution (p<0.01; n=6). Significantly enhanced CD45+ cell viability (p<0.01; n=3) was observed in C7 cultures compared to control cultures. Mitochondrial health was also improved as confirmed by lower mitochondrial superoxides and higher mitochondrial membrane potential in C7-cultured UCB-MNCs (p<0.05; n=3) that is reflective of reduced reactive oxygen species (ROS) (Fig 1B). As C7 is a chemical analogue of a p38 inhibitor (SB203580), p-p38 (T180/Y182) was significantly reduced in C7-expanded CD34+ culture compared to controls. Significant increases in HIF-1α in C7 expanded CD34+ cells were also observed, potentially indicative of a hypoxic state induced by C7 to benefit HSPC together with reduced levels of ROS. Kinase inhibition profiling studies on C7 identified CK1δ, a kinase involved in Wnt and p53 signalling pathways, as one of the activation targets directly activated by C7. CK1δ was boosted in C7-expanded cells and this was further validated with western blots on C7 expanded pooled CD34+. CK1δ plays a putative role in p53 activation which is stipulated to promote HSC quiescence favouring long-term maintenance of HSPCs. Western blot analysis also revealed that p53 and BCL-2 were increased with C7 expanded CD34+. GSK3β was also boosted in C7 expanded pooled CD34+ cultures possibly as a result of CK1δ signalling leading to increased proliferation via the Wnt signalling pathway. Multiple safety studies were conducted on C7 to ensure its safety in clinical applications. This included the BIOMAP phenotypic safety and toxicology tests on human primary cells, AMES test and fluctuation assays for bacteria and SAFETYscan47 TM In Vitro Pharmacological Profiling for biochemical safety, all of which were negative for any cytotoxicity or off-targets associated with acute toxicity. No changes were detected in cytogenetics and fluorescence in situ hybridization with UCB-MNC for major leukemia-related genetics as well when they were cultured with C7. As a small molecule capable of significant HSPC expansion from both UCB and PB sources, C7 is promising for the future of HSPC transplantation. The mechanism behind C7 is likely to be a multi-pronged one with p38 inhibition, increase in HIF-1α, CK1δ activation, improved mitochondrial health, and induction of pro-survival pathways for expanding HSPC populations.

Suppression of endoplasmic reticulum stress by 4-PBA enhanced atherosclerotic plaque stability via up-regulating CLOCK expression

Endoplasmic reticulum (ER) stress refers to a condition where the normal functioning of the ER is disrupted due to a variety of cellular stress factors. As a result, there is an accumulation of unfolded and misfolded proteins within the ER. Numerous studies have shown that ER stress can exacerbate inflammatory reactions and contribute to the development of various inflammatory diseases. However, the role of ER stress in the stability of atherosclerotic plaques remains poorly understood. In this study, we aimed to explore the potential impact of a specific ER stress inhibitor known as 4-phenyl butyric acid (4-PBA) on atherosclerosis in mice. The mice were fed a high-fat diet, and treatment with 4-PBA significantly improved the stability of the atherosclerotic plaques. This was evidenced by a reduction in oxidative stress and an increase in circadian locomotor output cycles kaput (CLOCK) protein and mRNA expression within the plaques. Additionally, 4-PBA reduced the expression of ER stress-related proteins and decreased apoptosis in the atherosclerotic plaques. In vitro investigation, we observed the effect of 4-PBA on vascular smooth muscle cells (VSMCs) that were exposed to oxidized low-density lipoprotein (ox-LDL), a significant contributor to the development of atherosclerosis. 4-PBA reduced reactive oxygen species (ROS) production and attenuated apoptosis, GRP78 and CHOP protein expression in ox-LDL-Induced VSMCs via up-regulating CLOCK expression. However, when the short hairpin RNA against CLOCK (sh-CLOCK) was introduced to the VSMCs, the protective effect of 4-PBA was abolished. This suggests that the up-regulation of CLOCK expression is crucial for the beneficial effects of 4-PBA on atherosclerotic plaque stability. This finding suggests that targeting ER stress and modulating CLOCK protein levels might be a promising way to enhance the stability of atherosclerotic plaques.

PVB exerts anti-inflammatory effects by inhibiting the activation of MAPK and NF-κB signaling pathways and ROS generation in neutrophils

Pinaverium bromide (PVB) has been shown to protect mice against sepsis, which is predominantly attributed to PVB-mediated anti-inflammatory effects by inhibiting primed neutrophils to produce proinflammatory cytokines. However, the underlying mechanism(s) by which PVB affects neutrophils remains unknown. In this study, we report that treatment with PVB either before or after LPS stimulation attenuated IL-1β and TNF-α expression at both mRNA and protein levels in LPS-activated murine neutrophils. Further experiments revealed that PVB inhibited the phosphorylation of ERK, JNK, and IκBα in LPS-stimulated murine neutrophils. Moreover, PVB reduced reactive oxygen species (ROS) levels via regulating NADPH oxidase 2 (NOX2) activity, as represented by inhibiting p47phox translocation from the cytoplasm to the cellular membrane. Importantly, PVB significantly attenuated IL-1β, TNF-α, IL-6, CXCL1 production in both LPS-stimulated low density neutrophils (LDNs) and normal density neutrophils (NDNs) isolated from septic patients. Collectively, we demonstrated that PVB exerts anti-inflammatory effect by attenuating ROS generation and suppressing the activation of MAPK and NF-κB signaling pathways, suggesting that PVB may act as a potential therapeutic agent for sepsis by inhibiting neutrophil priming and activation.