Elevated Levels Of Reactive Oxygen Species Research Articles

Lipid Variability Induces Endothelial Dysfunction by Increasing Inflammation and Oxidative Stress.

This study investigates the impact of fluctuating lipid levels on endothelial dysfunction. Human aortic and umbilical vein endothelial cells were cultured under varying palmitic acid (PA) concentrations: 0, 50, and 100 μM, and in a variability group alternating between 0 and 100 μM PA every 8 hours for 48 hours. In the lipid variability group, cells were exposed to 100 μM PA during the final 8 hours before analysis. We assessed inflammation using real-time polymerase chain reaction, Western blot, and cytokine enzyme-linked immunosorbent assay (ELISA); reactive oxygen species (ROS) levels with dichlorofluorescin diacetate assay; mitochondrial function through oxygen consumption rates via XF24 flux analyzer; and endothelial cell functionality via wound healing and cell adhesion assays. Cell viability was evaluated using the MTT assay. Variable PA levels significantly upregulated inflammatory genes and adhesion molecules (Il6, Mcp1, Icam, Vcam, E-selectin, iNos) at both transcriptomic and protein levels in human endothelial cells. Oscillating lipid levels reduced basal respiration, adenosine triphosphate synthesis, and maximal respiration, indicating mitochondrial dysfunction. This lipid variability also elevated ROS levels, contributing to a chronic inflammatory state. Functionally, these changes impaired cell migration and increased monocyte adhesion, and induced endothelial apoptosis, evidenced by reduced cell viability, increased BAX, and decreased BCL2 expression. Lipid variability induce endothelial dysfunction by elevating inflammation and oxidative stress, providing mechanistic insights into how lipid variability increases cardiovascular risk.

Circulating monocyte differentiation-activated nanoprodrugs for reprogramming macrophage immunity in atherosclerotic plaques

Atherosclerosis (AS) is characterized by the differentiation of monocytes and macrophages within AS plaques, coupled with the dysregulation of the immune microenvironment. Therefore, therapies targeting the immune microenvironment within plaques represent a promising strategy for AS treatment. However, the delivery efficiency of nanomedicine systems targeting plaques remains unsatisfactory, inevitably leading to off-target effects and premature leakage of active payloads. Based on the distinctive rise in intracellular reactive oxygen species (ROS) levels following differentiation of circulating monocytes within plaques, we designed a nano-prodrug system for spatiotemporally controllable activation of drug potency. We initially developed an inactive prodrug molecule (denoted as Citopril) with specific reactivity to ROS, and then Citopril was assembled into nanomicelles (denoted as Citop-NMs). After intravenous injection, "Trojan horse" Citop-NMs, facilitated by the polyethylene glycol (PEG) coating, can hitchhike into circulating monocytes. Moreover, Citop-NMs loaded in monocytes can efficiently accumulate within plaques via the inflammatory recruitment property of monocytes, thus achieving spatially targeted distribution. Upon the differentiation of monocyte vehicles, the elevated intracellular ROS level can trigger the temporal drug potency activation of the loaded Citopril. Treatment evaluation in an AS murine model demonstrates that Citop-NMs alleviate plaque burden without eliciting adverse reactions. Analysis of aortic macrophage phenotype unveils that Citop-NMs can regulate macrophage immunity in the lesions. The circulating monocyte differentiation-activated nanoprodrugs not only achieve targeted delivery of therapeutics to pathological sites but also effectively avoid nonspecific activation of off-target drugs in non-pathological regions. This spatiotemporally controllable strategy offers a novel approach for clinical precision medicine.

Interval training suppresses nod-like receptor protein 3 inflammasome activation to improve cardiac function in myocardial infarction rats by hindering the activation of the transforming growth factor-β1 pathway

ObjectiveMyocardial infarction (MI) -induced cardiac dysfunction can be attenuated by aerobic exercises. This study explored the mechanism of interval training (IT) regulating cardiac function in MI rats, providing some theoretical basis for clarifying MI pathogenesis and new ideas for clinically treating MI.MethodsRats were subjected to MI modeling, IT intervention, and treatments of the Transforming growth factor-β1 (TGF-β1) pathway or the nod-like receptor protein 3 (NLRP3) activators. Cardiac function and hemodynamic indicator alterations were observed. Myocardial pathological damage and fibrosis, reactive oxygen species (ROS) level, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities, MDA content, inflammasome-associated protein levels, and inflammatory factor levels were assessed. The binding between TGF-β1 and receptor was detected.ResultsMI rats exhibited decreased left ventricle ejection fraction (LVEF), left ventricle fractional shortening (LVFS), left ventricular systolic pressure (LVSP), positive and negative derivates max/min (dP/dt max/min) and increased left ventricular end-systolic pressure (LVEDP), a large number of scar areas in myocardium, disordered cell arrangement and extensive fibrotic lesions, increased TGF-β1 and receptor binding, elevated ROS level and MDA content and weakened SOD, CAT and GSH-Px activities, and up-regulated NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and cleaved-caspase-1 levels, while IT intervention caused ameliorated cardiac function. IT inactivated the TGF-β1 pathway to decrease oxidative stress in myocardial tissues of MI rats and inhibit NLRP3 inflammasome activation. Activating NLRP3 partially reversed IT-mediated improvement on cardiac function in MI rats.ConclusionIT diminished oxidative stress in myocardial tissues and suppressed NLRP3 inflammasome activation via inactivating the TGF-β1 pathway, thus improving the cardiac function of MI rats.

IRE1α regulates ROS and immune responses after UVB irradiation

Objective UV irradiation of the skin induces photo damage and generates cytotoxic intracellular reactive oxygen species (ROS), activating the unfolded protein response (UPR) to adapt or reduce these UVB-mediated damages. This study was designed to understand the role of the UPR mediator IRE1α in the antioxidant response following UVB irradiation of mouse skin and keratinocytes. Methods We used mice with an epidermal deletion of IRE1α and primary mouse keratinocytes to examine effects of UV on different parameters of the antioxidant response in the presence and absence of functional IRE1α. Results In the absence of IRE1α, PERK activity and protein levels are significantly compromised following UVB irradiation. Additionally, the loss of IRE1α suppressed phosphorylation of the PERK target, nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2-dependent antioxidant gene expression after UVB irradiation. Interestingly, IRE1α-deficient keratinocytes exhibit elevated basal ROS levels, while a robust ROS induction upon UVB exposure is abolished. Because UVB-induced ROS plays an essential role in regulating skin inflammation, we analyzed recruited immune cell populations and the expression of pro-inflammatory cytokines, Il-6 and Tnfα, in mice with epidermally targeted deletion of Ire1α. Following UVB irradiation, there was significantly less recruitment of neutrophils and leukocytes and reduced expression of pro-inflammatory cytokine genes in the skin of mice lacking IRE1α. Furthermore, keratinocyte proliferation was also significantly reduced after chronic UVB exposure in the skin of these mice. Conclusion Collectively, our findings indicate that IRE1α is essential for basal and UVB-induced oxidative stress response, UV-induced skin immune responses, and keratinocyte proliferation. Significance statement These findings shed new light on the protective function of IRE1α in the response to UV. IRE1α plays an important role in the regulation of ROS, PERK stability, and antioxidant gene expression in response to UVB in mouse keratinocytes and epidermis.

Gibberellin Positively Regulates Tomato Resistance to Tomato Yellow Leaf Curl Virus (TYLCV).

Tomato yellow leaf curl virus (TYLCV) is a prominent viral pathogen that adversely affects tomato plants. Effective strategies for mitigating the impact of TYLCV include isolating tomato plants from the whitefly, which is the vector of the virus, and utilizing transgenic lines that are resistant to the virus. In our preliminary investigations, we observed that the use of growth retardants increased the rate of TYLCV infection and intensified the damage to the tomato plants, suggesting a potential involvement of gibberellic acid (GA) in the conferring of resistance to TYLCV. In this study, we employed an infectious clone of TYLCV to inoculate tomato plants, which resulted in leaf curling and growth inhibition. Remarkably, this inoculation also led to the accumulation of GA3 and several other phytohormones. Subsequent treatment with GA3 effectively alleviated the TYLCV-induced leaf curling and growth inhibition, reduced TYLCV abundance in the leaves, enhanced the activity of antioxidant enzymes, and lowered the reactive oxygen species (ROS) levels in the leaves. Conversely, the treatment with PP333 exacerbated TYLCV-induced leaf curling and growth suppression, increased TYLCV abundance, decreased antioxidant enzyme activity, and elevated ROS levels in the leaves. The analysis of the gene expression profiles revealed that GA3 up-regulated the genes associated with disease resistance, such as WRKYs, NACs, MYBs, Cyt P450s, and ERFs, while it down-regulated the DELLA protein, a key agent in GA signaling. In contrast, PP333 induced gene expression changes that were the opposite of those caused by the GA3 treatment. These findings suggest that GA plays an essential role in the tomato's defense response against TYLCV and acts as a positive regulator of ROS scavenging and the expression of resistance-related genes.

Cell Membrane-Targeting Type I/II Photodynamic Therapy Combination with FSP1 Inhibition for Ferroptosis-Enhanced Photodynamic Immunotherapy.

An imbalance in reactive oxygen species (ROS) levels in tumor cells can result in the accumulation of lipid peroxide (LPO) which can induce ferroptosis. Moreover, elevated ROS levels in tumors present a chance to develop ROS-based cancer therapeutics including photodynamic therapy (PDT) and ferroptosis. However, their anticancer efficacies are compromised by insufficient oxygen levels and inherent cellular ROS regulatory mechanism. Herein, a cell membrane-targeting photosensitizer, TBzT-CNQi, which can generate 1O2, •OH, and O2 •- via type I/II process to induce a high level of LPO for potent ferroptosis and photodynamic therapy is developed. The FSP1 inhibitor (iFSP1) is incorporated with TBzT-CNQi to downregulate FSP1 expression, lower the intracellular CoQ10 content, induce a high level of LPO, and activate initial tumor immunogenic ferroptosis. In vitro and in vivo experiments demonstrate that the cell membrane-targeting type I/II PDT combination with FSP1 inhibition can evoke strong ICD and activate the immune response, which subsequently promotes the invasion of CD8+ T cells infiltration, facilitates the dendritic cell maturation, and decreases the tumor infiltration of tumor-associated macrophages. The study indicates that the combination of cell membrane-targeting type I/II PDT and FSP1 inhibition holds promise as a potential strategy for ferroptosis-enhanced photodynamic immunotherapy of hypoxia tumors.

Activation of PPARδ in bone marrow endothelial progenitor cells improves their hematopoiesis-supporting ability after myelosuppressive injury

Dysfunctional bone marrow (BM) endothelial progenitor cells (EPCs) with high levels of reactive oxygen species (ROS) are responsible for defective hematopoiesis in poor graft function (PGF) patients with acute leukemia or myelodysplastic neoplasms post-allotransplant. However, the underlying mechanism by which BM EPCs regulate their intracellular ROS levels and the capacity to support hematopoiesis have not been well clarified. Herein, we demonstrated decreased levels of peroxisome proliferator-activated receptor delta (PPARδ), a lipid-activated nuclear receptor, in BM EPCs of PGF patients compared with those with good graft function (GGF). In vitro assays further identified that PPARδ knockdown contributed to reduced and dysfunctional BM EPCs, characterized by the impaired ability to support hematopoiesis, which were restored by PPARδ overexpression. Moreover, GW501516, an agonist of PPARδ, repaired the damaged BM EPCs triggered by 5-fluorouracil (5FU) in vitro and in vivo. Clinically, activation of PPARδ by GW501516 benefited the damaged BM EPCs from PGF patients or acute leukemia patients in complete remission (CR) post-chemotherapy. Mechanistically, we found that increased expression of NADPH oxidases (NOXs), the main ROS-generating enzymes, may lead to elevated ROS level in BM EPCs, and insufficient PPARδ may trigger BM EPC damage via ROS/p53 pathway. Collectively, we found that defective PPARδ contributes to BM EPC dysfunction, whereas activation of PPARδ in BM EPCs improves their hematopoiesis-supporting ability after myelosuppressive therapy, which may provide a potential therapeutic target not only for patients with leukemia but also for those with other cancers.

Hydrogen Sulfide Regulates Erectile Function through Stimulation of Antioxidant Defense in the Corpus Cavernosum

Hydrogen sulfide (H2S) is a gaseous cell signaling molecule with vasodilatory properties. Diminished H2S production has been implicated in the pathology of several cardiovascular diseases. H2S may protect against oxidative stress through scavenging of reactive oxygen species (ROS) or through stimulation of the Nrf2-mediated cellular antioxidant defense systems. We have previously demonstrated that mice fed a western diet for 12 weeks develop elevated penile ROS levels and impaired erectile function, which coincides with diminished corpus cavernosal expression of cystathionine γ-lyase (CSE), a major H2S producing enzyme. The objective of this study was to investigate the role of H2S in the regulation of erectile function and penile redox balance. We hypothesized that H2S levels will be positively associated with cellular antioxidant defense and erectile function. Young male C57Bl/6J mice (n = 120) were fed a control diet (CD) or western diet (WD) ad libitum for 18 weeks. For the final six weeks of the dietary intervention, half of the mice were switched to an equivalent diet containing the H2S pro-drug SG1002 at approximately 20 mg/kg/day. Separately, male CSE knockout mice (n = 26) and wild type (WT) littermate controls (n = 26) were fed a standard diet and studied at 6 months. Following the interventions, erectile function was assessed by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) during cavernous nerve stimulation (1, 2, 4 V). In separate mice, penile in vivo ROS production was measured via microdialysis. Relative expression of 18 genes related to cellular antioxidant defense were assessed from naïve corpus cavernosum tissue by qRT-PCR. Group differences were determined by two-way ANOVA, with an α-level of 0.05. Erectile function was impaired in CSE KO mice (2V ICP/MAP - WT: 0.70±0.039 vs. KO: 0.55±0.043; p<0.05). Erectile function was similarly impaired in the untreated WD-fed mice (CD: 0.68±0.035 vs. WD: 0.49±0.012; p<0.05), while SG1002 induced a 54% functional restoration in WD mice (WD+SG1002: 0.59±0.019; p<0.05). Penile production of the ROS hydrogen peroxide and superoxide were elevated in CSE KO mice (WT: 0.73±0.04 vs. KO: 1.19±0.13; p<0.01). Penile ROS were also elevated in the WD mice (CD: 0.71±0.07 vs. WD: 1.09±0.15; p<0.01), which were normalized by SG1002 treatment (WD+SG1002: 0.83±0.07; p<0.01 vs. WD). Five antioxidant genes (Gclc, Gpx1, Gpx4, Prdx3, and Hmox1) that were stimulated by SG1002 treatment were also downregulated in CSE KO cavernosal tissues. Conversely, Txnip, a negative regulator of the thioredoxin antioxidant system was suppressed by SG1002 treatment and increased in CSE KO tissues. These results indicate that H2S is a key regulator of the cellular antioxidant systems and ROS balance in the penis, which may also regulate erectile function. Grant number K01DK115540 from the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ISOCITRATE DEHYDROGENASE 2 INHIBITION INDUCES ENDOTHELIAL SENESCENCE BY SUPPRESSION OF MITOPHAGY AND INCREASED ROS PRODUCTION

Objective: Endothelial senescence impairs vascular function and thus is a primary event of age-related vasculature diseases. Isocitrate dehydrogenase 2 (IDH2) plays an important role in inducing alpha-ketoglutarate (alpha-KG) production and preserving mitochondrial function. However, the mechanism and regulation of IDH2 in endothelial senescence have not been elucidated. Design and method: We confirmed vascular endothelial senescence using human umbilical vein endothelial cells(HUVECs) and IDH2 Knockout mouse. Results: We demonstrated that downregulation of IDH2 induced accumulation of miR-34b/c, which impaired mitophagy and elevated mitochondrial reactive oxygen species (ROS) levels by inhibiting mitophagy-related markers (PTEN-induced putative kinase 1 (PINK1), Parkin, LC-II/LC3-I, and p62) and attenuating Sirtuin deacetylation 3 (Sirt3) expression. The mitochondrial dysfunction induced by IDH2 deficiency disrupted cell homeostasis and the cell cycle and led to endothelial senescence. However, miR-34b/c inhibition or alpha-KG supplementation restored Sirt3, PINK1, Parkin, LC-II/LC3-I, p62, and mitochondrial ROS levels, subsequently alleviating endothelial senescence. Conclusions: This study demonstrates that IDH2 deficiency is critical for the suppression of alpha-KG levels, which induced endothelial senescence via miR-34b/c inhibition of mitophagy and mitochondrial ROS elevation in endothelial cells (Figure 7). Therefore, the IDH2/alpha-KG/miR-34b/c axis may provide a potential therapeutic target for age-related vascular diseases.

Telomerase Reverse Transcriptase Prevents Doxorubicin-induced Microvascular Endothelial Dysfunction

Introduction & Objective: Doxorubicin (Dox) is a frequently used chemotherapeutic known to induce cardiovascular (CV) complications by impairing microvascular- and cardiac function. We previously submitted an abstract showing that Dox-induced microvascular dysfunction preceded Dox-induced cardiac dysfunction and thus microvascular dysfunction might contribute to the damage of the heart itself.>Microvascular function specifically relies upon the adequate release of the vasodilator nitric oxide (NO). Our previous work showed that Telomerase Reverse Transcriptase (TERT), the catalytic subunit of telomerase, is necessary for physiological release of NO to mediate vasodilation. NO suppresses reactive oxygen species (ROS) levels. While Dox correlates with increased ROS production and suppressed TERT activity, attenuated TERT activity may explain the elevated levels of ROS after Dox exposure. The objective of this study was to determine whether TERT transcriptional activation is suffcient to protect against Dox-induced microvascular endothelial- and cardiac dysfunction. We utilized cycloastragenol (CAG) as transcriptional activator. Hypothesis: We hypothesized that TERT transcriptional activation is suffcient to prevent or attenuate Dox-induced endothelial dysfunction. Methods: WT or TERT loss-of-function rats on a Sprague Dawley background were assigned to untreated control-, CAG-, Dox and CAG+Dox groups. Animals were ~14 weeks of age to start the six-week protocol. Dox and CAG+Dox animals received one weekly IP Dox-injection (3x 4mg/kg*BW). CAG administer via diet with 50-60mg of CAG/day*BW. Microvascular function was assessed six-weeks after the first dose of Dox via videomicroscopy. Vasodilator responses to flow (flow-mediated dilation; FMD) and mediators of dilation (NO, H2O2) via fluorescent probes were evaluated. Cardiac function was evaluated using echocardiography. Human microvessels were treated either with 100nM Dox or 1μM CAG or a combination of both. Results: Dox treatment resulted in reduction in FMD in both WT and TERT mutant animals. Smooth-muscle dependent vasodilation was not impaired indicating endothelial impairment. CAG prevented Dox-induced endothelial dysfunction in WT but not TERT mutant rats. AGS499 another activator of TERT showed similar results suggestion a TERT mediated phenotype. Dox-treated WT animals exhibited an increase in H2O2 but not NO production. This phenotype was opposite in CAG+Dox WT animals. TERT mutant animals showed an increase in H2O2 but not NO production even without Dox. To assess which vasodilator mediates dilation to flow, we used L-NAME and PEG-Catalase to block NO- and H2O2, respectively. In CAG+Dox WT animals, L-NAME blocked the vasodilatory response to flow, while PEG-Cat blocked the vasodilatory response non-significantly. Similarly, we observed this protective effect of TERT in the human microcirculation. Human adipose microvessels treated over-night with Dox showed no vasodilatory response to flow while CAG+Dox treated vessels dilated normally similar to untreated control vessels. Smooth muscle function was not affected in neither group. Conclusion: We show evidence that TERT prevents Dox-induced endothelial dysfunction in an in vivo model and using human tissue. We highlight the specificity to TERT by a) using a genetic model lacking normal TERT activity and b) showing similar results using two different compounds which have been shown to activate TERT/promote TERT transcriptional activation. Ultimately, the present data shows that CAG is a potential therapeutic to counteract the detrimental effects of Dox treatment in the CV system via TERT signaling. RO1 HL133029-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exosomes loaded a smart bilayer-hydrogel scaffold with ROS-scavenging and macrophage-reprogramming properties for repairing cartilage defect

Enhancing the regeneration of cartilage defects remains challenging owing to limited innate self-healing as well as acute inflammation arising from the overexpression of reactive oxygen species (ROS) in post-traumatic microenvironments. Recently, stem cell-derived exosomes (Exos) have been developed as potential cell-free therapy for cartilage regeneration. Although this approach promotes chondrogenesis, it neglects the emerging inflammatory microenvironment. In this study, a smart bilayer-hydrogel dual-loaded with sodium diclofenac (DC), an anti-inflammatory drug, and Exos from bone marrow-derived mesenchymal stem cells was developed to mitigate initial-stage inflammation and promote late-stage stem-cell recruitment and chondrogenic differentiation. First, the upper-hydrogel composed of phenylboronic-acid-crosslinked polyvinyl alcohol degrades in response to elevated levels of ROS to release DC, which mitigates oxidative stress, thus reprogramming macrophages to the pro-healing state. Subsequently, Exos are slowly released from the lower-hydrogel composed of hyaluronic acid into an optimal microenvironment for the stimulation of chondrogenesis. Both in vitro and in vivo assays confirmed that the dual-loaded bilayer-hydrogel reduced post-traumatic inflammation and enhanced cartilage regeneration by effectively scavenging ROS and reprogramming macrophages. The proposed platform provides multi-staged therapy, which allows for the optimal harnessing of Exos as a therapeutic for cartilage regeneration.

Beta hydroxybutyrate induces lung cancer cell death, mitochondrial impairment and oxidative stress in a long term glucose-restricted condition.

Metabolic plasticity gives cancer cells the ability to shift between signaling pathways to facilitate their growth and survival. This study investigates the role of glucose deprivation in the presence and absence of beta-hydroxybutyrate (BHB) in growth, death, oxidative stress and the stemness features of lung cancer cells. A549 cells were exposed to various glucose conditions, both with and without beta-hydroxybutyrate (BHB), to evaluate their effects on apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) levels using flow cytometry, and the expression of CD133, CD44, SOX-9, and β-Catenin through Quantitative PCR. The activity of superoxide dismutase, glutathione peroxidase, and malondialdehyde was assessed using colorimetric assays. Treatment with therapeutic doses of BHB triggered apoptosis in A549 cells, particularly in cells adapted to glucose deprivation. The elevated ROS levels, combined with reduced levels of SOD and GPx, indicate that oxidative stress contributes to the cell arrest induced by BHB. Notably, BHB treatment under glucose-restricted conditions notably decreased CD133 expression, suggesting a potential inhibition of cell survival through the downregulation of CD133 levels. Additionally, the simultaneous decrease in mitochondrial membrane potential and increase in ROS levels indicate the potential for creating oxidative stress conditions to impede tumor cell growth in such environmental settings. The induced cell death, oxidative stress and mitochondria impairment beside attenuated levels of cancer stem cell markers following BHB administration emphasize on the distinctive role of metabolic plasticity of cancer cells and propose possible therapeutic approaches to control cancer cell growth through metabolic fuels.

Reactive Oxygen Species Activate a Ferritin-Linked TRPV4 Channel under a Static Magnetic Field.

Magnetogenetics has shown great potential for cell function and neuromodulation using heat or force effects under different magnetic fields; however, there is still a contradiction between experimental effects and underlying mechanisms by theoretical computation. In this study, we aimed to investigate the role of reactive oxygen species (ROS) in mechanical force-dependent regulation from a physicochemical perspective. The transient receptor potential vanilloid 4 (TRPV4) cation channels fused to ferritin (T4F) were overexpressed in HEK293T cells and exposed to static magnetic fields (sMF, 1.4-5.0 mT; gradient: 1.62 mT/cm). An elevation of ROS levels was found under sMF in T4F-overexpressing cells, which could lead to lipid oxidation. Compared with the overexpression of TRPV4, ferritin in T4F promoted the generation of ROS under the stimulation of sMF, probably related to the release of iron ions from ferritin. Then, the resulting ROS regulated the opening of the TRPV4 channel, which was attenuated by the direct addition of ROS inhibitors or an iron ion chelator, highlighting a close relationship among iron release, ROS production, and TRPV4 channel activation. Taken together, these findings indicate that the produced ROS under sMF act on the TRPV4 channel, regulating the influx of calcium ions. The study would provide a scientific basis for the application of magnetic regulation in cellular or neural regulation and disease treatment and contribute to the development of the more sensitive regulatory technology.

Protective Effect of N-Acetylcysteine on Rooster Semen Cryopreservation.

Cryopreservation of avian semen is a useful reproductive technique in the poultry industry. However, during cooling, elevated reactive oxygen species (ROS) levels have destructive effects on both quality and function of thawed sperm. The aim of the current study is to investigate the antioxidant effects of N-acetylcysteine (NAC) during rooster semen cryopreservation. Semen samples were collected from ten Ross 308 broiler breeder roosters (32 weeks) and mixed. The mixed samples were divided into five equal parts and cryopreserved in Lake Buffer extender that contained different concentrations (0, 0.01, 0.1, 1, and 10 mM) of NAC. The optimum concentration of NAC was determined based on quality parameters of mobility, viability, membrane integrity, acrosome integrity, lipid peroxidation, and mitochondrial membrane potential after the freeze-thaw process. There was a higher percentage (p < 0.05) of total motility (TM) (60.9 ± 2.4%) and progressive motility (PM) (35.6 ± 1.9%) observed with the NAC-0.1 group compared to the other groups. Significantly higher percentages of viability (74.4 ± 2.3% and 71 ± 2.3%), membrane integrity (76.4 ± 1.5% and 74.7 ± 1.5%) and mitochondrial membrane potential (67.1 ± 1.6% and 66.3 ± 1.6%) were observed in the NAC-0.1 and NAC-1 groups compared to the other frozen groups (p < 0.05). The lowest percentage of lipid peroxidation and nonviable sperm was found in the NAC-0.1 and NAC-1 groups compared to the other groups (p < 0.05). The average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and acrosome integrity, were not affected by different concentrations of NAC in the thawed sperm (p > 0.05). Both NAC-0.1 and NAC-1 appear to be beneficial for maintaining the quality of rooster sperm after thawing.

Ros-responsive nano-platform for lipid-specific fluorescence imaging of atherosclerosis

Fluorescent probes that specifically targeting Lipid droplets (LDs) have shown potential in biological imaging. Albeit, their in vivo applications are limited due to the hydrophobicity, low signal-to-noise ratio (SNR) and LDs-specificity. Thus, we designed a novel probe namely MeOND, and a reactive oxygen species (ROS)-responsive nano-platform to improve in vivo LDs-specific imaging. MeOND exhibits a remarkable twisted intramolecular charge transfer (TICT) effect with a strongly enhanced near-infrared emission in low-polarity lipid environment. Also, MeOND demonstrates satisfactory biocompatibility and superior intracellular LDs imaging capabilities. MeOND encapsulated nano-platform (MeOND@PMM) presented favorable water solubility and biocompatibility. MeOND@PMM remains stable in physiological conditions but quickly degrades in the environment of elevated ROS level. The released MeOND could then light up the intracellular LDs in atherosclerotic plaques. The design of the probe and nano-platform is expected to provide a better tool for the scientific research of LDs and LDs-related diseases.

Orostachys malacophylla (pall.) fisch extracts alleviate intestinal inflammation in Drosophila

Ethnopharmacological relevanceOrostachys malacophylla (Pall.) Fisch (O. malacophylla) is a succulent herbaceous plant that is the Orostachys genus of Crassulaceae family. O. malacophylla has been widely used as a traditional Chinese medicine with antioxidant, anti-inflammatory, anti-febrile, antidote, anti-Toxoplasma gondii properties. However, the biological function of alleviating intestinal inflammation and key bioactive compounds were still unknown. Aim of the studyWe used a Drosophila model to study the protective effects and bioactive compounds of O. malacophylla water extract (OMWE) and butanol extract (OMBE) on intestinal inflammation. Materials and methodsDrosophila intestinal inflammation was induced by oral invasion of dextran sodium sulfate (DSS) or Erwinia carotovora carotovora 15 (Ecc15). We revealed the protective effects of two extracts by determining intestinal reactive oxygen species (ROS) and antimicrobial peptide (AMP) levels and intestinal integrity, and using network pharmacology analysis to identify bioactive compounds. ResultsWe demonstrated that both OMWE and OMBE could ameliorate the detrimental effects of DSS, including a decreased survival rate, elevated ROS levels, increased cell death, excessive proliferation of ISCs, acid-base imbalance, and disruption of intestinal integrity. Moreover, the overabundance of lipid droplets (LDs) and AMPs by Ecc15 infection is mitigated by these extracts, thereby enhancing the flies' resistance to adverse stimuli. In addition, we used widely targeted metabolomics and network pharmacology analysis to identify bioactive compounds associated with IBD healing that are present in OMWE and OMBE. ConclusionsIn summary, our research indicates that OMWE and OMBE significantly mitigate intestinal inflammation and have the potential to be effective therapeutic agents for IBD in humans.

Discovery of Dolutegravir Derivative against Liver Cancer via Inducing Autophagy and DNA Damage.

We introduced a terminal alkyne into the core structure of dolutegravir, resulting in the synthesis of 34 novel dolutegravir-1,2,3-triazole compounds through click chemistry. These compounds exhibited remarkable inhibitory activities against two hepatocellular carcinoma cell lines, Huh7 and HepG2. Notably, compounds 5e and 5p demonstrated exceptional efficacy, particularly against Huh7 cells, with IC50 values of 2.64 and 5.42 μM. Additionally, both compounds induced apoptosis in Huh7 cells, suppressed tumor cell clone formation, and elevated reactive oxygen species (ROS) levels, further promoting tumor cell apoptosis. Furthermore, compounds 5e and 5p activated the LC3 signaling pathway, inducing autophagy, and triggered the γ-H2AX signaling pathway, resulting in DNA damage in tumor cells. Compound 5e exhibited low toxicity, highlighting its potential as a promising anti-tumor drug.

FoxO1 as the critical target of puerarin to inhibit osteoclastogenesis and bone resorption.

Elevated reactive oxygen species levels promote excessive osteoclastogenesis and bone resorption. Puerarin, a natural antioxidant, can prevent bone loss through its antioxidant effects; however, the underlying molecular mechanism remains unclear. This study aimed to explore the effects of puerarin on osteoclast differentiation and bone resorption by regulating the PI3K/AKT/FoxO1 signaling pathway. An ovariectomized (OVX) rat model of osteoporosis and H2O2-induced oxidative cell model of RAW 264.7 cells were established. The following indices were measured including bone μ-CT scanning and the protein expression levels of FoxO1, p-FoxO1, and catalase were detected using western blotting. Puerarin strongly alleviated oxidative stress-induced bone loss in OVX rats in vivo owing to its antioxidant effects. Puerarin improved the oxidative stress status of cells and inhibited osteoclast formation in vitro. Moreover, the protein expression of FoxO1 and its downstream target, catalase, was upregulated by puerarin. Puerarin improved the OPG/RANKL ratio, upregulated the protein expression and transcriptional activity of FoxO1, and suppressed the differentiation of RAW264.7 cells into osteoclasts. FoxO1 is a pivotal target of puerarin to confer anti-osteoporosis effects.

Amyloid Beta and Mitochondrial Perturbations: An Update on Mechanisms and Methods

Alzheimer’s disease (AD) is a debilitating mental disorder that causes a gradual decline in cognitive function and memory loss. The disease is associated with the accumulation of amyloid beta (Aβ) peptide-containing plaques outside the cells and the formation of neurofibrillary tangles (NFTs) inside the neurons due to tau protein hyperphosphorylation. AD is considered a hypometabolic disease at the cellular level, characterized by decreased adenosine triphosphate (ATP) levels and elevated reactive oxygen species (ROS) levels. Mitochondria, the cellular powerhouse, provide energy for cellular metabolism and protect cells against excessive oxidative stress. During disease progression, toxic Aβ causes significant disruptions in mitochondria bioenergetics, proteolytic systems, electron transport chains, mitophagy, and mitochondria DNA (mtDNA), leading to an accumulation of damaged organelles. This results in a disruption of the cellular energy demands, ROS scavenging pathways, and autophagy. The compromised mitochondria function caused by toxic Aβ exacerbates the development and progression of AD. This review aimed to provide current insights into the interactions between mitochondria and Aβ in AD pathogenesis, highlighting the pivotal role of mitochondrial dysfunction in the disease’s progression.

Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide

Ethnopharmacological relevanceAristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. Aim of the studyTo identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. Materials and methodsThe O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. ResultsAAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-β-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 μM, 9.05 μM, 3.87 μM, and 7.00 μM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. ConclusionCollectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.