Reduced Lactate Dehydrogenase Release Research Articles

Norwogonin aids in fighting MRSA-induced pneumonia by targeting agrAC to inhibit α-hemolysin production.

The increasing prevalence of infections related to methicillin-resistant Staphylococcus aureus (MRSA) necessitates the exploration of innovative therapeutic strategies that diverge from conventional antibiotic treatments. This is imperative to effectively combat resistance and manage these infections. The adoption of antivirulence strategies has emerged as a particularly promising avenue. This approach applies a heightened selective pressure on pathogens, thereby diminishing the likelihood of bacteria evolving resistance to antibiotics. In our pursuit of novel therapeutics for treating MRSA infections, we have focused on agents that inhibit the virulence of S. aureus without impeding its growth, aiming to minimize the development of drug resistance. α-Hemolysin, a critical virulence factor encoded by the hla gene, is a cytotoxin that forms pores in host cell membranes and plays a pivotal role in the progression of disease during bacterial infections. Herein, we identified that norwogonin could effectively inhibit Hla production via targeting agrAC, a crucial protein in quorum sensing, resulting in dose-dependent inhibition of hemolytic activity without suppressing S. aureus growth. In vitro assays illustrated that norwogonin decreased the thermal stability of agrAC, providing evidence of interaction between norwogonin and agrAC. Meanwhile, norwogonin alleviated Hla-mediated A549 cell damage and reduced lactate dehydrogenase release. In vivo studies suggested that norwogonin treatment blocked the establishment of a mouse model of pneumonia caused by S. aureus USA300. Notably, norwogonin enhanced the antibacterial potency of oxacillin. In conclusion, norwogonin is a promising candidate for treating S. aureus infections, offering a novel alternative to traditional antibiotics by targeting virulence factors and enhancing the efficacy of existing treatments.

Arnicolide D Inhibits Oxidative Stress-induced Breast Cancer Cell Growth and Invasion through Apoptosis, Ferroptosis, and Parthanatos.

Breast cancer is the most common malignant tumor in women, and its pathogenesis is very complicated. More and more studies have found that Traditional Chinese Medicine plays an important role in tumor prevention. To investigate the mechanism of arnicolide D isolated from Centipeda minima in breast cancer. Cell Counting Kit-8 (CCK-8), western blot, RT-qPCR, ELISA, flow cytometry, and Transwell were used to detect the effect of arnicolide D on the biological function of breast cancer cells. Arnicolide D promoted reactive oxygen species (ROS) production and induced a decrease in mitochondrial membrane potential in breast cancer cells, thereby inhibiting cell viability and increasing lactate dehydrogenase (LDH) release. Arnicolide D activated the classical apoptosis pathway to induce cell apoptosis; it significantly promoted PARP-1 expression, enhanced the nuclear translocation of apoptosis-inducing factor (AIF), and reduced the expression of AIF in mitochondria, indicating that it can induce the occurrence of parthanatos in a ROS dependent manner. In addition, arnicolide D down-regulated glutathione peroxidase 4 (GPX4) expression and increased the accumulation of Fe2+ and malondialdehyde (MDA), thereby activating ferroptosis. Apoptosis inhibitor, ferroptosis inhibitor, PARP inhibitor, PARP-1 siRNA, AIF siRNA and GPX4 overexpression vector significantly attenuated the inhibitory effect of arnicolide D on cell viability and reduced LDH release, which indicates that arnicolide D inhibits breast cancer cell growth by inducing apoptosis, parthanatos and ferroptosis. Arnicolide D also reduced breast cancer cell invasion and inhibited the expression of matrix metallopeptidase (MMP)-2 and MMP-9. Arnicolide D can activate a variety of cell death modes by inducing oxidative stress, thereby inhibiting the growth and invasion of breast cancer cells, indicating that arnicolide D has a good anti-tumor effect.

Exosomal long non-coding RNA AU020206 alleviates macrophage pyroptosis in atherosclerosis by suppressing CEBPB-mediated NLRP3 transcription

Recent studies have suggested exosomes (EXO) as potential therapeutic tools for cardiovascular diseases, including atherosclerosis (AS). This study investigates the function of bone marrow stem cell (BMSC)-derived exosomes (EXO) on macrophage pyroptosis in AS and explores the associated mechanism. BMSC-EXO were isolated from healthy mice and identified. RAW264.7 cells (mouse macrophages) were exposed to oxLDL to simulate an AS condition. BMSC-EXO treatment enhanced viability and reduced lactate dehydrogenase release of macrophages. An animal model of AS was established using ApoE−/− mice. BMSC-EXO treatment suppressed plaque formation as well as macrophage and lipid infiltration in mouse aortic tissues. Moreover, BMSC-EXO decreased concentrations of pyroptosis-related markers interleukin (IL)-1β, IL-18, cleaved-caspase-1 and gasdermin D in vitro and in vivo. Long non-coding RNA AU020206 was carried by the BMSC-EXO, and it bound to CCAAT enhancer binding protein beta (CEBPB) to block CEBPB-mediated transcriptional activation of NLR family pyrin domain containing 3 (NLRP3). Functional assays revealed that silencing of AU020206 aggravated macrophage pyroptosis and exacerbated AS symptoms in mice. These exacerbations were blocked upon CEBPB silencing but then restored after NLRP3 overexpression. In conclusion, this study demonstrates that AU020206 delivered by BMSC-EXO alleviates macrophage pyroptosis in AS by blocking CEBPB-mediated transcriptional activation of NLRP3.

Selenomethionine Alleviates Deoxynivalenol-Induced Oxidative Injury in Porcine Intestinal Epithelial Cells Independent of MAPK Pathway Regulation.

Deoxynivalenol (DON) is a prevalent contaminant in feed and food, posing a serious threat to the health of both humans and animals. The pig stands as an ideal subject for the study of DON due to its recognition as the most susceptible animal to DON. In this study, the IPEC-J2 cells were utilized as an in vitro model to explore the potential of SeMet in alleviating the intestinal toxicity and oxidative injury in intestinal epithelial cells when exposed to DON. Cells were treated either with or without 4.0 μM SeMet, in combination with or without a simultaneous treatment with 0.5 μg/mL DON, for a duration of 24 h. Then, cells or related samples were analyzed for cell proliferation, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, gene expressions, and protein expressions. The results showed that SeMet mitigated the cellular toxicity caused by DON, evidenced by elevated cell proliferation and the reduced LDH release of IPEC-J2 cells in the SeMet + DON group vs. the DON group. Moreover, the SeMet treatment markedly promoted antioxidant functions and decreased the oxidative injury in IPEC-J2 cell, which is indicated by the decreased ROS level and up-regulated mRNA levels of GPX1, TXNRD1, Nrf2, and GCLC in IPEC-J2 cells in the SeMet + DON group vs. the DON group. However, in both the absence and presence of exposure to DON, the SeMet treatment did not affect the protein expression of MAPK (JNK, Erk1/2, and P38) and phosphorylated MAPK (p-JNK, p-Erk1/2, and p-P38) in IPEC-J2 cells. Collectively, SeMet alleviated the DON-induced oxidative injury in porcine intestinal epithelial cells independent of the MAPK pathway regulation.

Propofol modulates Nrf2/NLRP3 signaling to ameliorate cigarette smoke-induced damage in human bronchial epithelial cells

Cigarette smoke extract (CSE) is known as a significant contributor to chronic obstructive pulmonary disease (COPD). Propofol, an anesthetic agent, has been studied for its potential protective effects against lung damage. This study aimed to elucidate the protective mechanisms of propofol against CSE-induced damage in human bronchial epithelial 16HBE cells. In CSE-induced 16HBE cells treated by propofol with or without transfection of nuclear factor erythroid 2-related factor 2 (Nrf2) interference plasmids, CCK-8 assay and lactate dehydrogenase (LDH) assay evaluated cytotoxicity. TUNEL assay and Western blot appraised cell apoptosis. ELISA and relevant assay kits severally measured inflammatory and oxidative stress levels. DCFH-DA fluorescent probe detected intracellular reactive oxygen species (ROS) activity. Immunofluorescence staining and Western blot estimated pyroptosis. Also, Western blot analyzed the expression of Nrf2/NLR family pyrin domain containing 3 (NLRP3) signaling-related proteins. Propofol was found to enhance the viability, reduce LDH release, and alleviate the apoptosis, inflammatory response, oxidative stress and pyroptosis in CSE-induced 16HBE cells in a concentration-dependent manner. Meanwhile, propofol decreased NLRP3 expression while raised Nrf2 expression. Further, after Nrf2 was silenced, the impacts of propofol on Nrf2/NLRP3 signaling, LDH release, apoptosis, inflammatory response, oxidative stress and pyroptosis in CSE-exposed 16HBE cells were eliminated. Conclusively, propofol may exert protective effects against CSE-induced damage in 16HBE cells, partly through the modulation of the Nrf2/NLRP3 signaling pathway, suggesting a potential therapeutic role for propofol in CSE-induced bronchial epithelial cell damage.

Inhibition of LINC00958 hinders the progression of osteoarthritis through regulation of the miR-214-3p/FOXM1 axis

ObjectiveWe investigated the impact of the long noncoding RNA LINC00958 on cellular activity and oxidative stress in osteoarthritis (OA).MethodsWe performed bioinformatics analysis via StarBase and luciferase reporter assays to predict and validate the interactions between LINC00958 and miR-214-3p and between miR-214-3p and FOXM1. The expression levels of LINC00958, miR-214-3p, and FOXM1 were measured by qRT-PCR and western blotting. To assess effects on CHON-001 cells, we performed MTT proliferation assays, evaluated cytotoxicity with a lactate dehydrogenase (LDH) assay, and examined apoptosis through flow cytometry. Additionally, we measured the levels of apoptosis-related proteins, including BAX and BCL2, using western blotting. The secretion of inflammatory cytokines (IL-6, IL-8, and TNF-α) was measured using ELISA.ResultsOur findings confirmed that LINC00958 is a direct target of miR-214-3p. LINC00958 expression was upregulated but miR-214-3p expression was downregulated in both OA cells and IL-1β-stimulated CHON-001 cells compared to the corresponding control cells. Remarkably, miR-214-3p expression was further reduced after miR-214-3p inhibitor treatment but increased following LINC00958-siRNA stimulation. Silencing LINC00958 significantly decreased its expression, and this effect was reversed by miR-214-3p inhibitor treatment. Notably, LINC00958-siRNA transfection alleviated the IL-1β-induced inflammatory response, as evidenced by the increased cell viability, reduced LDH release, suppression of apoptosis, downregulated BAX expression, and elevated BCL2 levels. Moreover, LINC00958 silencing led to reduced secretion of inflammatory factors from IL-1β-stimulated CHON-001 cells. The opposite results were observed in the miR-214-3p inhibitor-transfected groups. Furthermore, in CHON-001 cells, miR-214-3p directly targeted FOXM1 and negatively regulated its expression.ConclusionOur findings suggest that downregulating LINC00958 mitigates IL-1β-induced injury in CHON-001 cells through the miR-214-3p/FOXM1 axis. These results imply that LINC00958 plays a role in OA development and may be a valuable therapeutic target for OA.

EPCs-exos combined with tanshinone Ⅱ_A protect vascular endothelium cells from oxidative damage via PI3K/Akt pathway

This study aims to investigate the molecular mechanism of tanshinone Ⅱ_(A )(TaⅡ_A) combined with endothelial progenitor cells-derived exosomes(EPCs-exos) in protecting the aortic vascular endothelial cells(AVECs) from oxidative damage via the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt) pathway. The AVECs induced by 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine(POVPC) were randomly divided into model, TaⅡ_A, EPCs-exos, and TaⅡ_A+EPCs-exos groups, and the normal cells were taken as the control group. The cell counting kit-8(CCK-8) was used to examine the cell proliferation. The lactate dehydrogenase(LDH) cytotoxicity assay kit, Matrigel assay, DCFH-DA fluorescent probe, and laser confocal microscopy were employed to examine the LDH release, tube-forming ability, cellular reactive oxygen species(ROS) level, and endothelial cell skeleton morphology, respectively. The enzyme-linked immunosorbent assay was employed to measure the expression of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to determine the mRNA and protein levels, respectively, of PI3K and Akt. Compared with the control group, the model group showed decreased cell proliferation and tube-forming ability, increased LDH release, elevated ROS level, obvious cytoskeletal disruption, increased expression of IL-1β, IL-6, and TNF-α, and down-regulated mRNA and protein levels of PI3K and Akt. Compared with the model group, TaⅡ_A or EPCs-exos alone increased the cell proliferation and tube-forming ability, reduced LDH release, lowered the ROS level, repaired the damaged skeleton, decreased the expression of IL-1β, IL-6, and TNF-α, and up-regulated the mRNA and protein levels of PI3K and Akt. TaⅡ_A+EPCs-exos outperformed TaⅡ_A or EPCs-exos alone in regulating the above indexes. The results demonstrated that TaⅡ_A and EPCs-exos exerted a protective effect on POVPC-induced AVECs by activating the PI3K/Akt pathway, and the combination of the two had stronger therapeutic effect.

Abstract 176: The Pathological Role of Aldose Reductase in Isolated Cardiomyocytes Undergoing Hypoxia / Reoxygenation After Prior Exposure to High Glucose Concentrations

Hyperglycemia increases the vulnerability of the cardiovascular system to ischemia/reperfusion (IR) injury. There are currently no effective therapeutic strategies. We hypothesized that hyperglycemia would increase glucose flux through the polyol pathway. Aldose reductase (AR) catalyzes the first step of this pathway by converting glucose to sorbitol. This reaction generates more oxidative stress due to the depletion of NADPH and osmotic stress caused by sorbitol accumulation, making the heart more susceptible to IR injury. This study aimed to better understand the injury mechanism in cardiomyocytes after high glucose exposure and IR injury. Confluent cardiomyocytes were exposed for 24 hours to 25, 50, or 100 mM glucose or 25 mM glucose with 25 or 75 mM sucrose concentrations added. The non-metabolizable sugar sucrose was used as an osmolarity control. Epalrestat, an AR inhibitor, was added at 0, 1, or 10 μM per well (n = 18 per group) during glucose or sucrose exposure. Cells were then subjected to 24 hours of hypoxic conditions (glucose-, sucrose- and serum-free media; 0.01% O 2 , 37 °C; hypoxic chamber) or normoxic conditions (complete media; 21% O 2 , 37 °C), followed by a 2-hour reoxygenation period in normoxic conditions. Cell injury was quantified by measurement of lactate dehydrogenase (LDH) release. Data were normalized to 25 mM glucose after control/normoxic conditions without AR inhibitor. A three-way ANOVA was conducted to compare groups, followed by the Holm-Šidák test. Increasing glucose concentrations lead to increased cell damage after hypoxia/reoxygenation. Adding the AR inhibitor during the glucose exposure period reduced LDH release after hypoxia/reoxygenation in a dose-dependent manner. The addition of sucrose did not increase cell injury. The AR inhibitor did not affect cells exposed to sucrose. The polyol pathway may play a dominant role in increased cell injury by hyperglycemia after hypoxia/reoxygenation.

Antimicrobial effects of XF drugs against Candida albicans and its biofilms.

Compared with antibiotics for treating bacterial infections, there are a limited number of antifungal agents. This is due to several factors, including the difficulties of identifying suitable antifungals that target the fungal cell without damaging host cells, and the reduced rates of diagnosis of fungal infections compared with those caused by bacteria. The problem of treating fungal infections is exacerbated by an increasing incidence of antifungal resistance among human fungal pathogens. Three XF drugs (XF-73, XF-70, and DPD-207) have previously displayed innate bactericidal effects and a low propensity for microbial resistance, with XF-73 and XF-70 having a second, light-activated mechanism of action [known as photodynamic therapy (PDT)]. In an effort to expand the repertoire of antifungal agents, this research assessed the in vitro activity of XF drugs via both mechanisms of action against six strains of the fungal pathogen Candida albicans in both planktonic and biofilm cultures. In addition, this research examined the effects of XF drug treatment on biofilms of C. albicans in a reconstituted human oral epithelium model. All C. albicans strains tested were susceptible to XF-73 and XF-70, with minimum inhibitory concentrations (MICs) between 0.25 µg/mL and 2 µg/mL; DPD-207 was less potent, with MICs between 4 µg/mL and 16 µg/mL, and light activation did not enhance these MICs. Complete biofilm eradication was not reported at the tested XF drug concentrations. However, live and dead staining of C. albicans cells in biofilms after XF drug treatment demonstrated that XF-73 and XF-70 were active against most Candida biofilms tested from 64 µg/mL; again, light activation did not enhance anti-biofilm activity. Candida biofilms were more resistant to DPD-207, with fungicidal effects occurring from 256 µg/mL. XF-73 and XF-70 reduced penetration of C. albicans biofilm into reconstituted human oral epithelium (RHOE) and resulted in less damage (as determined by reduced lactate dehydrogenase release) than untreated biofilms. Overall, the results highlight the potential of XF drugs as new drugs for the management of topical infections caused by C. albicans. Further studies are warranted on the development of XF drugs as antifungals, particularly for XF-73 and XF-70.

Toxicity assessment of CeO₂ and CuO nanoparticles at the air-liquid interface using bioinspired condensational particle growth

CeO2 and CuO nanoparticles (NPs) are used as additives in petrodiesel to enhance engine performance leading to reduced diesel combustion emissions. Despite their benefits, the additive application poses human health concerns by releasing inhalable NPs into the ambient air. In this study, a bioinspired lung cell exposure system, Dosimetric Aerosol in Vitro Inhalation Device (DAVID), was employed for evaluating the toxicity of aerosolized CeO2 and CuO NPs with a short duration of exposure (≤10 min vs. hours in other systems) and without exerting toxicity from non-NP factors. Human epithelial A549 lung cells were cultured and maintained within DAVID at the air-liquid interface (ALI), onto which aerosolized NPs were deposited, and experiments in submerged cells were used for comparison. Exposure of the cells to the CeO2 NPs did not result in detectable IL-8 release, nor did it produce a significant reduction in cell viability based on lactate dehydrogenase (LDH) assay, with a marginal decrease (10%) at the dose of 388 μg/cm2 (273 cm2/cm2). In contrast, exposure to CuO NPs resulted in a concentration dependent reduction in LDH release based on LDH leakage, with 38% reduction in viability at the highest dose of 52 μg/cm2 (28.3 cm2/cm2). Cells exposed to CuO NPs resulted in a dose dependent cellular membrane toxicity and expressed IL-8 secretion at a global dose five times lower than cells exposed under submerged conditions. However, when comparing the ALI results at the local cellular dose of CuO NPs to the submerged results, the IL-8 secretion was similar. In this study, we demonstrated DAVID as a new exposure tool that helps evaluate aerosol toxicity in simulated lung environment. Our results also highlight the necessity in choosing the right assay endpoints for the given exposure scenario, e.g., LDH for ALI and Deep Blue for submerged conditions for cell viability.

Thiopental sodium attenuates hypoxia/reoxygenation-induced injury in osteoblasts by modulating AKT signaling.

Thiopental sodium (TPTS) is a barbiturate general anesthetic, while its effects on hypoxia/reoxygenation (H/R)-induced injury are still unclear. This study aimed to investigate whether TPTS exerts protective effects against the H/R-induced osteoblast cell injury and explore the underlying mechanisms. Osteoblast cell injury model was induced by the H/R condition, which was treated with or without TPTS. Cell viability and lactate dehydrogenase (LDH) release were determined by the corresponding commercial kits. The levels of oxidative stress were determined in the experimental groups. Cell apoptosis and Caspase-3 activities were determined by propidium iodide staining and substrate-based assay, respectively. Western blotting and qRT-PCR were performed to measure the mRNA and protein levels, respectively. Treatment with TPTS was able to increase cell viability and reduce LDH release in H/R-induced osteoblasts. Additionally, TPTS regulated oxidative stress in H/R-induced osteoblasts by suppressing malondialdehyde (MDA) and reactive oxygen species (ROS) as well as boosting superoxide dismutase (SOD). TPTS was able to suppress cell apoptosis by suppressing Caspase-3 activity and cleavage. TPTS exerted protective effects against cell injury and apoptosis induced by the H/R conditions, which were associated with its regulation of Akt signaling. Moreover, TPTS induced osteoblast differentiation under the H/R condition. In summary, TPTS attenuates H/R-induced injury in osteoblasts by regulating AKT signaling.

Salvianolic acid B inhibits atherosclerosis and TNF-α-induced inflammation by regulating NF-κB/NLRP3 signaling pathway

BackgroundInflammation is critical in the pathophysiology of atherosclerosis (AS). The aim of this study was to investigate the protective effect of salvianolic acid B (Sal B) on AS and to explore the molecular mechanism of tumor necrosis factor-α (TNF-α)-induced damage in human umbilical vein endothelial cells (HUVECs). MethodsIn vivo studies, LDLR−/− mice were fed a high-fat diet (HFD) for 14 weeks to establish an AS model to evaluate the protective effect of Sal B on the development of AS. Total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels were determined in the blood serum. En face and cross section lipid deposits were measured and quantified with Oil Red O staining. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to quantify atherosclerotic plaque size and collagen fiber content in aortic root sections. Reactive oxygen species (ROS) were detected in aortic root using dihydroethylenediamine (DHE) staining. Apoptosis rate was determined by TdT-mediated dUTP nick end labeling (TUNEL) staining. Immunofluorescence (IF) staining was used to detect the expression of the nuclear factor kappa-B (NF-κB) p65 and NOD-like receptor family pyrin domain containing 3 (NLRP3). To further investigate the protective effect of Sal B, we used TNF-α induced HUVECs inflammation model. We examined cell viability, lactate dehydrogenase (LDH) content, and ROS production. The transcription of NF-κB was evaluated by immunofluorescence. The mRNA levels of NLRP3, caspase-1, and IL-1β were detected by RT-PCR. Pyroptosis related proteins were detected by Western blot. ResultsThe change in the weight of the mice over time was an indication that Sal B had an effect on weight gain. In vivo studies: we were able to show that the serum lipids TC, TG and LDL-C were increased in the model group and that the treatment with Sal B reduced the levels of serum lipids. Histological staining showed that the LDLR−/− mice had a large amount of foam cell deposition accompanied by inflammatory cell infiltration and the formation of atherosclerotic plaques in theMOD group. The pathological abnormalities were significantly improved by Sal B treatment. ROS release and apoptosis were significantly increased after HFD in aortic root, which was attenuated by Sal B. IF results showed that the expression of NF-κB p65 and NLRP3 was significantly increased in the MOD group and significantly decreased in the Sal B group, suggesting that Sal B may act through the NF-κB/NLRP3 pathway. And in vitro studies: inflammatory damage of HUEVCs was induced by TNF-α, and Sal B treatmented significantly increased cell viability and reduced LDH release. It was also found that Sal B inhibited ROS level increase after TNF-α-induced HUEVCs. Activation of NF-κB p65 by TNF-α stimulation, NF-κB p65 is transferred to the nucleus. Sal B treatment could reverse this effect. RT-PCR and Western blot showed that Sal B affected NF-κB transcription and NLRP3 inflammasome activation and could significantly inhibit TNF-α-induced NLRP3 inflammasome activation. These results suggest that Sal B may participate in antiatherosclerotic and inflammatory responses through the NF-κB/NLRP3 pathway. ConclusionsThis study shows that Sal B ameliorates the development of AS lesions in HFD-induced LDLR−/− mice. Furthermore, under TNF-α conditions, Sal B reduced ROS release and reversed nuclear translocation of NF-κB, and inhibited atherosclerosis and inflammation by modulating the NF-κB/NLRP3 pathway.

DDX3X Deficiency attenuates Pyroptosis Induced by Oxygen-Glucose Deprivation/Reoxygenation in N2a Cells.

NOD‑like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis is strongly related to cerebral ischemia/reperfusion (I/R) injury. DDX3X, the DEAD-box family's ATPase/RNA helicase, promotes NLRP3 inflammasome activation. However, whether DDX3X deficiency attenuates NLRP3 inflammasome-mediated pyroptosis induced by cerebral I/R injury. This study investigated whether DDX3X deficiency attenuates NLRP3 inflammasome-mediated pyroptosis in N2a cells after oxygen-glucose deprivation/ reoxygenation (OGD/R) treatment. In vitro model of cerebral I/R injury, mouse neuro2a (N2a) cells subjected to OGD/R were treated with the knockdown of DDX3X. Cell counting kit-8 (CCK-8) assay and Lactate dehydrogenase (LDH) cytotoxicity assay were conducted to measure cell viability and membrane permeability. Double immunofluorescence was performed to determine the pyroptotic cells. Transmission electron microscopy (TEM) was used to observe morphological changes of pyroptosis. Pyroptosis-associated proteins were analyzed by Western blotting. The OGD/R treatment reduced cell viability, increased pyroptotic cells and released LDH compared to the control group. TEM showed membrane pore formation of pyroptosis. Immunofluorescence showed that GSDMD was translocated from the cytoplasm to the membrane after OGD/R treatment. Western blotting showed that the expression of DDX3X, and pyroptosis-related proteins (NLRP3, cleaved-Caspase1, and GSDMD-N) were increased after OGD/R treatment. Nevertheless, DDX3X knockdown markedly improved cell viability and reduced LDH release, expression of pyroptosis-related proteins, and N2a cells pyroptosis. DDX3X knockdown significantly inhibited membrane pore formation and GSDMD translocation from cytoplasm to membrane. This research demonstrates for the first time that DDX3X knockdown attenuates OGD/R‑induced NLRP3 inflammasome activation and pyroptosis, which implies that DDX3X may become a potential therapeutic target for cerebral I/R injury.

Tartaric acid induces toxicity in Madin–Darby canine kidney cells, but not human kidney‐2 cells in vitro, and is prevented by organic anion transporter inhibition and human OAT‐4 transfection

To evaluate the effect of tartaric acid (TTA) on Madin-Darby canine kidney (MDCK) cells compared to human kidney (HK)-2 cells. Secondarily, to evaluate the effects of probenecid, an organic anion transporter (OAT)-1 inhibitor, as well as human (h)OAT-4 transfection into MDCK cells to prevent TTA-induced cytotoxicity through decreasing accumulation via OAT-1 uptake inhibition or increasing OAT-4-mediated TTA efflux. Seventy-two-hour TTA concentration response and inhibitor studies in immortalized cell lines. School of Pharmacy biomedical research laboratory and tissue culture facility. MDCK and HK-2 immortalized cell lines. Both cell lines were treated with increasing concentrations of TTA for 72hours. Additionally, MDCK cells were co-incubated with TTA and increasing concentrations of probenecid or had been transfected with hOAT-4 and subsequently treated with TTA for 72hours. Media and samples were collected and lactate dehydrogenase (LDH) release was measured. LDH release was measured to assess TTA-induced cytotoxicity after 72hours. LDH was not significantly increased in the HK-2 cells at any concentration but was significantly increased in the MDCK cells from 10 to 100mM. LDH concentrations were significantly decreased (61%) in MDCK cells incubated with 50mM TTA and probenecid when compared to TTA alone. hOAT-4 MDCK cell transfection also significantly reduced LDH release (57%) when comparing the transfected MDCK cells to the nontransfected MDCK cells treated with 50mM TTA. TTA is a species-specific nephrotoxicant in dogs due to an interspecies difference in OAT-4 expression. Inhibiting TTA uptake in MDCK cells in vitro using the OAT-specific inhibitor, probenecid, prevents TTA-induced cytotoxicity.

Rutaecarpine attenuates high glucose-induced damage in AC16 cardiomyocytes by suppressing the MAPK pathway.

Diabetic cardiomyopathy is a common diabetic complication, resulting in heart failure. Rutaecarpine is an active compound with cardiovascular protective effects. However, the function of rutaecarpine in diabetic cardiomyopathy is largely unknown. The aim of this research was to study the effect and action mechanism of rutaecarpine in high glucose (HG)-induced cardiomyocyte damage. The overlapping genes of diabetic cardiomyopathy and rutaecarpine were analyzed according to GeneCards, DisGeNet, and SwissTargetPrediction. Cell damage was investigated by determining apoptosis, oxidative stress and inflammatory response in HG-stimulated AC16 cells. The expression of proteins involved in the mitogen-activated protein kinase (MAPK) signaling was measured using Western blotting. Totally 7 overlapping genes of diabetic cardiomyopathy and rutaecarpine were screened out and predicted to be associated with the MAPK signaling. Rutaecarpine protected against HG-induced cardiomyocyte damage by enhancing cell viability and reducing cell spoptosis, caspase-3 activity, and lactate dehydrogenase (LDH) release. Rutaecarpine mitigated HG-induced oxidative stress in cardiomyocytes through decreasing reactive oxygen species (ROS) formation and malondialdehyde (MDA) level and elevating superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) level. Rutaecarpine alleviated HG-induced inflammatory response via reducing the level of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-8. Moreover, rutaecarpine inhibited HG-induced activation of the MAPK pathway. Treatment with MAPK signaling agonist reversed the suppressive effect of rutaecarpine on HG-induced damage. In conclusion, rutaecarpine alleviated HG-induced cardiomyocyte damage through decreasing apoptosis, oxidative stress, and inflammatory response by inactivating the MAPK pathway.

BDNF mimetic 7,8-dihydroxyflavone rescues rotenone-induced cytotoxicity in cardiomyocytes by ameliorating mitochondrial dysfunction.

The relationship between mitochondrial dysfunction and cardiovascular disease pathogenesis is well recognized. 7,8-Dihydroxyflavone (7,8-DHF), a mimetic of brain-derived neurotrophic factor, inhibits mitochondrial impairments and improves cardiac function. However, the regulatory role of 7,8-DHF in the mitochondrial function of cardiomyocytes is not fully understood. To investigate the potential mito-protective effects of 7,8-DHF in cardiomyocytes, we treated H9c2 or HL-1cells with the mitochondrial respiratory complex I inhibitor rotenone (Rot) as an in vitro model of mitochondrial dysfunction. We found that 7,8-DHF effectively eliminated various concentrations of Rot-induced cell death and reduced lactate dehydrogenase release. 7,8-DHF significantly improved mitochondrial membrane potential and inhibited mitochondrial reactive oxygen species. Moreover, 7,8-DHF decreased routine and leak respiration, restored protein levels of mitochondrial complex I-IV, and increased ATP production in Rot-treated H9c2 cells. The protective role of 7,8-DHF in Rot-induced damage was validated in HL-1cells. Nuclear phosphorylation protein expression of signal transducer and activator of transcription 3 (STAT3) was significantly increased by 7,8-DHF. The present study suggests that 7,8-DHF rescues Rot-induced cytotoxicity by inhibiting mitochondrial dysfunction and promoting nuclear translocation of p-STAT3 in cardiomyocytes, thus nominating 7,8-DHF as a new pharmacological candidate agent against mitochondrial dysfunction in cardiac diseases.

Monoammonium glycyrrhizinate improves antioxidant capacity of calf intestinal epithelial cells exposed to heat stress in vitro.

Dairy calves are highly susceptible to the negative effects of heat stress, which can cause organ hypoxia after blood redistribution, damage the intestinal barrier, and trigger intestinal oxidative stress. This study aimed to investigate the antioxidant effects of monoammonium glycyrrhizinate (MAG) on calf small intestinal epithelial cells under heat stress in vitro. Small intestinal epithelial cells were isolated from a 1-d-old healthy calf and purified by differential enzymatic detachment. The purified cells were divided into seven groups. The control group was cultured with DMEM/F-12 at 37 °C for 6 h, and the treatment groups were cultured with 0, 0.1, 0.25, 0.5, 1, or 5 μg/mL MAG at 42 °C for 6 h. Heat stress causes oxidative damage to cells. Adding MAG to the medium can significantly improve cell activity and reduce cellular oxidative stress. MAG significantly increased the total antioxidant capacity and superoxide dismutase activity caused by heat stress, and significantly decreased malondialdehyde and nitric oxide levels. The MAG treatment also reduced lactate dehydrogenase release, increased mitochondrial membrane potential, and decreased apoptosis under heat stress. MAG also upregulated the expression of the antioxidant-related genes, Nrf2 and GSTT1, in heat-stressed intestinal epithelial cells and significantly downregulated the expression of the heat shock response-related proteins, MAPK, HSP70, HSP90, and HSP27. From the above results, we conclude that 0.25 μg/mL MAG improves the capability of the antioxidant system in small intestinal epithelial cells to eliminate reactive oxygen species by activating antioxidant pathways, improving the oxidant/antioxidant balance, lowering excessive heat shock responses, and reducing intestinal oxidative stress.

Irbesartan eases lipopolysaccharide-induced lung injury In Vitro and In Vivo.

Acute lung injury (ALI) is classified as a devastating pulmonary disorder contributing to significant incidence and fatality rate. Irbesartan (IRB) is an angiotensin II receptor blocker that has been proposed to protect against oleic acid-induced ALI. To this end, the current study is concentrated on ascertaining the role of IRB in ALI and figuring out the probable action mechanism. First, cell counting kit-8 (CCK-8) appraised the viability of human pulmonary microvascular endothelial cells (HPMVECs) exposed to ascending concentrations of IRB. HPMVEC injury model and a mouse model of ALI induced by lipopolysaccharide (LPS) were pretreated by IRB. In vitro, cell viability was estimated by CCK-8 assay, and lactate dehydrogenase (LDH) release was tested by LDH assay kit. Enzyme-linked immunosorbent assay (ELISA) and Western blotting estimated the expression levels of inflammatory factors. Fluorescein isothiocyanate-dextran was used to assess HPMVEC permeability. Western blotting examined the expression of adherent and tight junction proteins. In vivo, hematoxylin and eosin staining evaluated lung tissue damage and lung wet/dry (W/D) weight was measured. ELISA analyzed the levels of inflammatory factors in the serum and bronchoalveolar lavage fluid (BALF), and Western blotting examined the expression of inflammatory factors. The total cell, neutrophil, and macrophage numbers in BALF were determined using a cell counter. Lung capillary permeability was assayed by Evans blue albumin and total protein concentration in BALF was measured using bicinchoninic acid method. Immunofluorescence assay and Western blotting examined the expression of adherent and tight junction proteins in lung tissues. It was observed that IRB dose-dependently enhanced the viability while reduced LDH release, inflammatory response as well as permeability in LPS-challenged HPMVECs in vitro. In addition, LPS-stimulated lung tissue damage, pulmonary edema, inflammatory response as well as lung capillary permeability in vivo were all reversed following IRB treatment. Collectively, IRB treatment might elicit protective behaviors against LPS-triggered ALI.

Necrostatin-1 prevents skeletal muscle ischemia reperfusion injury by regulating Bok-mediated apoptosis.

Receptor interacting serine/threonine kinase 1 (RIPK1) mediates apoptosis by regulating the classic proapoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Although Bcl-2-related ovarian killer (Bok) is structurally similar to Bak and Bax, it is unclear whether it mediates apoptosis in skeletal muscle ischemia reperfusion (IR) injury. We hypothesized that by regulating Bok-mediated apoptosis, inhibiting RIPK1 with necrostatin-1 would reduce skeletal muscle IR injury. Rats were randomized into four groups: sham (SM), IR, IR treated with necrostatin-1 (NI), or vehicle dimethyl sulfoxide (DI). For the IR group, the right femoral artery was clamped for 4 hours and then reperfused for 4 hours, and for the NI and DI groups, necrostatin-1 (1.65 mg/kg) and the equal volume of dimethyl sulfoxide were intraperitoneally administered prior to IR induction. The structural damage of muscle tissue and protein expression of Bok, Bcl-2, and cleaved caspase-3 were investigated, and apoptotic cells were identified with terminal dUTP nick-end labeling (TUNEL) staining. In vitro, human skeletal muscle cells (HSMCs) were exposed to 6 hours of oxygen-glucose deprivation followed by normoxia for 6 hours to establish an oxygen-glucose deprivation/reoxygenation (OGD/R) model. To determine the role of Bok, cell viability, lactate dehydrogenase (LDH) release, and flow cytometry were examined to demonstrate the effects of necrostatin-1 and Bok knockdown on the OGD/R insult of HSMCs. Necrostatin-1 pretreatment markedly reduced IR-induced muscle damage and RIPK1, Bok, and cleaved caspase-3 expression, whereas upregualted Bcl-2 expression (p < 0.05). Furthermore, necrostatin-1 prevented mitochondrial damage and decreased TUNEL-positive muscle cells (p < 0.05). In vitro, HSMCs treated with necrostatin-1 showed reduced Bok expression, increased cell viability, and reduced LDH release in response to OGD/R (p < 0.05), and Bok knockdown significantly blunted the OGD/R insult in HSMCs. Necrostatin-1 prevents skeletal muscle from IR injury by regulating Bok-mediated apoptosis.

Sex Differences in X-ray-Induced Endothelial Damage: Effect of Taurine and N-Acetylcysteine.

Ionizing radiation (IR) can induce some associated pathological conditions due to numerous cell damages. The influence of sex is scarcely known, and even less known is whether the effect of antioxidants is sex-dependent. Given the increased use of IR, we investigated whether male human umbilical vein endothelial cells (MHUVECs) and female human umbilical vein endothelial cells (FHUVECs) respond differently to IR exposure and whether the antioxidants 10 mM taurine (TAU) and 5 mM N-acetylcysteine (NAC) can prevent IR-induced damage in a sex-dependent way. In untreated cells, sex differences were observed only during autophagy, which was higher in FHUVECs. In non-irradiated cells, preincubation with TAU and NAC did not modify viability, lactate dehydrogenase (LDH) release, migration, or autophagy, whereas only NAC increased malondialdehyde (MDA) levels in FHUVECs. X-ray irradiation increased LDH release and reduced viability and migration in a sex-independent manner. TAU and NAC did not affect viability while reduced LDH release in irradiated cells: they have the same protective effect in FHUVECs, while, TAU was more protective than NAC in male cells.. Moreover, TAU and NAC significantly promoted the closure of wounds in both sexes in irradiated cells, but NAC was more effective at doing this in FHUVECs. In irradiated cells, TAU did not change autophagy, while NAC attenuated the differences between the sexes. Finally, NAC significantly decreased MDA in MHUVECs and increased MDA in FHUVECs. In conclusion, FHUVECs appear to be more susceptible to IR damage, and the effects of the two antioxidants present some sex differences, suggesting the need to study the influence of sex in radiation mitigators.