Reactive Oxygen Species Scavenger NAC Research Articles

Inhibition of Caspase-1-dependent pyroptosis alleviates myocardial ischemia/reperfusion injury during cardiopulmonary bypass (CPB) in type 2 diabetic rats

Cardiovascular complications pose a significant burden in type 2 diabetes mellitus (T2DM), driven by the intricate interplay of chronic hyperglycemia, insulin resistance, and lipid metabolism disturbances. Myocardial ischemia/reperfusion (MI/R) injury during cardiopulmonary bypass (CPB) exacerbates cardiac vulnerability. This study aims to probe the role of Caspase-1-dependent pyroptosis in global ischemia/reperfusion injury among T2DM rats undergoing CPB, elucidating the mechanisms underlying heightened myocardial injury in T2DM. This study established a rat model of T2DM and compared Mean arterial pressure (MAP), heart rate (HR), and hematocrit (Hct) between T2DM and normal rats. Myocardial cell morphology, infarction area, mitochondrial ROS and caspase-1 levels, NLRP3, pro-caspase-1, caspase-1 p10, GSDMD expressions, plasma CK-MB, cTnI, IL-1β, and IL-18 levels were assessed after reperfusion in both T2DM and normal rats. The role of Caspase-1-dependent pyroptosis in myocardial ischemia/reperfusion injury during CPB in T2DM rats was examined using the caspase-1 inhibitor VX-765 and the ROS scavenger NAC. T2DM rats demonstrated impaired glucose tolerance but stable hemodynamics during CPB, while showing heightened vulnerability to MI/R injury. This was marked by substantial lipid deposition, disrupted myocardial fibers, and intensified cellular apoptosis. The activation of caspase-1-mediated pyroptosis and increased reactive oxygen species (ROS) production further contributed to tissue damage and the ensuing inflammatory response. Notably, myocardial injury was mitigated by inhibiting caspase-1 through VX-765, which also attenuated the inflammatory cascade. Likewise, NAC treatment reduced oxidative stress and partially suppressed ROS-mediated caspase-1 activation, resulting in diminished myocardial injury. This study proved that Caspase-1-dependent pyroptosis significantly contributes to the inflammation and injury stemming from global MI/R in T2DM rats under CPB, which correlate with the surplus ROS generated by oxidative stress during reperfusion.

Neuroprotective effects of chlorogenic acid: Modulation of Akt/Erk1/2 signaling to prevent neuronal apoptosis in Parkinson's disease

As a prevalent neurodegenerative disorder, Parkinson's disease is associated with oxidative stress. Our recent investigations revealed that reactive oxygen species (ROS) and PD-toxins like 6-hydroxydopamine (6-OHDA) can induce neuronal apoptosis through over-activation of Akt signaling. Chlorogenic acid (CGA), a natural acid phenol abundant in the human diet, is well-documented for its ability to mitigate intracellular ROS. In this study, we utilized CGA to treat experimental models of PD both in vitro and in vivo. Our study results demonstrated that SH-SY5Y and primary neurons exhibited cell apoptosis in response to 6-OHDA. Pretreatment with CGA significantly attenuated PD toxins-induced large amount of ROS, inhibiting Erk1/2 activation, preventing Akt inhibition, and hindering neuronal cell death. Combining the Erk1/2 inhibitor U0126 with CGA could reverse 6-OHDA-induced Akt inhibition, ROS, and apoptosis in the cells. Crucially, the Akt activator SC79 and ROS scavenger NAC both could eliminate excessive ROS via Akt and Erk1/2 signaling pathways, and CGA further potentiated these effects in PD models. Behavioral experiments revealed that CGA could alleviate gait abnormalities in PD model mice. The neuroprotective effects have been demonstrated in several endocrine regions and in the substantia nigra tissue, which shows the positive tyrosine hydroxylase (TH). Overall, our results suggest that CGA prevents the activation of Erk1/2 and inactivation of Akt by removing excess ROS in PD models. These findings propose a potential strategy for mitigating neuronal degeneration in Parkinson's disease by modulating the Akt/Erk1/2 signaling pathway through the administration of CGA and/or the use of antioxidants to alleviate oxidative stress.

Ilex pubescens inhibits pyroptosis post-myocardial infarction through suppression of the ROS/NLRP3 pathway

IntroductionIlex pubescens (IPES), a traditional Chinese herb widely used in cardiovascular diseases, has shown potential anti-inflammatory capabilities in myocardial infarction. Reactive Oxygen Species (ROS) and the NOD-like Receptor Protein 3 (NLRP3) pathway are significant contributors to aseptic inflammation in heart diseases. This study aims to elucidate the primary mechanism by which IPES inhibits pyroptosis post-myocardial infarction. MethodsBy ligating the left coronary artery in C57BL/6 mice, a myocardial infarction model was established to be conducted in vivo. To establish pyroptosis in vitro, primary neonatal cardiomyocytes, extracted from the hearts of Sprague-Dawley rats, were treated in an oxygen-glucose deprivation way. ROS scavenger, NLRP3 inhibitor, and NLRP3 was overexpressed by adenovirus to confirm IPES inhibiting myocardial pyroptosis through the ROS/NLRP3 pathway. ResultsIn vivo, IPES exerted significant cardioprotective effects, as evidenced by reducing heart injury, improving cardiac function, and decreasing serum markers of cardiac damage. Furthermore, IPES treatment significantly inhibits ROS generation and reduces the expression levels of NLRP3 and its downstream pyroptosis-related proteins. In vitro, IPES therapy significantly decreased cell damage and pyroptosis in a concentration-dependent manner in an oxygen-glucose deprivation/re-oxygenation (OGD/R) cell model. Additionally, IPES demonstrates synergistic cardiomyocyte protection with the ROS scavenger NAC, whereas its inhibition of pyroptosis is not significantly different from that of the NLRP3 inhibitor. More importantly, the inhibitory impacts of IPES on pyroptosis were partially reversed by NLRP3 overexpression. The active components of IPES exhibit the ability to stably and efficiently bind with NLRP3. DiscussionThese results demonstrate that IPES inhibit pyroptosis post-MI by suppressing the ROS/NLRP3 pathway, providing a new insight into its potential application in treating MI.

Luteolin Alleviates Cadmium-Induced Kidney Injury by Inhibiting Oxidative DNA Damage and Repairing Autophagic Flux Blockade in Chickens.

Chickens are a major source of meat and eggs in human food and have significant economic value. Cadmium (Cd) is a common environmental pollutant that can contaminate feed and drinking water, leading to kidney injury in livestock and poultry, primarily by inducing the generation of free radicals. It is necessary to develop potential medicines to prevent and treat Cd-induced nephrotoxicity in poultry. Luteolin (Lut) is a natural flavonoid compound mainly extracted from peanut shells and has a variety of biological functions to defend against oxidative damage. In this study, we aimed to demonstrate whether Lut can alleviate kidney injury under Cd exposure and elucidate the underlying molecular mechanisms. Renal histopathology and cell morphology were observed. The indicators of renal function, oxidative stress, DNA damage and repair, NAD+ content, SIRT1 activity, and autophagy were analyzed. In vitro data showed that Cd exposure increased ROS levels and induced oxidative DNA damage and repair, as indicated by increased 8-OHdG content, increased γ-H2AX protein expression, and the over-activation of the DNA repair enzyme PARP-1. Cd exposure decreased NAD+ content and SIRT1 activity and increased LC3 II, ATG5, and particularly p62 protein expression. In addition, Cd-induced oxidative DNA damage resulted in PARP-1 over-activation, reduced SIRT1 activity, and autophagic flux blockade, as evidenced by reactive oxygen species scavenger NAC application. The inhibition of PARP-1 activation with the pharmacological inhibitor PJ34 restored NAD+ content and SIRT1 activity. The activation of SIRT1 with the pharmacological activator RSV reversed Cd-induced autophagic flux blockade and cell injury. In vivo data demonstrated that Cd treatment caused the microstructural disruption of renal tissues, reduced creatinine, and urea nitrogen clearance, raised MDA content, and decreased the activities or contents of antioxidants (GSH, T-SOD, CAT, and T-AOC). Cd treatment caused oxidative DNA damage and PARP-1 activation, decreased NAD+ content, decreased SIRT1 activity, and impaired autophagic flux. Notably, the dietary Lut supplement observably alleviated these alterations in chicken kidney tissues induced by Cd. In conclusion, the dietary Lut supplement alleviated Cd-induced chicken kidney injury through its potent antioxidant properties by relieving the oxidative DNA damage-activated PARP-1-mediated reduction in SIRT1 activity and repairing autophagic flux blockade.

Decitabine combined with cold atmospheric plasma induces pyroptosis via the ROS/Caspase-3/GSDME signaling pathway in Ovcar5 cells

BackgroundHigh methylation of the DFNA5 gene results in the absence of GSDME, a key protein that mediates pyroptosis, while decitabine demethylates the DFNA5 gene, resulting in high expression of the GSDME protein. Cold atmospheric plasma (CAP) is a novel anti-cancer method that induces tumor cell death. MethodsThe pyroptosis induced by decitabine in combination with CAP in Ovcar5 cells was evaluated. In particular, mitochondrial membrane potential was estimated by JC-1 staining, dehydrogenase (LDH) release was assessed by ELISA, Annexin V/PI staining was detected by flow cytometry, the cell cycle changes were evaluated using PI staining followed by detection by flow cytometry, and Caspase-9 cleavage, Caspase-3 cleavage and GSDME expression were evaluated by western blot. ResultsDecitabine resulted in high expression of the GSDME in Ovcar5 in a concentration-dependent manner and increased tumor cell sensitivity to CAP. CAP induced mitochondrial damage and activated the Caspase-9/Caspase-3 pathway. Therefore, decitabine combined with CAP induced Ovcar5 cell pyroptosis through Caspase-3 mediated GSDME cleavage. Reactive oxygen species (ROS) generated by CAP treatment played an important role in the CAP/decitabine combination-induced production of ROS, activation of Caspase-9/Caspase-3, GSDME cleavage and pyroptosis that ROS scavenger NAC inhibited all these processes. ConclusionsCAP combined with decitabine induced Caspase-3 activation, which cleaved decitabine-upregulated GSDME and ediated pyroptosis.

Human umbilical cord mesenchymal stem cell-derived exosomes attenuate neuroinflammation and oxidative stress through the NRF2/NF-κB/NLRP3 pathway.

We investigated whether human umbilical cord mesenchymal stem cell (hUC-MSC)-derived exosomes bear therapeutic potential against lipopolysaccharide (LPS)-induced neuroinflammation. Exosomes were isolated from hUC-MSC supernatant by ultra-high-speed centrifugation and characterized by transmission electron microscopy and western blotting. Inflammatory responses were induced by LPS in BV-2 cells, primary microglial cultures, and C57BL/6J mice. H2 O2 was also used to induce inflammation and oxidative stress in BV-2 cells. The effects of hUC-MSC-derived exosomes on inflammatory cytokine expression, oxidative stress, and microglia polarization were studied by immunofluorescence and western blotting. Treatment with hUC-MSC-derived exosomes significantly decreased the LPS- or H2 O2 -induced oxidative stress and expression of pro-inflammatory cytokines (IL-6 and TNF-α) in vitro, while promoting an anti-inflammatory (classical M2) phenotype in an LPS-treated mouse model. Mechanistically, the exosomes increased the NRF2 levels and inhibited the LPS-induced NF-κB p65 phosphorylation and NLRP3 inflammasome activation. In contrast, the reactive oxygen species scavenger NAC and NF-κB inhibitor BAY 11-7082 also inhibited the LPS-induced NLRP3 inflammasome activation and switched to the classical M2 phenotype. Treatment with the NRF2 inhibitor ML385 abolished the anti-inflammatory and anti-oxidative effects of the exosomes. hUC-MSC-derived exosomes ameliorated LPS/H2 O2 -induced neuroinflammation and oxidative stress by inhibiting the microglial NRF2/NF-κB/NLRP3 signaling pathway.

The Apoptotic and Anti-Warburg Effects of Brassinin in PC-3 Cells via Reactive Oxygen Species Production and the Inhibition of the c-Myc, SIRT1, and β-Catenin Signaling Axis.

Though Brassinin is known to have antiangiogenic, anti-inflammatory, and antitumor effects in colon, prostate, breast, lung, and liver cancers, the underlying antitumor mechanism of Brassinin is not fully understood so far. Hence, in the current study, the apoptotic mechanism of Brassinin was explored in prostate cancer. Herein, Brassinin significantly increased the cytotoxicity and reduced the expressions of pro-Poly ADP-ribose polymerase (PARP), pro-caspase 3, and B-cell lymphoma 2 (Bcl-2) in PC-3 cells compared to DU145 and LNCaP cells. Consistently, Brassinin reduced the number of colonies and increased the sub-G1 population and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL)-positive cells in the PC-3 cells. Of note, Brassinin suppressed the expressions of pyruvate kinase-M2 (PKM2), glucose transporter 1 (GLUT1), hexokinase 2 (HK2), and lactate dehydrogenase (LDH) as glycolytic proteins in the PC-3 cells. Furthermore, Brassinin significantly reduced the expressions of SIRT1, c-Myc, and β-catenin in the PC-3 cells and also disrupted the binding of SIRT1 with β-catenin, along with a protein-protein interaction (PPI) score of 0.879 and spearman's correlation coefficient of 0.47 being observed between SIRT1 and β-catenin. Of note, Brassinin significantly increased the reactive oxygen species (ROS) generation in the PC-3 cells. Conversely, ROS scavenger NAC reversed the ability of Brassinin to attenuate pro-PARP, pro-Caspase3, SIRT1, and β-catenin in the PC-3 cells. Taken together, these findings support evidence that Brassinin induces apoptosis via the ROS-mediated inhibition of SIRT1, c-Myc, β-catenin, and glycolysis proteins as a potent anticancer candidate.

ROS-mediated mitophagy and necroptosis regulate osteocytes death caused by TCP particles in MLO-Y4 cells

Our previous data have revealed TCP particles caused cell death of osteocytes, comprising over 95 % of all bone cells, which contribute to periprosthetic osteolysis, joint loosening and implant failure, but its mechanisms are not fully understood. Here, we reported that TCP particles inhibited cell viability of osteocytes MLO-Y4, and caused cell death. TCP particles caused mitochondrial impairment and increased expressions of LC-3 II, Parkin and PINK 1, accompanied by the elevation of autophagy flux and intracellular acidic components, the accumulation of LC-3II, PINK1 and Parkin in damaged mitochondria, and p62 reduction. The increased LC-3II expression and cell death extent were significantly enhanced by the autophagy inhibitor Baf A1, compared with Baf A1 (or TCP particles) alone, indicating that TCP particles increase autophagic flux and lead to cell even death of MLO-Y4 cells, closely associated with mitophagy. Furthermore, TCP particles induced propidium iodide (PI) uptake and the phosphorylation of RIP1, RIP3 and MLKL, thereby increasing necroptosis in MLO-Y4 cells. The pro-necroptotic effect was alleviated by the RIP1 inhibitor Nec-1 or the MLKL inhibitor NSA. Additionally, TCP particles promoted the production of intracellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS), and increased TXNIP expression, but decreased protein levels of TRX1, Nrf2, HO-1 and NQO1, leading to oxidative stress. The ROS scavenger NAC remarkably reversed mitophagy and necroptosis caused by TCP particles, suggesting that ROS is responsible for mitophagy and necroptosis. Collectively, ROS-mediated mitophagy and necroptosis regulate osteocytes death caused by TCP particles in MLO-Y4 cells, which enhances osteoclastogenesis and periprosthetic osteolysis.

The efficient induction of human retinal ganglion-like cells provides a platform for studying optic neuropathies

Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (hiPSCs) by the overexpression of ATOH7, BRN3B, and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers by whole transcriptome profiling. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome–autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention.

TiO2 nanoparticles affect spermatogenesis and adhesion junctions via the ROS-mediated mTOR signalling pathway in Eriocheir sinensis testes

Recent findings found that TiO2 nanoparticles (TiO2-NPs) have male reproductive toxicity. However, few reports have studied the toxicity of TiO2-NPs in crustaceans. In this study, we first chose the freshwater crustacean Eriocheir sinensis (E. sinensis) to explore the male toxicity of TiO2-NP exposure and the underlying mechanisms. Three nm and 25 nm TiO2-NPs at a dose of 30 mg/kg bw induced apoptosis and damaged the integrity of the haemolymph-testis-barrier (HTB, a structure similar to the blood-testis-barrier) and the structure of the seminiferous tubule. The 3-nm TiO2-NPs caused more severe spermatogenesis dysfunction than the 25-nm TiO2-NPs. We initially confirmed that TiO2-NP exposure affected the expression patterns of adherens junctions (α-catenin and β-catenin) and induced tubulin disorganization in the testis of E. sinensis. TiO2-NP exposure caused reactive oxygen species (ROS) generation and an imbalance of mTORC1-mTORC2 (mTORC1/rps6/Akt levels were increased, while mTORC2 activity was not changed). After using the ROS scavenger NAC to inhibit ROS generation, both the mTORC1-mTORC2 imbalance and alterations in AJs were rescued. More importantly, the mTORC1 inhibitor rapamycin abolished mTORC1/rps6/Akt hyperactivation and partially restored the alterations in AJs and tubulin. Collectively, the mTORC1-mTORC2 imbalance induced by TiO2-NPs was involved in the mechanism of AJ and HTB disruption, resulting in spermatogenesis in E. sinensis.

Dihydromyricetin functions as a tumor suppressor in hepatoblastoma by regulating SOD1/ROS pathway.

Hepatoblastoma has an unsatisfactory prognosis, and traditional chemotherapy has strong side effects. Dihydromyricetin is a flavonoid extracted from a woody vine of the genus Serpentine in the family Vitaceae, with effects such as preventing alcoholic liver and reducing the incidence of liver cancer. However, the effect of DHM on hepatoblastoma and its specific pathway are still unclear. The purpose of this study was to investigate the effects of DHM on children's hepatoblastoma and its related mechanisms. CCK-8 assays were used to measure proliferation. Apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. Apoptotic cells were observed using Hoechst 33342 staining and fluorescence microscopy. Protein expression levels in HuH-6 and HepG2 cells were determined by western blotting. We found that DHM was able to inhibit the growth and increase cellular mortality in HuH-6 and HepG2 cells. Furthermore, DHM decreased the intracellular ROS level and increased the expression of SOD1. ROS scavenger NAC promoted apoptosis, while the use of SOD1 inhibitor LCS-1 weakened the ROS scavenging effect of DHM , and to some extent reduced the killing effect of DHM on hepatoblastoma cells. These results suggest that regulating SOD1/ROS pathway to induce apoptosis is one of the potential mechanisms of DHM as a tumor suppressor in hepatoblastoma. Therefore, DHM may be a novel candidate for inhibiting hepatoblastoma growth and deserves further study.

Activation of CncC pathway is involved in the regulation of P450 genes responsible for clothianidin resistance in Bradysia odoriphaga.

Insect cytochrome P450 monooxygenases (P450s) play a key role in the detoxification metabolism of insecticides and their overexpression is often associated with insecticide resistance. Our previous research showed that the overexpression of four P450 genes is responsible for clothianidin resistance in B. odoriphaga. In this study, we characterized another P450 gene CYP6FV21 associated with clothianidin resistance. However, the molecular basis for the overexpression of P450 genes in clothianidin resistant strain remains obscure in B. odoriphaga. In this study, the CYP6FV21 gene was significantly overexpressed in the clothianidin resistant (CL-R) strain. Clothianidin exposure significantly increased the expression level of CYP6FV21. Knockdown of CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to clothianidin. The transcription factor Cap 'n' Collar isoform-C (CncC) was highly expressed in the midgut of larvae in B. odoriphaga. The expression level of CncC was higher in the CL-R strain compared with the susceptible (SS) strain. Clothianidin exposure caused reactive oxygen species (ROS) accumulation and significantly increased the expression level of CncC. Knockdown of CncC cause a significant decrease in the expression of CYP3828A1 and CYP6FV21 and P450 enzyme activity, and leads to a significant increase in mortality after exposure to LC30 clothianidin. After treatment with CncC agonist curcumin, the P450 activity and the expression levels of CYP3828A1 and CYP6FV21 significantly increased, and larval sensitivity to clothianidin decreased. The ROS scavenger NAC treatment significantly inhibited the expression levels of CncC, CYP3828A1 and CYP6FV21 in response to clothianidin exposure and increased larval sensitivity to clothianidin. Taken together, these results indicate that activation of the CncC pathway by the ROS burst plays a critical role in clothianidin resistance by regulating the expression of CYP3828A1 and CYP6FV21 genes in B. odoriphaga. This study provides more insight into the mechanisms underlying B. odoriphaga larval resistance to clothianidin.

ROS/JNK-mediated lysosomal injury in rat intestinal epithelial-6 cells during heat stress

Injury to the intestinal epithelial cells and loss of the intestinal barrier are critical to heatstroke. To reveal the mechanism through which heatstroke leads to intestinal epithelial injury, the relationship between reactive oxygen species (ROS), c-Jun NH2-terminal kinase (JNK), and lysosomes were studied in intestinal epithelial cells subjected to heat stress. Cells of heat stress groups were incubated at 43 °C for 1 h, then incubated at 37 °C as indicated. Control group cells were incubated at 37 °C. Cell-counting kit-8 assay was used to assess cell viability. Cells were labeled with 2′-7′dichlorofluorescin diacetate and acridine orange (AO) staining, respectively, the total ROS and AO were detected by confocal laser scanning microscopy and flow cytometry. Apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate/prodium iodide staining, the expressions of mitogen-activated protein kinases were detected by western blotting. Heat stress induced apoptosis and inhibited cell viability, the production of ROS, and lysosomal injury in IEC-6 cells. After pretreatment with the lysosomal cathepsin inhibitor E64, the JNK inhibitor SP600125, or the ROS scavenger NAC, the effect of heat stress on apoptosis or lysosomal injury was significantly attenuated. In conclusion, heat stress induced apoptosis, lysosomal injury, and the accumulation of ROS in IEC-6 cells; mechanistically, this occurred through the ROS-induced activation of JNK signaling, which mediated the lysosomal injury and ultimately apoptosis.

Kayadiol exerted anticancer effects through p53-mediated ferroptosis in NKTCL cells

BackgroundExtranodal natural killer/T cell lymphoma (NKTCL) is a highly aggressive type of non-Hodgkin lymphoma that facing the treatment challenges. Natural compounds are important sources for drug development because of their diverse biological and chemical properties, among which terpenoids have strong anticancer activities.MethodsThe human NK/T cell lymphoma cell line YT and peripheral blood lymphocytes isolated from NKTCL patients were treated with different concentrations of kayadiol. Then, the following experiments were performed: CCK-8 assay for cell viability, reactive oxygen species (ROS) and glutathione (GSH) assay and co-treatment with NAC, reduced GSH, or ferrostatin-1 for ferroptosis, the proteome profiling for elucidating signaling pathways, and western blot for the expression of p53, SCL7A11, and GPX4. siRNA and CRISPR/Cas9 plasmid for p53 knockout was designed and transfected into YT cells to evaluate the causal role of p53 in kayadiol-induced ferroptosis. The synergistic effect was evaluated by CCK8 assay after co-treatment of kayadiol with L-asparaginase or cisplatin.ResultsIn this study, we found that kayadiol, a diterpenoid extracted from Torreya nucifera, exerted significant killing effect on NKTCL cells without killing the healthy lymphocytes. Subsequently, we observed that kayadiol treatment triggered significant ferroptosis events, including ROS accumulation and GSH depletion. ROS scavenger NAC, GSH, and ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed kayadiol-induced cell death in NKTCL cells. Furthermore, kayadiol decreased the expression of SLC7A11 and GPX4, the negative regulatory proteins for ferroptosis. We then demonstrated that p53 was the key mediator of kayadiol-induced ferroptosis by SLC7A11/GPX4 axis through p53 knockout experiments. In addition, kayadiol exerted a synergistic effect with L-asparaginase and cisplatin in NKTCL cells.ConclusionTaken together, our results suggested that the natural product kayadiol exerted anticancer effects through p53-mediated ferroptosis in NK/T cell lymphoma cells. Hence, it can serve as an effective alternative in the treatment of NK/T cell lymphoma, especially for patients exhibiting chemoresistance.

Curcumin sensitizes carboplatin treatment in triple negative breast cancer through reactive oxygen species induced DNA repair pathway.

As patients with triple-negative breast cancer (TNBC) have a very weak response to hormone inhibition or anti-HER2 therapy, traditional chemotherapy is commonly used in these patients. Recently, carboplatin has been approved for the clinical treatment of TNBC. However, several patients exhibit resistance to carboplatin treatment. Therefore, strategies to enhance the antitumor effect of carboplatin need to be explored. In our study, we investigated the function of curcumin in increasing the response to carboplatin. MTT and colony formation assays were used to evaluate cell viability after carboplatin and curcumin treatment. In addition, we conducted flow cytometric and Western blot analyses to examine cellular apoptosis. Subsequently, molecular and biochemical experiments were used to explore the mechanism by which curcumin sensitized TNBC to carboplatin treatment. We demonstrated that different TNBC cells responded differently to carboplatin. Low-dose carboplatin killed CAL-51 cells but barely influenced CAL-51-R and MDA-MB-231 cells. To improve the sensitivity of resistant TNBC cells to carboplatin, combined treatment with curcumin was applied and was found to inhibit proliferation and induce apoptosis. Mechanistically, curcumin exerted its anticancer effect by increasing reactive oxygen species (ROS) production, which downregulated the DNA repair protein RAD51, leading to upregulation of γH2AX. As expected, ROS scavenger NAC reversed the inhibitory effect on growth and DNA repair pathway activity mediated by curcumin. Collectively, our data demonstrate that curcumin sensitizes TNBC to the anticancer effect of carboplatin by increasing ROS-induced DNA damage, thus providing an effective combination treatment strategy for TNBC.

Taraxasterol attenuates melanoma progression via inactivation of reactive oxygen species-mediated PI3K/Akt signaling pathway.

Background: Taraxasterol (TX), a pentacyclic triterpene, is one of the main active constituents isolated from Taraxacum officinale. A growing number of studies have reported that TX exhibits a wide range of biological activities such as anti-oxidative, anti-inflammatory, and neuro-protective effects. Recently, TX has been demonstrated to be a potential drug candidate for treatment of some types of cancers. However, the specific role of TX in melanoma remains unclear.Purpose: In this study, we aimed at exploration of the effect of TX on melanoma cell viability, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT) as well as the underlying mechanisms.Research design: A375 and SK-MEL-28 cells were treated with various concentrations of TX for different times. Cell viability was measured using CCK-8 assay. Cell apoptosis was determined by flow cytometry. Transwell assays were performed to measure cell migration and invasion. The expression of E-cadherin, α-catenin, N-cadherin, vimentin, p-PI3K, PI3K, p-Akt and Akt was detected using western blot.Results: The study showed that TX induced A375 and SK-MEL-28 cell apoptosis. Furthermore, exposure to TX inhibited A375 and SK-MEL-28 cell migration and invasion. Besides, the EMT process was reversed in A375 and SK-MEL-28 cells after TX treatment. We also observed that TX reduced the protein expression of p-PI3K and p-Akt; thus, inhibiting activity of the PI3K/Akt pathway in A375 and SK-MEL-28 cells. In addition, TX treatment increased the levels of reactive oxygen species (ROS) in A375 and SK-MEL-28 cells, and treatment with the ROS scavenger NAC significantly rescued TX-induced down-regulation of p-PI3K and p-Akt in A375 and SK-MEL-28 cells.Conclusions: In conclusion, our study demonstrated that TX induced ROS accumulation followed by inactivation of the PI3K/Akt pathway and subsequently attenuated melanoma progression, suggesting that TX may be a potential candidate for treatment of melanoma.

Auriculasin enhances ROS generation to regulate colorectal cancer cell apoptosis, ferroptosis, oxeiptosis, invasion and colony formation

Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system, and Chinese herbal medicine plays an important role in tumor treatment. The in-depth study of auriculasin isolated from Flemingia philippinensis showed that auriculasin promoted reactive oxygen species (ROS) generation in a concentration-dependent manner; when ROS scavenger NAC was added, the effects of auriculasin in promoting ROS generation and inhibiting cell viability were blocked. Auriculasin induced CRC cell apoptosis, led to mitochondrial shrinkage, and increased the intracellular accumulation of Fe2+ and MDA. When auriculasin and NAC were added simultaneously, the levels of apoptosis, Fe2+ and MDA returned to the control group levels, indicating that auriculasin activated apoptosis and ferroptosis by inducing ROS generation. In addition, auriculasin promoted the expression of Keap1 and AIFM1, but significantly reduced the phosphorylation level of AIFM1, while NAC significantly blocked the regulation of Keap1 and AIFM1 by auriculasin, which indicates that auriculasin can also induce oxeiptosis through ROS. When Z-VAD-FMK, Ferrostatin-1, Keap1 siRNA, PGAM5 siRNA and AIFM1 siRNA were added respectively, the inhibitory effect of auriculasin on cell viability was significantly weakened, indicating that auriculasin inhibits cell viability by inducing apoptosis, ferroptosis and oxeiptosis. Auriculasin also inhibited the invasion and clone forming ability of CRC cells, while NAC blocked the above effects of auriculasin. Therefore, auriculasin can promote CRC cell apoptosis, ferroptosis and oxeiptosis by inducing ROS generation, thereby inhibiting cell viability, invasion and clone formation, indicating that auriculasin has a significant antitumor effect.

Restoring Dysfunctional Bone Marrow Endothelial Cell Alleviates Aplastic Anemia

BackgroundAplastic anemia (AA) is a life-threatening disease characterized by bone marrow (BM) failure and pancytopenia. Immunosuppressive therapy can rescue most patients with AA. However, the pathogenesis of AA is still not well elucidated and the new strategies need to be developed for AA patients. Increasing evidences suggested the dysfunctional BM microenvironment may be involved in the pathogenesis of AA. As important components of the BM microenvironment, endothelial cells (ECs) play a crucial role in supporting hematopoiesis and regulating immune. However, whether BM ECs are involved in the occurrence of AA and whether repairing BM ECs could improve the hematopoietic and immune status of AA patients remain to be elucidated.AimsTo evaluate the quantity and function of BM ECs from AA patients. Moreover, to determine whether the dysfunctional BM ECs are involved in the occurrence of AA by affecting hematopoiesis and regulating immunity in vitro and in vivo. Finally, to uncover the therapeutic potential of repairing dysfunctional BM ECs to alter the hematopoietic and immunological status in AA patients.MethodsThis study enrolled 30 patients with AA and 30 healthy donors(HD). Flow cytometry and BM in situ immunofluorescence staining were used to analyze the proportion of ECs in BM of the two groups. The level of intracellular reactive oxygen species(ROS) and the proportion of apoptosis were detected by flow cytometry. The functions of BM ECs were evaluated by double-positive staining, migration and tube formation assays. To determine the effect of BM ECs on hematopoiesis and immunity, primary human BM ECs were separately cocultured with CD34 + and CD3 + cells. To further validate the role of BM ECs in the occurrence of AA, a classical AA mice model and VE-cadherin blocking antibody that could antagonize the function of BM ECs were used. Moreover, to explore potential approach of targeting the dysfunctional BM ECs, the exogenous EC infusion or N-acetyl-L-cysteine (NAC, a ROS scavenger) for repairing BM ECs were administrated to the AA mice. To further explore the repairing effect of NAC on BM ECs, the primary BM ECs from AA patients were treated by NAC in vitro and then the functions of BM ECs were evaluated.ResultsCompared with HD, BM ECs in AA patients were decreased and dysfunctional, which characterized by higher levels of ROS and apoptosis, impaired abilities of migration and angiogenesis. Furthermore, dysfunctional BM ECs from AA patients not only impaired their hematopoiesis-supporting ability but also promoted co-cultured T cells to polarize towards pro-inflammatory T cells in vitro, which resulted in an unbalanced T cell subsets. Consistently, AA mice demonstrated decreased BM ECs with increased level of intracellular ROS. Moreover, hematopoietic failure and immune imbalance in AA mice became more severe when the function of BM ECs was antagonized, whereas the administration of NAC or infusion of exogenous EC improved the hematopoietic and immunological status of AA mice via repairing BM ECs in vivo. In addition, we found the NAC treatment also restored the hematopoiesis-supporting ability and immunity-regulating ability of the primary ECs derived from AA patients in vitro.Summary/ConclusionOur study demonstrates for the first time that dysfunctional BM ECs with impaired hematopoiesis-supporting ability and abnormal immunomodulatory ability are involved in the pathogenesis of AA. Although further validation is required, restoring dysfunctional BM ECs via EC infusion or administration of ROS scavenger NAC might be a potential therapeutic approach for AA patients. DisclosuresNo relevant conflicts of interest to declare.

Fractionated Irradiation of Right Thorax Induces Abscopal Damage on Bone Marrow Cells via TNF-α and SAA.

Radiation-induced abscopal effect (RIAE) outside of radiation field is becoming more attractive. However, the underlying mechanisms are still obscure. This work investigated the deleterious effect of thoracic irradiation (Th-IR) on distant bone marrow and associated signaling factors by irradiating the right thorax of mice with fractionated doses (8 Gy × 3). It was found that this localized Th-IR increased apoptosis of bone marrow cells and micronucleus formation of bone marrow polychromatic erythrocytes after irradiation. Tandem mass tagging (TMT) analysis and ELISA assay showed that the concentrations of TNF-α and serum amyloid A (SAA) in the mice were significantly increased after Th-IR. An immunohistochemistry assay revealed a robust increase in SAA expression in the liver rather than in the lungs after Th-IR. In vitro experiments demonstrated that TNF-α induced SAA expression in mouse hepatoma Hepa1–6 cells, and these two signaling factors induced DNA damage in bone marrow mesenchymal stem cells (BMSCs) by increasing reactive oxygen species (ROS). On the other hand, injection with TNF-α inhibitor before Th-IR reduced the secretion of SAA and attenuated the abscopal damage in bone marrow. ROS scavenger NAC could also mitigated Th-IR/SAA-induced bone marrow damage in mice. Our findings indicated that Th-IR triggered TNF-α release from lung, which further promoted SAA secretion from liver in a manner of cascade reaction. Consequently, these signaling factors resulted in induction of abscopal damage on bone marrow of mice.

Astragaloside prevents UV-induced keratinocyte injury by regulating TLR4/NF-κB pathway.

Ultraviolet (UV) radiation is a key risk factor of environment to contribute photoaging and skin cancer through production of reactive oxygen species (ROS) and inflammatory responses. Astragaloside IV (AS-IV) is an active component from Astragalus membranaceus, and shows various pharmacological effects on inflammation, oxidative stress and apoptosis. However, whether AS-IV shows protective effect on UVB-induced injury in epidermal keratinocytes remain unknown. To explored the effects of AS-IV on UVB-induced oxidative injury and inflammatory response in human epidermal keratinocytes. HaCaT keratinocytes were exposed to UVB irradiation, followed by AS-IV incubation. The cell viability, intracellular ROS level, oxidative stress, and apoptosis were determined. The regulatory effects of AS-IV on toll-like receptor 4 (TLR4) pathway in UVB-exposed HaCaT cells were also investigated. Astragaloside IV pretreatment (10, 25, 50, 100 and 150 μM) increased cell viability in UVB-exposed HaCaT cells. AS-IV (50 μM) significantly reduced intracellular ROS level and lipid oxidation product malondialdehyde (MDA) content, and increased a ROS-scavenging enzyme superoxide dismutase (SOD) in HaCaT cells with UVB irradiation. In addition, AS-IV pretreatment suppressed apoptosis, increased Bax protein, caspase-3 and 9, and decreased BCL-2 protein in contrast to HaCaT cells with UVB-irradiation. AS-IV suppressed proinflammatory cytokine production, inhibited TLR4 and its downstream signaling molecules NF-κB, iNOS and cyclooxygenase-2 (COX-2) protein expression. We also found that the effects of AS-IV on cell viability and TLR4 expression was reversed by NAC. The protective of AS-IV on UVB-induced damage and TLR4 expression was dependent on ROS, as the increase in viability and decrease in TLR4 protein by AS-IV was significantly attenuated by ROS scavenger NAC (1 mM). Astragaloside IV prevent UVB-induced oxidative damage and inflammation by inhibiting TLR4 expression.