N-acetylcysteine Pretreatment Research Articles

N-acetylcysteine Attenuates Cigarette Smoke-induced Alveolar Epithelial Cell Apoptosis through Reactive Oxygen Species Depletion and Glutathione Replenish In vivo and In vitro.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. N-acetylcysteine (NAC) is well known for its antioxidant properties, along with potential protective effects on COPD. However, the molecular mechanism of NAC against the apoptosis of alveolar epithelial cells (AECs) in COPD remains unclear. This study aimed to explore the anti-apoptosis effect of NAC in COPD mice and alveolar epithelial cells. In the present study, the mouse model of COPD was established by cigarette smoke (CS), and mouse alveolar epithelial (MLE-12) cells were treated with cigarette smoke extract (CSE). TdT-mediated dUTP nick-end labeling (TUNEL) assay, reverse transcription polymerase chain reaction (RT-PCR), and western blot were performed to evaluate the effects of NAC on apoptosis, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction. Meanwhile, Lbuthionine- sulfoximine (BSO), a glutathione (GSH) inhibitor, was used to uncover the mechanism of COPD treatment by NAC. We found that NAC pretreatment could attenuate the protein levels of apoptosis, ER stress, and mitochondrial dysfunction-related genes caused by CS in vivo. Meanwhile, CSE could decrease MLE-12 cell viability, which was prevented by apoptosis inhibitor ZVAD-FMK but not necroptosis inhibitor necrostatin-1. Pretreatment of MLE-12 cells with NAC increased cellular GSH levels, inhibited cellular and mitochondrial reactive oxygen species (ROS) accumulation, and decreased protein level of apoptosis, ER stress, and mitochondrial dysfunction-related genes. Moreover, experiment results showed that BSO could completely reverse the beneficial effects of NAC. Our study confirmed that NAC can attenuate CS-induced AEC apoptosis via alleviating ROS-mediated ER stress and mitochondrial dysfunction pathway, and the mechanism was found to be related to replenishing the cellular GSH content.

XPC Protects against Carcinogen-Induced Histologic Progression to Lung Squamous Cell Carcinoma by Reduced Basal Epithelial Cell Proliferation.

Lung squamous cell carcinoma (LUSC) is the second leading cause of lung cancer. Although characterized by high DNA mutational burdens and genomic complexity, the role of DNA repair in LUSC development is poorly understood. We sought to better understand the role of the DNA repair protein Xeroderma Pigmentosum Group C (XPC) in LUSC development. XPC knock-out (KO), heterozygous, and wild-type (WT) mice were exposed topically to N-nitroso-tris-chloroethylurea (NTCU), and lungs were evaluated for histology and pre-malignant progression in a blinded fashion at various time-points from 8-24 weeks. High-grade dysplasia and LUSC were increased in XPC KO compared with XPC WT NTCU mice (56% vs. 34%), associated with a higher mean LUSC lung involvement (p < 0.05). N-acetylcysteine pre-treatment decreased bronchoalveolar inflammation but did not prevent LUSC development. Proliferation, measured as %Ki67+ cells, increased with NTCU treatment, in high-grade dysplasia and LUSC, and in XPC deficiency (p < 0.01, ANOVA). Finally, pre-LUSC dysplasia developed earlier and progressed to higher histologic classification sooner in XPC KO compared with WT mice. Overall, this supports the protective role of XPC in squamous dysplasia progression to LUSC. Mouse models of early LUSC development are limited; this may provide a valuable model to study mechanisms of LUSC development and progression.

Pubertal exposure to Microcystin-LR arrests spermatogonia proliferation by inducing DSB and inhibiting SIRT6 dependent DNA repair in vivo and in vitro

The reproduction toxicity of pubertal exposure to Microcystin-LR (MC-LR) and the underlying mechanism needs to be further investigated. In the current study, pubertal male ICR mice were intraperitoneally injected with 2 μg/kg MC-LR for four weeks. Pubertal exposure to MC-LR decreased epididymal sperm concentration and blocked spermatogonia proliferation. In-vitro studies found MC-LR inhibited cell proliferation of GC-1 cells and arrested cell cycle in G2/M phase. Mechanistically, MC-LR exposure evoked excessive reactive oxygen species (ROS) and induced DNA double-strand break in GC-1 cells. Besides, MC-LR inhibited DNA repair by reducing PolyADP-ribosylation (PARylation) activity of PARP1. Further study found MC-LR caused proteasomal degradation of SIRT6, a monoADP-ribosylation enzyme which is essential for PARP1 PARylation activity, due to destruction of SIRT6-USP10 interaction. Additionally, MG132 pretreatment alleviated MC-LR-induced SIRT6 degradation and promoted DNA repair, leading to the restoration of cell proliferation inhibition. Correspondingly, N-Acetylcysteine (NAC) pre-treatment mitigated the disturbed SIRT6-USP10 interaction and SIRT6 degradation, causing recovered DNA repair and subsequently restoration of cell proliferation inhibition in MC-LR treated GC-1 cells. Together, pubertal exposure to MC-LR induced spermatogonia cell cycle arrest and sperm count reduction by oxidative DNA damage and simultaneous SIRT6-mediated DNA repair failing. This study reports the effect of pubertal exposure to MC-LR on spermatogenesis and complex mechanism how MC-LR induces spermatogonia cell proliferation inhibition.

Heme Oxygenase-1 Regulates Zearalenone-Induced Oxidative Stress and Apoptosis in Sheep Follicular Granulosa Cells.

Zearalenone (ZEA) is a common non-steroidal estrogenic mycotoxin found in a range of animal feeds and poses a serious threat to the reproductive health of farm animals and humans. However, the mechanism underlying ZEA-induced reproductive toxicity in sheep remains unknown. Granulosa cells are crucial for egg maturation and the fertility of female sheep. In this study, we aimed to examine the impact of different ZEA concentrations on sheep follicular granulosa cells and to elucidate the potential molecular mechanism underlying ZEA-induced toxicity using transcriptome sequencing and molecular biological approaches. Treating primary sheep follicular granulosa cells with different concentrations of ZEA promoted the overproduction of reactive oxygen species (ROS), increased lipid peroxidation products, led to cellular oxidative stress, decreased antioxidant enzyme activities, and induced cell apoptosis. Using transcriptome approaches, 1395 differentially expressed genes were obtained from sheep follicular granulosa cells cultured in vitro after ZEA treatment. Among them, heme oxygenase-1 (HMOX1) was involved in 11 biological processes. The protein interaction network indicated interactions between HMOX1 and oxidative and apoptotic proteins. In addition, N-acetylcysteine pretreatment effectively reduced the ZEA-induced increase in the expression of HMOX1 and Caspase3 by eliminating ROS. Hence, we suggest that HMOX1 is a key differential gene involved in the regulation of ZEA-induced oxidative stress and apoptosis in follicular granulosa cells. These findings provide novel insights into the prevention and control of mycotoxins in livestock.

Lactate induces C2C12 myoblasts differentiation by mediating ROS/p38 MAPK signalling pathway

Lactate serves not merely as an energy substrate for skeletal muscle but also regulates myogenic differentiation, leading to an elevation of reactive oxygen species (ROS) levels. The present study was focused on exploring the effects of lactate and ROS/p38 MAPK in promoting C2C12 myoblasts differentiation. Our results demonstrated that lactate increased C2C12 myoblasts differentiation at a range of physiological concentrations, accompanied by enhanced ROS contents. We used n-acetylcysteine (NAC, a ROS scavenger) pretreatment and found that it delayed lactate-induced C2C12 myoblast differentiation by upregulating Myf5 expression on days 5 and 7 and lowering MyoD and MyoG expression. The finding implies that lactate accompanies ROS-dependent manner to promote C2C12 myoblast differentiation. Additionally, lactate significantly increased p38 MAPK phosphorylation to promote C2C12 cell differentiation, but pretreatment with SB203580 (p38 MAPK inhibitor) reduced lactate-induced C2C12 myoblasts differentiation. whereas lactate pretreatment with NAC inhibited p38 MAPK phosphorylation in C2C12 cells, demonstrating that lactate mediated ROS and regulated the p38 MAPK signalling pathway to promote C2C12 cell differentiation. In conclusion, our results suggest that the promotion of C2C12 myoblasts differentiation by lactate is dependent on ROS and the p38 MAPK signalling pathway. These observations reveal a beneficial role for lactate in increasing myogenesis through ROS-sensitive mechanisms as well as providing new ideas regarding the positive impact of ROS in improving the function of skeletal muscle.

Jaceosidin induces apoptosis and inhibits migration in AGS gastric cancer cells by regulating ROS-mediated signaling pathways

ABSTRACT Jaceosidin (JAC) is a natural flavonoid with anti-oxidant and other pharmacological activities; however, its anti-cancer mechanism remains unclear. We investigated the mechanism of action of JAC in gastric cancer cells. Cytotoxicity and apoptosis assays showed that JAC effectively killed multiple gastric cancer cells and induced apoptosis in human gastric adenocarcinoma AGS cells via the mitochondrial pathway. Network pharmacological analysis suggested that its activity was linked to reactive oxygen species (ROS), AKT, and MAPK signaling pathways. Furthermore, JAC accumulated ROS to up-regulate p-JNK, p-p38, and IκB-α protein expressions and down-regulate the p-ERK, p-STAT3, and NF-κB protein expressions. Cell cycle assay results showed that JAC accumulated ROS to up-regulate p21 and p27 protein expressions and down-regulate p-AKT, CDK2, CDK4, CDK6, Cyclin D1, and Cyclin E protein expressions to induce G0/G1 phase arrest. Cell migration assay results showed JAC accumulated ROS to down-regulate Wnt-3a, p-GSK-3β, N-cadherin, and β-catenin protein expressions and up-regulate E-cadherin protein expression to inhibit migration. Furthermore, N-acetyl cysteine pre-treatment prevented the change of these protein expressions. In summary, JAC induced apoptosis and G0/G1 phase arrest and inhibited migration through ROS-mediated signaling pathways in AGS cells.

Sodium p-perfluorous nonenoxybenzene sulfonate induces ROS-mediated necroptosis by directly targeting catalase in HepG2 cells

Sodium p-perfluorous nonenoxybenzene sulfonate (OBS) has been widely used as a substitute for perfluorooctane sulfonic acid (PFOS) because of its high surface activity and low cost, but the knowledge of its biological effects is still limited. In this study, we compared the toxic effects of OBS and PFOS on human hepatoma cells (HepG2). OBS resulted in lower cell viability, higher ROS levels, and more severe necrosis than PFOS, indicating that OBS caused higher cytotoxicity than PFOS. In this process, OBS induced a burst of ROS and downregulation of catalase (CAT). OBS-induced oxidative stress was recovered after the CAT overexpression, but the CAT levels were not reversed after N-acetylcysteine (NAC) pretreatment. This indicates that the downregulated CAT is an upstream signal of the ROS burst. Moreover, drug affinity targeting assay, spectroscopic analysis and molecular docking were conducted, showing that OBS directly targeted CAT and therefore downregulated CAT. In addition, we found that OBS-induced necrosis is RIP1/RIP3-dependent programmed necroptosis. In summary, OBS directly targets CAT to reduce CAT levels and induces oxidative stress and necroptosis. Our findings are helpful to understand the toxicity of OBS and to evaluate the safety of OBS as a substitute for PFOS.

Enhancing Gastric Cancer Therapeutic Efficacy through Synergistic Cotreatment of Linderae Radix and Hyperthermia in AGS Cells.

Gastric cancer remains a global health threat, particularly in Asian countries. Current treatment methods include surgery, chemotherapy, and radiation therapy. However, they all have limitations, such as adverse side effects, tumor resistance, and patient tolerance. Hyperthermia therapy uses heat to selectively target and destroy cancer cells, but it has limited efficacy when used alone. Linderae Radix (LR), a natural compound with thermogenic effects, has the potential to enhance the therapeutic efficacy of hyperthermia treatment. In this study, we investigated the synergistic anticancer effects of cotreatment with LR and 43 °C hyperthermia in AGS gastric cancer cells. The cotreatment inhibited AGS cell proliferation, induced apoptosis, caused cell cycle arrest, suppressed heat-induced heat shock responses, increased reactive oxygen species (ROS) generation, and promoted mitogen-activated protein kinase phosphorylation. N-acetylcysteine pretreatment abolished the apoptotic effect of LR and hyperthermia cotreatment, indicating the crucial role of ROS in mediating the observed anticancer effects. These findings highlight the potential of LR as an adjuvant to hyperthermia therapy for gastric cancer. Further research is needed to validate these findings in vivo, explore the underlying molecular pathways, and optimize treatment protocols for the development of novel and effective therapeutic strategies for patients with gastric cancer.

Role of ROS-mediated PERK/ATF4 signaling activation in extracorporeal tube formation injury of human umbilical vein endothelial cells induced by cooking oil fume PM2.5 exposure

Cooking oil fume-derived PM2.5 (COF-PM2.5) is a major source of indoor air contamination in China, which has been demonstrated to be a hazard factor of cardiovascular and cerebrovascular diseases. This study aimed to investigate the role of ROS-mediated PERK/ATF4 signaling activation in COF-PM2.5-inhibited extracorporeal tube formation in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with 100 μg/mL COF-PM2.5 at different times, with or without 100 nM PERK activity inhibitor GSK2606414 (GSK) or 200 μM antioxidant N-acetylcysteine (NAC) pretreatment. Our results showed that COF-PM2.5 exposure can inhibit extracorporeal tube formation and down-regulate VEGFR2 expression in HUVECs. Furthermore, our data indicated that COF-PM2.5 exposure can activate the PERK/ATF4 signaling in HUVECs. Mechanistically, pretreatment with GSK interdicted PERK/ATF4 signaling, thereby reversing COF-PM2.5-downregulated VEGFR2 protein expression in HUVECs. Furthermore, NAC reversed VEGFR2 expression downregulated induced by COF-PM2.5 by inhibiting the upregulation of intracellular ROS levels and PERK/ATF4 signaling in HUVECs. As above, COF-PM2.5 exposure could induce ROS release from HUVECs, which in turn activate the endoplasmic reticulum PERK/ATF4 signaling and inhibit tube formation of HUVECs.

Tranexamic acid improves psoriasis-like skin inflammation: Evidence from in vivo and in vitro studies

The chronic disease psoriasis is associated with severe inflammation and abnormal keratinocyte propagation in the skin. Tranexamic acid (TXA), a plasmin inhibitor, is used to cure serious bleeding. We investigated whether TXA ointment mitigated Imiquimod (IMQ)-induced psoriasis-like inflammation. Furthermore, this study investigated the effect of noncytotoxic concentrations of TXA on IL-17-induced human keratinocyte (HaCaT) cells to determine the status of proliferative psoriatic keratinocytes. We found that TXA reduced IMQ-induced psoriasis-like erythema, thickness, scaling, and cumulative scores (erythema plus thickness plus scaling) on the back skin of BALB/c mice. Additionally, TXA decreased ear thickness and suppressed hyperkeratosis, hyperplasia, and inflammation of the ear epidermis in IMQ-induced BALB/c mice. Furthermore, TXA inhibited IMQ-induced splenomegaly in BALB/c mouse models. In IL-17-induced HaCaT cells, TXA inhibited ROS production and IL-8 secretion. Interestingly, TXA suppressed the IL-17-induced NFκB signaling pathway via IKK-mediated IκB degradation. TXA inhibited IL-17-induced activation of the NLRP3 inflammasome through caspase-1 and IL1β expression. TXA inhibited IL-17-induced NLRP3 inflammasome activation by enhancing autophagy, as indicated by LC3-II accumulation, p62/SQSTM1 expression, ATG4B inhibition, and Beclin-1/Bcl-2 dysregulation. Notably, TXA suppressed IL-17-induced Nrf2-mediated keratin 17 expression. N-acetylcysteine pretreatment reversed the effects of TXA on NFκB, NLRP3 inflammasomes, and the Nrf2-mediated keratin 17 pathway in IL-17-induced HaCaT cells. Results further confirmed that in the ear skin of IMQ-induced mice, psoriasis biomarkers such as NLRP3, IL1β, Nrf2, and keratin 17 expression were downregulated by TXA treatment. TXA improves IMQ-induced psoriasis-like inflammation in vivo and psoriatic keratinocytes in vitro. Tranexamic acid is a promising future treatment for psoriasis.

Effects of N-acetylcysteine on CG8005 gene-mediated proliferation and apoptosis of Drosophila S2 embryonic cells

To investigate the effect of the antioxidant N-acetylcysteine (NAC) on the proliferation and apoptosis in CG8005 gene-interfering Drosophila S2 embryonic cells by scavenging intracellular reactive oxygen species (ROS). The interfering efficiency of CG8005 gene in Drosophila S2 embryonic cells was verified by real-time quantitative PCR (qRT-PCR). Different concentrations of NAC and phosphate buffered saline (PBS) were used to affect the Drosophila S2 embryonic cells. The growth state of Drosophila S2 embryonic cells was observed by light microscope. Two probes dihydroethidium (DHE) and 2,7-dichlorodihydrofluorescein-acetoacetate (DCFH-DA) were used to observe the ROS production in each group after immunofluorescence staining. TUNEL staining and flow cytometry were used to investigate the apoptosis level of Drosophila S2 embryos, and CCK-8 (Cell Counting Kit-8) was used to detect the cell viability of Drosophila S2 embryos. The knockdown efficiency of siCG8005-2 fragment was high and stable, which was verified by interference efficiency (P < 0.05). There was no significant change in the growth of Drosophila S2 embryonic cells after the treatment of NAC as compared to PBS group. Moreover, knockdowning CG8005 gene resulted in an increase in ROS and apoptosis in Drosophila S2 embryonic cells (P < 0.05) and a decrease in proliferation activity (P < 0.05). In addition, the pretreatment of antioxidant NAC could inhibit ROS production in Drosophila S2 embryonic cells (P < 0.05), reduce cell apoptosis (P < 0.05), and improve cell survival (P < 0.05). The CG8005 gene in Drosophila S2 embryonic cells could regulate the proliferation and apoptosis of S2 embryonic cells by disrupting the redox homeostasis, and antioxidant NAC could inhibit cell apoptosis and promotes cell proliferation by scavenging ROS in Drosophila S2 embryonic cells, which is expected to provide novel insights for the pathogenesis of male infertility and spermatogenesis.

N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells

Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson’s Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells.

Boesenbergia stenophylla-Derived Stenophyllol B Exerts Antiproliferative and Oxidative Stress Responses in Triple-Negative Breast Cancer Cells with Few Side Effects in Normal Cells.

Triple-negative breast cancer (TNBC) is insensitive to target therapy for non-TNBC and needs novel drug discovery. Extracts of the traditional herb Boesenbergia plant in Southern Asia exhibit anticancer effects and contain novel bioactive compounds but merely show cytotoxicity. We recently isolated a new compound from B. stenophylla, stenophyllol B (StenB), but the impact and mechanism of its proliferation-modulating function on TNBC cells remain uninvestigated. This study aimed to assess the antiproliferative responses of StenB in TNBC cells and examine the drug safety in normal cells. StenB effectively suppressed the proliferation of TNBC cells rather than normal cells in terms of an ATP assay. This preferential antiproliferative function was alleviated by pretreating inhibitors for oxidative stress (N-acetylcysteine (NAC)) and apoptosis (Z-VAD-FMK). Accordingly, the oxidative-stress-related mechanisms were further assessed. StenB caused subG1 and G2/M accumulation but reduced the G1 phase in TNBC cells, while normal cells remained unchanged between the control and StenB treatments. The apoptosis behavior of TNBC cells was suppressed by StenB, whereas that of normal cells was not suppressed according to an annexin V assay. StenB-modulated apoptosis signaling, such as for caspases 3, 8, and 9, was more significantly activated in TNBC than in normal cells. StenB also caused oxidative stress in TNBC cells but not in normal cells according to a flow cytometry assay monitoring reactive oxygen species, mitochondrial superoxide, and their membrane potential. StenB induced greater DNA damage responses (γH2AX and 8-hydroxy-2-deoxyguanosine) in TNBC than in normal cells. All these StenB responses were alleviated by NAC pretreatment. Collectively, StenB modulated oxidative stress responses, leading to the antiproliferation of TNBC cells with little cytotoxicity in normal cells.

ROS and DRP1 interactions accelerate the mitochondrial injury induced by polystyrene nanoplastics in human liver HepG2 cells

Microplastics have become a serious environmental pollutant and subsequently have harmful effects on human health. Thus, the impacts of microplastics on human cells need to be explored. In the present study, the cytotoxic effects at the subcellular-organelle levels to polystyrene nanoplastics (PS-NPs, diameter 21.5 ± 2.7 nm) were investigated in the human hepatocellular carcinoma (HepG2) cell line. The cell viability exposed to PS-NPs at the concentrations of 6.25, 12.5, 25 and 50 μg/mL for 24 h diminished in a concentration-dependent manner. The PS-NPs treatment induced mitochondrial injuries, including morphological changes, decreased adenosine triphosphate (ATP) production and the loss of mitochondrial membrane potentials (MMP). The PS-NPs treatment could further spark cell apoptosis by upregulating caspase 3, caspase 9, cytochrome c, and Bcl-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) in HepG2 cells, which is related to the mitochondrial dysfunction. PS-NPs exposure stimulated the excessive cellular reactive oxygen species (ROS) production and also induced mitochondrial fission by upregulating dynamin-related protein 1 (DRP1) and P-DRP1, but downregulating optic atrophy protein 1 (OPA1) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α) expression levels. The above effects on mitochondria damage induced by PS-NPs were reversed by the pretreatment of N-acetylcysteine (NAC), mitochondrial division inhibitor 1 (Mdivi-1) and DRP1 siRNA. The results suggested that the interaction between ROS and DRP1-dependent mitochondrial division could promote mitochondrial lesions and mitochondria-related apoptosis caused by PS-NPs. These findings on molecular mechanisms provide a theoretical basis for preventing the hazards caused by microplastics to human health.

N-acetyl cysteine: A new look at its effect on PTZ-induced convulsions

Introduction/AimEpilepsy is widely investigated as a common neurological disease requiring pharmacologically effective agents. N-acetyl cysteine (NAC), has become a remarkable molecule with its role in both antioxidant and glutaminergic modulation. There are many points and processes waiting to be revealed regarding the role of NAC in epilepsy. Materials and methodsPentylenetetrazole (PTZ) was administered to induce seizures in a total number of 48 Sprague-Dawley rats. 35 mg/kg PTZ dose as a sub-convulsive dose was administered to 24 animals to monitor EEG changes, while 70 mg/kg PTZ dose which was a convulsive dose was administered to 24 animals to determine seizure-related behavioral changes with the Racine's scale. 30 min before the seizure-induced procedure, NAC was administered at doses of 300 and 600 mg/kg as pretreatment to investigate anti-seizure and anti-oxidative effects. The spike percentage, the stage of convulsion, and the onset time of the first myoclonic jerk were evaluated to determine the anti-seizure effect. Furthermore, its effect on oxidative stress was determined by measuring both malondialdehyde (MDA) level and superoxide dismutase (SOD) enzyme activity. ResultsThere was a dose-dependent reduction in the seizure stage and prolonged onset time of the first myoclonic jerk in rats with NAC pretreatment. EEG recordings resulted in a dose-dependent decrease in spike percentages. Moreover, the same dose-dependent changes were observed in oxidative stress biomarkers, both 300 mg/kg NAC and 600 mg/kg decreased MDA levels and ameliorated SOD activity. ConclusionWe can report that 300 mg/kg and 600 mg/kg doses of NAC are promising with their reducing effect on convulsions and have a beneficial effect by preventing oxidative stress. In addition, NAC has been also determined that this effect is dose-dependent. Detailed and comparative studies are needed on the convulsion-reducing effect of NAC in epilepsy.

Rational Molecular Designing of Aggregation-Enhanced Emission (AEE) Active Red-Emitting Iridium(III) Complexes: Effect of Lipophilicity and Nanoparticle Encapsulation on Photodynamic Therapy Efficacy.

Two "aggregation-enhanced emission" (AEE) active cyclometalated phosphorescent iridium(III) complexes, SM2 and SM4, were synthesized to evaluate the influence of lipophilicity on photodynamic therapy efficacy. Compared to SM2, SM4 had a higher logP due to the presence of naphthyl groups. As observed by confocal microscopy, this increased lipophilicity of SM4 significantly enhanced its cellular uptake in breast cancer cells. Both the molecules were found to be noncytotoxic under nonirradiating conditions. However, with light irradiation, SM4 exhibited significant cytotoxicity at a 500 nM dose, whereas SM2 remained noncytotoxic, signifying the influence of lipophilicity on cellular internalization and cytotoxicity. Mechanistically, light-irradiated SM4-treated cancer cells exhibited a significant increase in the intracellular reactive oxygen species (ROS) level. Neutralizing ROS with N-acetylcysteine (NAC) pretreatment partly abolished the cytotoxic ability, indicating ROS as one of the major effectors of cell cytotoxicity. Two nanoparticle (NP) formulations of SM4 were developed to improve the intracellular delivery: a PLGA-based NP and a Soluplus-based micelle. Interestingly, PLGA and Soluplus NP formulations exhibited a 10- and 22-fold increased emission intensity, respectively, compared to SM4. There was also an increase in the excited-state lifetime. Additionally, the Soluplus-based micelles encapsulating SM4 exhibited enhanced cellular uptake and increased cytotoxicity compared to the PLGA NPs encapsulating SM4. Altogether, the current study indicates the importance of rational molecular designing and the significance of a proper delivery vector for improving photodynamic therapy efficacy.

Development of near-infrared lysosomal pH-activatable fluorescent probe for real-time visualization of autophagy progression

As the central role in maintaining physiological homeostasis, tracking the autophagy in living cells is highly desired in biological research and pharmacologic development. Herein, a lysosome-targeted pH-activatable probe (Lyso-QL) with emission at near-infrared region (NIR) was developed for monitoring the progression of autophagy. During autophagy progress, the double-membraned autophagosomes are engulfed into lysosomes to provide autolysosomes, exhibiting significantly enhanced acidification. Based on the characteristic, the real-time monitoring of autophagic flux were realized by evaluating the pH changes in these acidic compartments with Lyso-QL under nutrient-deprivation conditions as well as pharmacological induction. Importantly, through the NIR changes, the modulation of tamoxifen on autophagic levels in estrogen receptor-negative cancer cells was disclosed. Further, the elevated hepatic autophagy in mice during acetaminophen-induced liver injury was revealed, and the N-acetylcysteine pretreatment was demonstrated with a certain protective effect to restore the autophagic levels. The study demonstrates probe Lyso-QL would provide an edge for mechanistic insights into autophagy and evaluating the activities of autophagy modulators.

N-acetylcysteine rescues meiotic arrest during spermatogenesis in mice exposed to BDE-209.

Deca-bromodiphenyl ethers (BDE-209) has been widely used in electronic devices and textiles as additives to flame retardants. Growing evidence showed that BDE-209 exposure leads to poorer sperm quality and male reproductive dysfunction. However, the underlying mechanisms of BDE-209 exposure caused a decline in sperm quality remains unclear. This study aimed to evaluate the protective effects of N-acetylcysteine (NAC) on meiotic arrest in spermatocytes and decreased sperm quality in BDE-209-exposed mice. In the study, mice were treated with NAC (150mg/kg BW) 2h before administrated with BDE-209 (80mg/kg BW) for 2weeks. For the in vitro studies, spermatocyte cell line GC-2spd cells were pretreated with NAC (5mM) 2h before treated with BDE-209 (50μM) for 24h. We found that pretreatment with NAC attenuated the oxidative stress status induced by BDE-209 in vivo and in vitro. Moreover, pretreatment with NAC rescued the testicular histology impairment and decreased the testicular organ coefficient in BDE-209-exposed mice. In addition, NAC supplement partially promoted meiotic prophase and improved sperm quality in BDE-209-exposed mice. Furthermore, NAC pretreatment effectively improved DNA damage repair by recovering DMC1, RAD51, and MLH1. In conclusion, BDE-209 caused spermatogenesis dysfunction related to the meiotic arrest medicated by oxidative stress, decreasing sperm quality.

NADPH oxidase 4 regulate the glycolytic metabolic reprogramming of microglial cells to promote M1 polarization.

This work aimed to investigate the role and mechanism of NADPH oxidase 4 (NOX4) in the polarization of microglial cells.Microglial cells were transfected with the NOX4 overexpression plasmid (pGL3-NOX4), and later treated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to induce its M1 polarization. Later, the F4/80 + CD86 + cell proportion was detected by flow cytometry (FCM), the inflammatory factor expression levels were analyzed through enzyme-linked immunosorbent assay (ELISA), while ionized calcium binding adapter molecule 1(IBA-1) and PKM2 expression were measured by immunofluorescence (IF) staining. In addition, dichlorodihydrofluorescein diacetate probe was utilized to detect the reactive oxygen species(ROS) levels, glucose uptake, and glycolysis, as well as lactic acid level. The expression of glycolytic enzymes PKM2, HK2, and citrate (Si)-synthas(CS) was detected by Western-blot (WB) assay. Moreover, the polarization level of microglial cells was detected after ROS expression was suppressed by the ROS inhibitor N-acetylcysteine (NAC). In mouse experiments, LPS was applied in inducing central neuroinflammation in NOX4 knockdown mouse model (KO) and wild-type mice (WT). Thereafter, the inflammatory factor levels and lactic acid level in mouse tissues were detected; IBA-1 and CD86 expression in mice was measured by IF staining; and the expression of glycolytic enzymes PKM2, HK2, and CS in the central nervous system (CNS) was also detected.After NOX4 overexpression in microglial cells, the M1 polarization level was upregulated, the F4/80 + CD86 + cell proportion increased, and inflammatory factors were upregulated. At the same time, the expression of glycolytic enzymes PKM2, HK2, and CS was upregulated. NAC pretreatment suppressed the effects of NOX4, reduced the F4/80 + CD86 + cell proportion, and suppressed the expression of PKM2, HK2, and CS. In the mouse model, the expression levels of CD86 in KO group decreased, and the inflammatory factors were also downregulated.NOX4 promotes glycolysis of microglial cells via ROS, thus accelerating M1 polarization and inflammatory factor expression. In this regard, NOX4 is promising as a new target for the treatment of neuroinflammation.

Acacetin induces sustained ERK1/2 activation and RIP1-dependent necroptotic death in breast cancer cells

Acacetin (AC), a naturally occurring flavonoid has shown anticancer potential. Herein, we studied the mechanisms of cell death and growth inhibition by AC in breast carcinoma T-47D and MDA-MB-231 cells. AC (10–40 μM) significantly decreased the levels of G2/M phase cyclins and CDKs, simultaneously increasing the expression of CDK inhibitors including Cip1/p21. A concentration-dependent increase in cell death was noted in both breast cancer cell lines with no such considerable effects on MCF-10A non-tumorigenic breast cells. The cell death-inducing potential of AC was further confirmed using confocal microscopy and flow cytometry analysis. AC resulted in mitochondrial superoxide generation, DNA damage, and ROS generation. N-acetyl cysteine (NAC) pre-treatment inhibited ROS generation and partially reversed ERK1/2 activation as well as cell death by AC. Further, AC enhanced the expression of RIP1 and RIP3, which mediate necroptosis. RIP1-specific inhibitor Necrostatin-1 (NS-1) reversed the AC-induced DNA damage and cell death. Collectively, these findings, for the first time, suggested that AC exerts its antitumor potential through ROS induction and RIP1-dependent necroptosis in breast carcinoma cells.