Promoted ROS Production Research Articles

Design, synthesis and biological evaluation of novel benzimidazole-derived p21-activited kinase 4 (PAK4) inhibitors bearing a 4-(4-methylpiperazin-1-yl)phenyl scaffold as potential antitumor agents

A series of novel 6-(4-(4-methylpiperazin-1-yl)phenyl)-1H-benzo[d]imidazole-based p21-activited kinase 4 (PAK4) inhibitors were designed and synthesized based on the structure of lead compound GNE-2861 and that of anticancer inhibitors reported in our previous studies. All target compounds so designed were preliminarily screened in vitro for anti-tumor potency through kinase inhibitory assays and MTT assays, which revealed that most compounds exhibited significant inhibitory effects on PAK4 enzyme as well as prominent antiproliferative activities against four cancer cell models (A549, NCI–H1975, MDA-MB-231 and SK-BR-3) and low damage to healthy cells. In particular, the hit compound 12i was identified as the most effective and rather selective compound both at the enzyme and cellular level. Meanwhile, molecular docking and dynamic studies disclosed that compound 12i could bind to PAK4 stably via multiple interactions applied between the compound and the enzyme. Further mechanism studies indicated that compound 12i could inhibit the proliferation and suppress the migratory potential of MDA-MB-231 cells by inhibiting the phosphorylation of PAK4 and LIMK1, arresting cell cycle in the G0/G1 phase, inducing apoptosis and promoting ROS production. Notably, compound 12i could effectively inhibit triple-negative breast cancer (TNBC) growth with little toxicity in the MDA-MB-231 cell xenograft model. Taken together, in vitro and in vivo results demonstrated that compound 12i possessed high drug potential as an inhibitor of PAK4 to inhibit the growth and metastasis of TNBC.

Bisphenol S decreased lifespan and healthspan via insulin/IGF-1-like signaling-against mitochondrial stress in Caenorhabditis elegans

Bisphenol S (BPS) is widely presented and affects aging with unclear mechanisms. Here, we applied C. elegans to evaluate the effects of BPS on lifespan and healthspan and to investigate the underlying mechanisms. Both early-life and whole-life exposure to BPS at environmentally relevant doses (0.6, 6, 60 μg/L) significantly decreased lifespan, and healthspan (body bend, pharyngeal pumping, and lipofuscin accumulation). BPS exposure impaired mitochondrial structure and function, which promoted ROS production to induce oxidative stress. Furthermore, BPS increased expressions of the insulin/IGF-like signaling (IIS). Also, BPS inhibited expression of the IIS transcription factor daf-16 and its downstream anti-oxidative genes. Quercetin effectively improved BPS-induced oxidative stress byreversing BPS-regulated IIS/daf-16 pathway and anti-oxidative gene expressions. In daf-2 and daf-16 mutants, the effects of BPS and quercetin on lifespan, healthspan, oxidative stress, and anti-oxidative genes expressions were reversed, demonstrating the requirement of IIS/daf-16 for aging regulation. Molecular docking and molecular dynamics simulations confirmed the stable interaction between DAF-2 and BPS mainly via three residues (VAL1260, GLU1329, and MET1395), which was attenuated by quercetin. Our results highlighted that adverse effects of BPS on impairing lifespan and healthspan by affecting IIS/daf-16 function against mitochondrial stress, which could be inhibited by quercetin treatment. Thus, we first revealed the underlying mechanisms of BPS-induced aging and the potential treatment.

A new therapeutic strategy for infectious diseases against intracellular multidrug-resistant bacteria

Bacterial infections result in 7,700,000 deaths per year globally, with intracellular bacteria causing repeated and resistant infection. No drug is currently licenced for the treatment of intracellular bacteria. A new screening platform mimicking the host milieu has been established to explore phytochemical antibiotic adjuvants. Previously neglected isoprenylated flavonoids were found to be effective against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Specifically, the synergistic effect between glabrol and streptomycin against intracellular bacteria was observed for the first time. The glabrol-streptomycin combination targets bacterial inner membrane phospholipids, disrupts arginine biosynthesis, inhibits cell wall proteins and biofilm formation genes (agrA/B/C/D), and promotes ROS production, causing subsequent membrane and wall damage.To enhance the selective uptake of combination drug into infected cells, hyaluronic acid-streptomycin-lipoic acid-glabrol nanoparticles (HSLGS-S) were designed and synthesized to trigger the intracellular delivery of the glabrol-streptomycin combination. Thus, the treatment can be transported into the infected intracellular region and selectively release the glabrol-streptomycin combination to the bacterial at site. The bioactivity of HSLGS-S in clearing intracellular bacteria was 20-fold higher than that of the glabrol-streptomycin combination alone in vitro and 2- to 10-fold higher in vivo.

Sanguinarine induces ferroptosis of colorectal cancer cells by upregulating STUB1 and downregulating GPX4

To investigate the effect of sanguinarine (SAN) on proliferation and ferroptosis of colorectal cancer cells. SW620 and HCT-116 cells treated with different concentrations of SAN were examined for cell viability changes using CCK8 assay to determine the IC50 of SAN in the two cells. The inhibitory effects of SAN on proliferation, invasion and migration of the cells were evaluated using colony-forming assay and Transwell assays. ROS production in the treated cells was analyzed with flow cytometry, and lipid peroxide production was assessed by detecting malondialdehyde (MDA) level. Glutathione (GSH) levels in the cells were detected, and Western blotting was used to detect the expressions of ferroptosis-related proteins STUB1 and GPX4. SAN significantly inhibited the proliferation, invasion and migration of SW620 and HCT-116 cells. SAN treatment significantly promoted ROS production, increased intracellular MDA level, and lowered GSH level in the two cells (P<0.05). Western blotting showed that SAN significantly upregulated the expression of STUB1 and down-regulated the expression of its downstream protein GPX4 (P<0.05). SAN induces ferroptosis in colorectal cancer cells by regulating STUB1/GPX4, which may serve as a new therapeutic target for colorectal cancer.

Polyphenolic Nanomedicine Regulating Mitochondria REDOX for Innovative Cancer Treatment.

Cancer remains a highly lethal disease globally. The approach centered on REDOX-targeted mitochondrial therapy for cancer has displayed notable benefits. Plant polyphenols exhibit strong REDOX and anticancer properties, particularly by affecting mitochondrial function, yet their structural instability and low bioavailability hinder their utility. To overcome this challenge, researchers have utilized the inherent physical and chemical characteristics of polyphenols and their derivatives to develop innovative nanomedicines for targeting mitochondria. This review examines the construction strategies and anticancer properties of various types of polyphenol-based biological nanomedicine for regulating mitochondria in recent years, such as polyphenol self-assembly, metal-phenol network, polyphenol-protein, polyphenol-hydrogel, polyphenol-chitosan, and polyphenol-liposome. These polyphenolic nanomedicines incorporate enhanced features such as improved solubility, efficient photothermal conversion capability, regulation of mitochondrial homeostasis, and ion adsorption through diverse construction strategies. The focus is on how these polyphenol nanomedicines promote ROS production and their mechanism of targeting mitochondria to inhibit cancer. Furthermore, it delves into the benefits and applications of polyphenolic nanomedicine in cancer treatments, as well as the challenges for future research.

Using chitosan-coated magnetite nanoparticles as a drug carrier for opioid delivery against breast cancer

Over the past decades, opium derivatives have been discovered as new anticancer agents. In our study, Fe3O4 superparamagnetic nanoparticles (SPIONs) decorated with chitosan were loaded with papaverine or noscapine to surmount drug delivery-related obstacles. Modifying the magnetic nanoparticles (MNP) surface with polymeric materials such as chitosan prevents oxidation and provides a site for drug linkage, which renders them a great drug carrier. The obtained systems were characterized by DLS (20–40 nm were achieved for MNPs and drug- loaded MNPs), TEM (spherical with average size of 11–20 nm) FTIR, XRD, and VSM (71.3 − 42.8 emu/g). Contrary to noscapine, papaverine-MNPs attenuated 4T1 murine breast cancer cell proliferation (11.50 ± 1.74 µg/mL) effectively compared to the free drug (62.35 ± 2.88 µg/mL) while sparing L-929 fibroblast cells (138.14 ± 4.38 µg/mL). Furthermore, SPION and SPION-chitosan displayed no cytotoxic activity. Colony-formation assay confirmed the long-term cytotoxicity of nanostructures. Both developed formulations promoted ROS production accompanied by late apoptotic cell death. The biocompatible nanoparticle exerted an augmenting effect to deliver papaverine to metastatic breast cancer cells.

CRABP2 reduces the sensitivity of Olaparib in ovarian cancer by downregulating Caspase-8 and decreasing the production of reactive oxygen species

Poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors, such as Olaparib, have been pivotal in treating BRCA-deficient ovarian cancer. However, their efficacy is limited in over 40% of BRCA-deficient patients, with acquired resistance posing new clinical challenges. To address this, we employed bioinformatics methods to identify key genes impacting Olaparib sensitivity in ovarian cancer. Through comprehensive analysis of public databases including GEO, CPTAC, Kaplan Meier Plotter, and CCLE, we identified CRABP2 as significantly upregulated at both mRNA and protein levels in ovarian cancer, correlating with poor prognosis and decreased Olaparib sensitivity. Using colony formation and CCK-8 assays, we confirmed that CRABP2 knockdown in OVCAR3 and TOV112D cells enhanced sensitivity to Olaparib. Additionally, 4D label-free quantitative proteomics analysis, GSEA, and GO/KEGG analysis revealed CRABP2's involvement in regulating oxidation signals. Flow cytometry, colony formation assays, and western blotting demonstrated that CRABP2 knockdown promoted ROS production by activating Caspase-8, thereby augmenting Olaparib sensitivity and inhibiting ovarian cancer cell proliferation. Moreover, in xenograft models, CRABP2 knockdown significantly suppressed tumorigenesis and enhanced Olaparib sensitivity, with the effect being reversed upon Caspase-8 knockdown. These findings suggest that CRABP2 may modulate Olaparib sensitivity in ovarian cancer through the Caspase-8/ROS axis, highlighting its potential as a target for Olaparib sensitization.

Identification of a ferritinophagy inducer via sinomenine modification for the treatment of colorectal cancer

Ferritinophagy is a cellular process to release redox-active iron. Excessive activation of ferritinophagy ultimately results in ferroptosis characterized by ROS accumulation which plays important roles in the development and progression of cancer. Sinomenine, a main bioactive alkaloid from the traditional Chinese medicine Sinomenum acutum, inhibits the proliferation of cancer cells by promoting ROS production. Herein, new compounds were designed and synthesized through the stepwise optimization of sinomenine. Among them, D3-3 induced the production of lipid ROS, and significantly promoted colorectal cancer cells to release the ferrous ion in an autophagy-dependent manner. Moreover, D3-3 enhanced the interaction of FTH1-NCOA4, indicating the activation of ferritinophagy. In vivo experiments showed that D3-3 restrained tumor growth and promoted lipid peroxidation in the HCT-116 xenograft model. These findings demonstrated that D3-3 is an inducer of ferritinophagy, eventually triggering ferroptosis. Compound D3-3, as the first molecule to be definitively demonstrated to induce ferritinophagy, is worth further evaluation as a promising drug candidate in the treatment of colorectal cancer.

Discovery of 2,4-diarylaminopyrimidine derivatives bearing sulfonamide moiety as novel FAK inhibitors

Two series of 2,4-diarylaminopyrimidine derivatives containing sulfonamide moiety were designed and synthesized for screening as inhibitors of focal adhesion kinase (FAK). Most compounds significantly inhibited the enzymatic activities of FAK, and the best compound was 7b (IC50 = 0.27 nM). A majority of aminoethyl sulfonamide derivatives could effectively inhibit the proliferation of human cancer cell lines (HCT116, A549, MDA-MB-231 and Hela) expressing high levels of FAK. Particularly, compounds 7b, 7c, and 7o exhibited more significant efficacy against all of four cancer cell lines within concentrations of 1.5 μM. Furthermore, these three compounds displayed higher selectivity of cancer cells over normal cells (SI value > 14), compared to the positive control TAE226 (SI value = 1.63). Interestingly, introduction of dithiocarbamate moiety to the aminoethyl sulfonamide derivatives can indeed improve the antiproliferative activities against A549 cells. Especially, compound 8d demonstrated most significant cytotoxicity activity against A549 cells with an IC50 value of 0.08 μM, which is 20-fold superior to parent compound 7k. Additionally, compound 7b, which display the best anti-FAK potency, can inhibit the clone formation and migration of HCT-116 cells, and cause cell cycle arrest at G2/M phase, inducing apoptosis by promoting ROS production. Overall, these results suggest that 7b is a valuable FAK inhibitor that deserves further optimization to improve its druggability.

Cancer cell membrane-camouflaged CuPt nanoalloy boosts chemotherapy of cisplatin prodrug to enhance anticancer effect and reverse cisplatin resistance of tumor

The biotoxicity and chemotherapeutic resistance of cisplatin (CDDP) pose a challenge for tumor therapy. Practically, the change in the therapeutic response of tumor from resistance to sensitivity are impressive but challenging. To this end, we propose a strategy of “one stone, three birds” by designing a CuPt nanoalloy to simultaneously eliminate GSH, relieve hypoxia, and promote ROS production for effectively reversing the platinum (IV) (Pt(IV), (c,c,t-[Pt(NH3)2Cl2(OOCCH2CH2COOH)2)) resistance. Notably, the CuPt nanoalloy exhibits ternary catalytic capabilities including mimicking GSH oxidase, catalase and peroxidase. With the subsequent disguise of tumor cell membrane, the CuPt nanoalloy is conferred with homologous targeting ability, making it actively recognize tumor cells and then effectively internalized by tumor cells. Upon entering tumor cell, it gives rise to GSH depletion, hypoxia relief, and oxidative stress enhancement by catalyzing the reaction of GSH and H2O2, which mitigates the vicious milieu and ultimately reinforces the tumor response to Pt(IV) treatment. In vivo results prove that combination therapy of mCuPt and Pt(IV) realizes the most significant suppression on A549 cisplatin-resistant tumor. This study provides a potential strategy to design novel nanozyme for conquering resistant tumor.

Sulfur oxidation states manipulate excited state electronic configurations for constructing highly efficient organic type I photosensitizers.

The multiple relaxation processes of excited states are a bridge connecting molecular structures and properties, providing enormous application potential for organic luminogens. However, a systematic understanding and manipulation of the relationship between the molecular structure, excited state relaxation processes, and properties of organic luminogens is still lacking. Herein, we report a strategy for manipulating excited state electronic configurations through the regulation of the sulfur oxidation state to construct eminent organic type I PSs. Combined with the experimental results and theoretical calculations, we have successfully revealed the decisive role of high sulfur oxidation states in promoting ROS production capacity. Impressively, a higher sulfur oxidation state can reduce the singlet-triplet energy gap (ΔE ST), increase the matching degree of transition configurations, promote the changes of the excited state electronic configurations, and boost the effective ISC proportion by enhancing intramolecular interactions. Therefore, DBTS2O with the highest sulfur oxidation state exhibits the strongest type I ROS generation ability. Additionally, guided by our strategy, a water-soluble PS (2OA) is designed and synthesized, showing selective imaging capacity and photokilling ability against Gram-positive bacteria. This study broadens the horizons for both molecular design and mechanism study of high-performance organic type I PSs.

Selenium promotes immunogenic radiotherapy against cervical cancer metastasis through evoking P53 activation

Radiotherapy is still the recommended treatment for cervical cancer. However, radioresistance and radiation-induced side effects remain one of the biggest clinical problems. Selenium (Se) has been confirmed to exhibit radiation-enhancing effects for cancer treatment. However, Se species dominate the biological activities and which form of Se possesses better radiosensitizing properties and radiation safety remains elusive. Here, different Se species (the valence state of Se ranged from - 2, 0, +4 to + 6) synergy screen was carried out to identify the potential radiosensitizing effects and radiation safety of Se against cervical cancer. We found that the therapeutic effects varied with the changes in the Se valence state. Sodium selenite (+4) displayed strong cancer-killing effects but also possessed severe cytotoxicity. Sodium selenate (+6) neither enhanced the killing effects of X-ray nor possessed anticancer activity by its alone treatment. Although nano-selenium (0), especially Let-SeNPs, has better radiosensitizing activity, the - 2 organic Se, such as selenadiazole derivative SeD (-2) exhibited more potent anticancer effects and possessed a higher safe index. Overall, the selected Se drugs were able to synergize with X-ray to inhibit cell growth, clone formation, and cell migration by triggering G2/M phase arrest and apoptosis, and SeD (-2) was found to exhibit more potent enhancing capacity. Further mechanism studies showed that SeD mediated p53 pathway activation by inducing DNA damage through promoting ROS production. Additionally, SeD combined with X-ray therapy can induce an anti-tumor immune response in vivo. More importantly, SeD combined with X-ray significantly inhibited the liver metastasis of tumor cells and alleviated the side effects caused by radiation therapy in tumor-bearing mice. Taken together, this study demonstrates the radiosensitization and radiation safety effects of different Se species, which may shed light on the application of such Se-containing drugs serving as side effects-reducing agents for cervical cancer radiation treatment.

A Combination of EGFR Inhibitors and AE-PDT Could Synergistically Suppress Breast Cancer Progression.

Breast cancer is the most frequently diagnosed malignancy and the leading cause of cancerrelated deaths in women. Activation of EGFR by EC-secreted EGFR ligands promotes breast cancer progression. Current treatments provide limited benefits in triple-negative breast cancer (TNBC). Photodynamic therapy (PDT) has been proven effective for the treatment of TNBC through the EGFR pathway, but the underlying mechanism is still unclear. The purpose of this study was to determine the role of the EGFR pathway in the treatment of PDT on TNBC in a co-culture system. MB-231 and HUVEC were co-cultured for experiments (HU-231). Cell viability and ROS production were detected after AE-PDT, a combination of EGFR inhibitors (AEE788)with PDT to test angiogenesis, apoptosis, and pyroptosis. WB detects expression of EGFR. EGFR, P-EGFR, VEGF, caspase-1, capase-3, and GSDMD . AE-PDT inhibited HU-231 cell proliferation and tumor angiogenesis, and induced cell apoptosis and pyroptosis by promoting ROS production. AEE788, an inhibitor of the EGFR, enhanced HU-231 cell killing after AE-PDT. Our study suggested that the combination of EGFR inhibitors and AE-PDT could synergistically suppress breast cancer progression, providing a new treatment strategy.

SARS-CoV-2 primed platelets–derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation

BackgroundHyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive.MethodsWe explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting.ResultsCOVID-19 patient–derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet–derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1β/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement.ConclusionsWe identified SARS-CoV-2 triggered platelets–derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

Polystyrene nanoplastics induce apoptosis of human kidney proximal tubular epithelial cells via oxidative stress and MAPK signaling pathways.

Polystyrene nanoplastics (PS-NPs) have recently been found to be present in human blood and kidney. However, the renal toxicity of PS-NPs and the underlying mechanisms have not been fully elucidated. Here, we found that exposure of PS-NPs induced apoptosis of human renal proximal tubular epithelial cells (HK-2) in a size- and dose-dependent manner as revealed by AnnexinV-FITC assay. In addition, PS-NPs promoted ROS production and caused structure changes of mitochondrial and endoplasmic reticulum. Mechanistically, transcriptional sequencing indicated the involvement of MAPK pathway in apoptosis, which was further confirmed by the upregulation of p-p38, p-ERK, CHOP, BAX, cytochrome C, and caspase 3 expression. This study clarified the molecular mechanism underlying PS-NP-induced apoptosis in HK-2 cells and contributed to our risk estimation of PS-NPs in human kidney.

Platelet-mimetic nano-sensor for combating postoperative recurrence and wound infection of triple-negative breast cancer

Tumor recurrence mainly triggered by tumor residual cells significantly contributes to mortality following breast tumor resection, and meanwhile post-surgical bacterial wound infections may accelerate tumor recurrence due to a series of infection-related complications. In this study, a nano-sensor system, Van-ICG@PLT, is constructed by a membrane camouflage and small molecule drug self-assembly strategy. This nano-sensor harnesses the innate tropism of platelets (PLT) to deliver vancomycin (Van) and indocyanine green (ICG) to surgical incisions, effectively eliminating both residual tumor cells and bacterial infections. Our findings demonstrate that Van-ICG@PLT preferentially accumulates at surgical wound. Under near-infrared (NIR) laser irradiation, Van-ICG@PLT exhibits significant cytotoxicity against 4T1 cells. Additionally, it is found to significantly promote ROS production thus inhibiting Staphylococcus aureus (S. aureus) growth, underscoring the synergistic benefits of phototherapy in combination with antibiotic treatment. In the 4T1 post-surgery recurrence mice model, Van-ICG@PLT is shown to efficiently ablate tumors in tumor-bearing mice (tumor inhibition rate of about 83%), and it demonstrates an excellent anti-infective effect in mice abscess models. Taken together, Van-ICG@PLT represents a promising paradigm in post-surgical adjuvant therapy (PAT). Its dual benefit in inhibiting cancer growth and promoting antibacterial activity makes Van-ICG@PLT a valuable addition to the existing arsenal of therapeutic options available for breast cancer patients.

Curcumin suppresses proliferation, migration and invasion of papillary thyriod cancer B-CPAP cells through the Keap1-Nrf2 pathway

To observe the effects of curcumin on migration and invasion of papillary thyriod cancer B-CPAP cells. B-CPAP cells were treated with 5, 10, 15, or 20 μmol/L curcumin, and the changes in cell survival, migration and invasion were examined using MTT assay and Transwell assay. ROS levels in the treated cells were detected with a DCFH-DA probe. The expression levels of Nrf2 and Keap1 in the cells were determined using Western blotting and qRT-PCR. Treatment with curcumin dose- and time-dependently suppressed the viability of B-CPAP cells (P < 0.05 or P < 0.01). Curcumin inhibited the migration and invasion (P < 0.001) and promoted ROS production in B-CPAP cells in a dose-dependent manner, and application of NAC effectively reversed curcumin- induced increase of ROS. Curcumin at 20 μmol/L significantly decreased the protein and mRNA expressions of Nrf2 and increased the expressions of Keap1 protein and mRNA (P < 0.05 or P < 0.01), causing also significantly reduced expression of Nrf2 protein in the cell nuclei (P < 0.05) without obviously affecting its expression in the cytoplasm (P > 0.05). Curcumin inhibits the proliferation, migration and invasion of papillary thyriod cancer B-CPAP cells probably via the Keap1-Nrf2 signaling pathway.

Switch of phosphorylation to O-GlcNAcylation of AhR contributes to vascular oxidative stress induced by benzo[a]pyrene

Benzo[a]pyrene (B[a]P) is a food contaminant toxic for cardiovascular diseases. The nuclear translocation of Arylhydrocarbon receptor (AhR) plays an important role in B[a]P-induced oxidative stress and vascular diseases. We confirmed that B[a]P promoted ROS production in vascular smooth muscle cells (VSMCs) in vitro and in vivo, associated with the nuclear translocation of AhR. It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR. However, from the posttranslational modification level, the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear. Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation, and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs, suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR. Next, siRNAs of O-linked N-acetylglucosamine transferase (OGT), O-GlcNAcase (OGA) and OGA inhibitor PUGNAc were used. SiOGT blocks but siOGA and PUGNAc promote B[a]P -dependent AhR nuclear translocation and oxidative stress. Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR. Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress. Our findings highlight a new perspective for AhR nuclear translocation regulated by the competitive modification between phosphorylation and O-GlcNAcylation.

Polystyrene-microplastics and DEHP co-exposure induced DNA damage, cell cycle arrest and necroptosis of ovarian granulosa cells in mice by promoting ROS production

The joint pollution of microplastics (MPs) and di-(2-ethylhexyl) phthalic acid (DEHP) often occurs, and consequently poses a serious threat to human and animal health, which has attracted widespread attention. However, the damage to the female mammalian ovary caused by the single exposure and co-exposure of MPs and DEHP and its specific mechanisms are not clear. Here, we established mouse models of single and co-exposures to polystyrene-microplastics (PS-MPs) and DEHP. The results showed that exposed to 100 mg/L PS-MPs and 200 mg/kg DEHP for 35 days destroyed the ovarian granulosa cell layer of mice, leading to follicular fragmentation and atresia. We cultured ovary granulosa cells in vitro to perform further mechanism studies and found that PS-MPs and DEHP had synergistic effects. Both of them promoted the excessive production of ROS and induced oxidative stress by triggering the CNR1/CRBN/YY1/CYP2E1 signaling axis, which in turn caused DNA oxidative damage. Additionally, we provided compelling evidence that oxidative stress mediated-hippo signaling pathway played a critical role in PS-MPs and DEHP caused ovary damage, resulting in ovarian granulosa cell cycle arrest and necroptosis. Using oxidative stress inhibitor AM251 or DAS could reverse these changes markedly and alleviate the reproductive toxicity caused by PS-MPs and DEHP, effectively. Overall, these results demonstrated that co-exposure of PS-MPs and DEHP adversely affected the integrity of ovary granulosa cell layer, resulting in DNA oxidative damage, cell cycle arrest and increased necroptosis of mouse ovarian granulosa cells by inducing oxidative stress. Our study shed new light on the co-exposure toxicity of PS-MPs and DEHP, provided novel insights for the reproductive toxicity of PS-MPs combined exposure with DEHP in female animals from a new free radical generation pathway perspective.

Zearalenone induces oxidative stress and autophagy in goat Sertoli cells

Zearalenone (ZEA), one of the non-steroidal estrogen mycotoxin, can cause male reproductive damage and genotoxicity in mammals. Testicular oxidative injury is an important factor causing male sterility. Testicular Sertoli cells are essential for spermatogenesis and male fertility. At present, the mechanism of oxidative injury in dairy goat Sertoli cells after exposure to ZEA remains unclear. This study explored the effects of ZEA on oxidative stress and autophagy in dairy goat Sertoli cells. It was found that treatment of primary Sertoli cells with 25, 50 and 100 μmol/L ZEA for 24 h can promote ROS production, decrease cell viability, antioxidant enzyme activity and mitochondrial membrane potential, induce caspase-dependent cell apoptosis and autophagy activity. ZEA-induced autophagy was confirmed by LC3-I/LC3-II transformation. More importantly, N-acetylcysteine (NAC) pretreatment can remarkably inhibit ZEA-induced oxidative stress, apoptosis and autophagy in Sertoli cells by eliminating ROS. In conclusion, this study indicates that ZEA induces oxidative stress and autophagy in dairy goat Sertoli cells by promoting ROS production.