Zhen Zou

Zinc oxide nanoparticles harness autophagy to induce cell death in lung epithelial cells

Although zinc oxide nanoparticles (ZnONPs) are widely used, they have raised concerns of toxicity in humans. Previous studies have indicated that reactive oxygen species (ROS) and autophagy are involved in the cytotoxicity of ZnONPs, but the regulatory mechanisms between autophagy and ROS remain to be elucidated. Herein, we comprehensively investigated the regulatory mechanism of autophagy and the link between autophagy and ROS in ZnONPs-treated lung epithelial cells. We demonstrated that ZnONPs could induce autophagy, and this process could enhance the dissolution of ZnONPs in lysosomes to release zinc ions. Sequentially, zinc ions released from ZnONPs were able to damage not only lysosomes, leading to impaired autophagic flux, but also mitochondria. Impaired autophagic flux resulted in the accumulation of damaged mitochondria, which could generate excessive ROS to cause cell death. We further demonstrated that the inhibition of autophagy by either pharmacological inhibitors or small interfering RNA (siRNA)-mediated knockdown of Beclin-1 and AMP-activated protein kinase could ameliorate ZnONPs-induced cell death. Moreover, we found that lysosomal-associated membrane protein 1/2 (LAMP-1/2), which were the most abundant highly glycosylated protein in late endosomes/lysosomes, exhibited aberrant expression pattern upon treatment with ZnONPs. Intriguingly, LAMP-2 knockdown, but not LAMP-1 knockdown, could exacerbate the ROS generation and cell death induced by ZnONPs treatment. Meanwhile, LAMP-2 overexpression alleviated ZnONPs-induced cell death, suggesting that LAMP-2 was linked to this toxic phenotype induced by ZnONPs. Our results indicate that autophagic dysfunction could contribute to excessive ROS generation upon treatment with ZnONPs in lung epithelial cells, suggesting that modulating the autophagy process would minimize ZnONPs-associated toxicity.

LAMP-2 mediates oxidative stress-dependent cell death in Zn2+-treated lung epithelium cells

Zinc is an essential element for the biological system. However, excessive exogenous Zn2+ would disrupt cellular Zn2+ homeostasis and cause toxicity. In particular, Zinc salts or ZnO nanoparticles exposure could induce respiratory injury. Although previous studies have indicated that organelle damage (including mitochondria or lysosomes) and reactive oxygen species (ROS) production are involved in Zn2+-induced toxicity, the interplay between mitochondria/lysosomes damage and ROS production is obscure. Herein, we demonstrated that Zn2+ could induce deglycosylation of lysosome-associated membrane protein 1 and 2 (LAMP-1 and LAMP-2), which primarily locate in late endosomes/lysosomes, in A549 lung epithelium cells. Intriguingly, LAMP-2 knockdown further aggravated Zn2+-mediated ROS production and cell death, indicating LAMP-2 (not LAMP-1) was involved in Zn2+-induced toxicity. Our results provide a new insight that LAMP-2 contributes to the ROS clearance and cell death induced by Zn2+ treatment, which would help us to get a better understanding of Zn2+-induced toxicity in respiratory system.

The size of zinc oxide nanoparticles controls its toxicity through impairing autophagic flux in A549 lung epithelial cells

Zinc oxide nanoparticles (ZnONPs) widely used in various products, have been concerned with its impact on human health, in particular, on the risk of pulmonary toxicity. Our previous study indicated that ZnONPs could harness autophagy and impair the autophagic flux, which was positively linked to ZnONPs-induced toxicity. The objective of this study was to investigate whether ZnONPs-induced impairment of autophagic flux and cell death in lung epithelial cells is related to the size of ZnONPs. We demonstrate that ZnONPs with the average size of 50 nm could induce toxic effects in A549 lung epithelial cells, including accumulation of autophagosomes (the elevation of LC3B-II/LC3B-I ratio), impaired autophagic flux (the increase of p62 expression), the release of intracellular zinc ions (the increase of FluoZin-3 signal and ZnT1 mRNA expression), mitochondrial damage (the decrease of TMRE signal), lysosomal dysfunction (the aberrant expression of LAMP-2), oxidative stress (the increase of DCFH-DA signal and HO-1 expression) and cell death. Interestingly, ZnONPs with the average size of 200 nm failed to induce autophagy-mediated toxicity. Taken together, our results indicate that the size of ZnONPs is closely correlated with its toxicity, which is probably mediated by induction of impaired autophagic flux. This finding provides an insight into better understating of ZnONPs-associated toxicity, and mitigating the risk to humans and allowing the safer application.

Lysosomal deposition of copper oxide nanoparticles triggers HUVEC cells death

The increasing use of copper oxide nanoparticles (CuONPs) has led to major concerns regarding both the predominant physicochemical properties and the potential toxic effects on the environment and human health. The objective of this study is to explore the possible mechanisms underlying the toxicity of CuONPs in vascular endothelial cells. We found that CuONPs induced the cell death in human umbilical vein endothelial cells (HUVECs) through a caspase-independent pathway. Our results also demonstrated that CuONPs were prevalently deposited within lysosomes. The lysosomal deposition of CuONPs led to lysosomal dysfunction, resulting in the impairment of autophagic flux and the accumulation of undegraded autophagosomes. Nevertheless, blockage of the lysosomal deposition of CuONPs could significantly attenuate HUVEC cell death. Interestingly, we found that the inhibition of lysosomal deposition of CuONPs reduced the release of Cu ions, which has been considered as the crucial factor for the toxicity of CuONPs. In summary, our results indicate that the lysosomal deposition of CuONPs (along with the enhanced release of Cu ions form CuONPs) triggers CuONPs-induced HUVEC cell death. Our findings provide an insight into the mechanism of toxicity to the cardiovascular system induced by toxic metal oxide nanoparticles exposure.

Ferroptosis is newly characterized form of neuronal cell death in response to arsenite exposure

ABSTRACT Ferroptosis is a novel iron‐dependent form of cell death implicated in brain pathology. However, whether arsenite is an inducer of ferroptosis in the neuron remains completely unknown. In this study, the seven‐week‐old healthy C57BL/6J male mice were treated with environmental related doses (0.5, 5 and 50mg/L) of arsenite for 6 months via drinking water, and the ferroptosis‐related indicators were further determined. Our results demonstrated for the first time that, arsenite exposure significantly reduced the number of neuron and caused the pathological changes of mitochondria in the cerebral cortex of mice. We further revealed that arsenite induced ferroptotic cell death in neuron by accumulation of reactive oxygen species and lipid peroxidation products, disruption of Fe2+ homeostasis, depletion of glutathione and adenosine triphosphate, inhibition of cysteine/glutamate antiporter, activation of mitogen‐activated protein kinases and mitochondrial voltage‐dependent anion channels pathways, up‐regulation of endoplasmic reticulum stress, all of which were involved in the process of ferroptosis. These findings were also verified in the cultured PC‐12 cells by using ferropotosis inhibitor, desferoxamine. Taken together, our results not only reveal a novel mechanism that chronic arsenite exposure may trigger the new form of cell death, ferroptosis, but also shed a new light on a potential clue for the intervention and prevention against arsenite‐related neurodegenerative diseases. HIGHLIGHTSArsenite triggers new form of cell death, ferroptosis, in neuron of cerebral cortex.Arsenite induces ferroptosis by causing severe mitochondrial oxidative damage.Arsenite disrupts Fe2+ homeostasis and activates ferroptosis‐related pathways.

m6A Demethylase FTO Regulates Dopaminergic Neurotransmission Deficits Caused by Arsenite

Arsenite exposure is known to increase the risk of neurological disorders via alteration of dopamine content, but the detailed molecular mechanisms remain largely unknown. In this study, using both dopaminergic neurons of the PC-12 cell line and C57BL/6J mice as in vitro and in vivo models, our results demonstrated that 6 months of arsenite exposure via drinking water caused significant learning and memory impairment, anxiety-like behavior and alterations in conditioned avoidance and escape responses in male adult mice. We also were the first to reveal that the reduction in dopamine content induced by arsenite mainly resulted from deficits in dopaminergic neurotransmission in the synaptic cleft. The reversible N6- methyladenosine (m6A) modification is a novel epigenetic marker with broad roles in fundamental biological processes. We further evaluated the effect of arsenite on the m6A modification and tested if regulation of the m6A modification by demethylase fat mass and obesity-associated (FTO) could affect dopaminergic neurotransmission. Our data demonstrated for the first time that arsenite remarkably increased m6A modification, and FTO possessed the ability to alleviate the deficits in dopaminergic neurotransmission in response to arsenite exposure. Our findings not only provide valuable insight into the molecular neurotoxic pathogenesis of arsenite exposure, but are also the first evidence that regulation of FTO may be considered as a novel strategy for the prevention of arsenite-associated neurological disorders.

Mechanically induced autophagy is associated with ATP metabolism and cellular viability in osteocytes in vitro

Both mechanical loading and intracellular autophagy play important roles in bone homeostasis; however, their relationship remains largely unexplored. The objectives of this study were to determine whether osteocytes undergo autophagy upon fluid shear stress (FSS) loading and to determine the correlation between mechanically induced autophagy and ATP metabolism. Autophagic vacuoles were observed by transmission electron microscopy (TEM) in osteocyte-like MLO-Y4 cells subjected to FSS. Increased autophagic flux was further confirmed by the increased amount of the LC3-II isoform and the degradation of p62. Fluorescent puncta distributed in the cytoplasm were observed in the GFP-LC3 transformed cells subjected to FSS. Furthermore, FSS-induced ATP release and synthesis in osteocytes were attenuated by inhibiting autophagy with 3-MA. After FSS exposure, a high ratio of cell death was observed in cultures pretreated with 3-MA, an autophagy inhibitor, with no significantly different Caspase 3/7 activity. Our results indicated that FSS induces protective autophagy in osteocytes and that mechanically induced autophagy is associated with ATP metabolism and osteocyte survival. From the clinical perspective, it may be possible to enhance skeletal cell survival with drugs that modulate the autophagic state, and the autophagy-related pathway could be a potential target for the prevention of ageing-related bone disorders.

Autophagy-dependent release of zinc ions is critical for acute lung injury triggered by zinc oxide nanoparticles

Abstract Pulmonary exposure to zinc oxide nanoparticles (ZnONPs) could cause acute lung injury (ALI), but the underlying molecular mechanism remains unclear. Herein, we established a ZnONPs-induced ALI mouse model, characterized by the histopathological changes (edema and infiltration of inflammatory cells in lung tissues), and the elevation of total protein and cytokine interleukin-6 in bronchoalveolar lavage fluid in time- and dose-dependent manners. This model also exhibited features like the disturbance of redox-state (reduced of glutathione to glutathione disulfide ratio, elevation of heme oxygenase-1 and superoxide dismutase 2), the decrease of adenosine triphosphate synthesis and the release of zinc ions in the lung tissues. Interestingly, we found that ZnONPs exposure caused the accumulation of autophagic vacuoles and the elevation of microtubule-associated proteins 1A/1B light chain (LC)3B-II and p62, indicating the impairment of autophagic flux. Our data indicated that the above process might be regulated by the activation of AMP-activated protein kinase but not the mammalian target of rapamycin pathway. The association between ZnONPs-induced ALI and autophagy was further verified by a classical autophagy inhibitor, 3-methyladenine (3-MA). 3-MA administration reduced the accumulation of autophagic vacuoles, the expression of LC3B-II and p62, followed by a significant attenuation of histopathological changes, inflammation, and oxidative stress. More importantly, 3-MA could directly decrease the release of zinc ions in lung tissues. Taken together, our study provides the evidence that ZnONPs-induced pulmonary toxicity is autophagy-dependent, suggests that limiting the release of zinc ions by inhibiting autophagy could be a feasible strategy for the prevention of ZnONPs-associated pulmonary toxicity.

Novel osteogenic growth peptide C-terminal pentapeptide grafted poly(d,l-lactic acid) improves the proliferation and differentiation of osteoblasts: The potential bone regenerative biomaterial

Poly(d,l-lactic acid) (PDLLA) is widely used for bone regenerative engineering, because of its proven biocompatibility and biodegradability. However, the major limitation of PDLLA is its cell recognition and low hydrophilicity. The objective of this study was to develop a novel bioactive poly(d,l-lactic acid) tethered with osteogenic growth peptide (OGP), which has been confirmed as one of the important growth factors related to bone repair/regeneration. The biomimetic material modification methods were utilized that maleic anhydride-modified poly(d,l-lactic acid) (MPLA) as raw material, the active C-terminal pentapeptide OGP(10-14) were covalently grafted onto the side chain of MPLA through amide reaction using 1‑ethyl‑3‑(3‑dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N‑hydroxysuccinimide (NHS) as the condensing agent to produce a new biopolymer (OGP(10-14)-MPLA). The OGP(10-14)-MPLA were further characterized with the Fourier transform infrared spectrometry, amino acid analyzer, elementary analysis, X-ray photoelectron spectroscopy. The results revealed that OGP(10-14) was successfully modified MPLA and its coupling efficiency was 12.40%. The data from both contact angle and water absorption showed the better hydrophilicity of OGP(10-14)-MPLA, compared with MPLA. Also, we found that OGP(10-14)-MPLA could improve the proliferation, differentiation, and mineralization of osteoblasts, indicating that the novel OGP(10-14)-MPLA has the better biocompatibility and is more osteoinductive. In conclusion, the OGP(10-14) modified MPLA have the potential for bone regenerative engineering.

Disruption of the superoxide anions-mitophagy regulation axis mediates copper oxide nanoparticles-induced vascular endothelial cell death

&NA; Copper oxide nanoparticles (CuONPs) have been widely used in the industrial and pharmaceutical fields; however, their toxicity profile is deeply concerning. Currently, nanomaterials‐induced toxicity in the cardiovascular system is receiving increased attention. Our previous toxicological study found that lysosomal deposition of CuONPs triggered vascular endothelial cell death, indicating that the involvement of autophagic dysfunction was crucial for CuONPs‐induced toxicity in human umbilical vein endothelial cells (HUVECs). In the current study, we investigated the detailed mechanism underlying the autophagic dysfunction induced by CuONPs. We demonstrated that CuONPs exposure caused accumulation of superoxide anions, which likely resulted from mitochondrial dysfunctions. MnTBAP, a superoxide anions scavenger, alleviated CuONPs‐induced HUVECs death, indicating that excessive superoxide anions were directly related to the CuONPs cytotoxicity in HUVECs. Interestingly, we found that mitophagy (a protective mechanism for clearance of damaged mitochondria and excessive superoxide anions) was initiated but failed to be cleared in CuONPs‐treated cells, resulting in the accumulation of damaged mitochondria. Inhibition of mitophagy through Atg5 knockout or blocking of mitochondria fission with Mdivi‐1 significantly aggravated CuONPs‐induced superoxide anions accumulation and cell death, suggesting that mitophagy is a protective mechanism against CuONPs cytotoxicity in HUVECs. In summary, we demonstrate that superoxide anions (originating from damaged mitochondria) are involved in CuONPs‐associated toxicity and that impaired mitophagic flux aggravates the accumulation of excessive superoxide anions, which leads to HUVECs death. Our findings indicate that there are crucial roles for superoxide anions and mitophagy in CuONPs‐induced toxicity in vascular endothelial cells. Graphical abstract Figure. No caption available. HighlightsCuONPs induce mitochondrial dysfunctions and the accumulation of mitochondrial superoxide anions in HUVECs.Accumulation of superoxide anions is involved in CuONPs‐induced HUVECs death.CuONPs‐induced superoxide anions triggers mitophagy initiation in HUVECs.Impaired mitophagic flux due to lysosomal deposition of CuONPs causes accumulation of damaged mitochondria.Mitophagy inhibition aggravates CuONPs‐induced HUVECs death.