Elsevier

NanoImpact

Volume 21, January 2021, 100280
NanoImpact

Research Paper
Copper nanoparticles induce the formation of fatty liver in Takifugu fasciatus triggered by the PERK-EIF2α- SREBP-1c pathway

https://doi.org/10.1016/j.impact.2020.100280Get rights and content

Highlights

  • The effect of CuNPs on T. fasciatus were caused by CuNPs itself, not the released Cu ions.

  • ERS related gene sequence was first cloned from T. fasciatus.

  • CuNPs induced ERS and its mediated UPR signaling pathway in the livers of T. fasciatus.

  • SREBPs were up-regulated by the PERK-EIF2α pathway to promote fatty liver formation.

Abstract

Copper nanoparticles (CuNPs), a new pollutant in water environments, were widely used in various industrial and commercial applications. This study indicated that the presence of CuNPs exposure under environmental related concentration is an inducing factor that contributes to the fatty liver formation in Takifugu fasciatus. Furthermore, we explored the fatty liver formation mechanism. The results shown, (1) the cloned genes related to endoplasmic reticulum stress (ERS) (GRP78, IRE-1α, PERK, and ATF-6α) were highly expressed in the liver of T. fasciatus. (2) after 30-days exposure, CuNPs accumulated in the endoplasmic reticulum of liver and induced the appearance of ERS, then activated unfolded protein response (UPR) signaling pathway. Furthermore, the SREBP-1c pathway that plays a key role in lipid synthesis was activated. (3) by using 4-PBA and GSK inhibitors to respectively stimulate ERS and PKR-like ER kinase (PERK) through in vitro experiments, we confirmed that CuNPs induced the fatty liver formation in T. fasciatus triggered by the PERK-EIF2α pathway by activating the SREBP-1c pathway to promote fatty liver formation. This study provides a new perspective for identifying the pathogens of fatty liver formation, and adds to the knowledge of the ecological safety data service of CuNPs in water

Introduction

Nanomaterials exhibit novel physicochemical properties that determine their interactions with biological substrates and processes (Gillespie et al. 2010; Abdelkhaliq et al. 2020). As one of the most widely used nanotechnology products, copper nanoparticles (CuNPs) have many physical and chemical properties that differ from those of conventional substances. CuNPs are widely used in human life (such as textiles, boat antifouling paints, skin products, wood preservation, et al), and are terminated in water environments through migration, transportation, and other processes (Gomes et al. 2011; Liu et al. 2021). In addition, CuNPs have been applied in fish medicine and as feed additives in aquaculture (Basuini et al., 2016; Ostaszewska et al. 2018). The use of CuNPs bring convenience to human life. However, an increasing amount of attention is being focused on the safety of CuNPs (Buffet et al. 2013; Chan et al. 2016). At present, although there have been studies regarding the toxic effects of CuNPs on organisms (Shaw et al. 2012; Buffet et al. 2013; Song et al. 2016; Wang et al. 2017), its use is still in the early stages, and studies regarding its toxicological mechanisms are not currently sufficiently comprehensive.

Takifugu fasciatus is a species of marine freshwater breeding migratory fish. It is widespread in the East China Sea, the South China Sea, the Sea of Japan, and inland waters in China and Korea. T. fasciatus is inevitably affected by CuNPs due to breeding migration (Wang et al. 2019a). Interestingly, T. fasciatus can be applied as an ideal model organism to research the ecological influence of pollutants (Kim et al. 2010; Wang et al. 2018). In addition, the T. fasciatus can be regarded as a model organism in the field of medical research due to their peculiar biology, physiology, and genomics (pufferfish have the smallest currently known vertebrate genome, which is one-eighth the length of the human genome) (Yamanoue et al. 2009; Kim et al. 2010; Guo et al. 2010). In recent years, as a delicacy and commercially farmed puffer fish, the area for aquatic breeding of T. fasciatus has gradually expanded. Based on the 2017 data from puffer fish branch of the China Fisheries Association, the yearly output value is more than CNY 10 billion (Wang et al. 2019a). The annual consumption of T. fasciatus is approximately 10,000 tons in Korea. In Japan, T. fasciatus is considered to have a large value-added space (Yoo et al. 2018). However, the currently cultured T. fasciatus generally suffer from fatty liver disease, which seriously affects the healthy development of T. fasciatus and negatively impacts on the breeding industry. At present, most of the studies regarding fatty liver disease in animals focus on nutrition and feed. However, there are many causes for the induction of fatty liver disease but some are considered less important. For the first time, this research finds that exposure to CuNPs induces fatty liver formation in T. fasciatus, but its underlying mechanisms are currently unclear.

A state defined as endoplasmic reticulum stress (ERS), which activates a complex signaling network called unfolded protein response (UPR), is caused by the disruption to the homeostasis of endoplasmic reticulum (ER) (Chen et al. 2014). As an integrated endocellular signaling pathway, UPR induces translational inhibition and then causes the up-regulation of ER-resident chaperone (GRP78/BiP). UPR has a characteristic of downregulating the protein translation by three ER-transmembrane transducers; activating transcription factor (ATF)-6α, inositol-requiring enzyme (IRE)-1α, and PKR-like ER kinase (PERK) (Hu et al. 2019). The disturbance of ER homeostasis and the activation the UPR can be caused by many environmental factors (Han and Kaufman 2016). ERS and its signaling pathway play a key role in fat deposition in mammals (Schröder and Kaufman 2005; Bobrovnikova-Marjon et al. 2008; Malhi and Kaufrnan 2011; Basseri and Austin 2012). In addition, the liver is a significant organ in systemic lipid homeostasis (Basseri and Austin 2012). In this study, we combine in vitro and in vivo experiments to explore whether exposure to CuNPs induces ERS in the liver of T. fasciatus and activates its downstream signaling pathway. Secondly, we investigate the role that ERS and its signaling pathway play in lipid deposition of the liver of T. fasciatus induced by CuNPs. The results of these studies may assist in revealing the lipid deposition mechanism induced by CuNPs in T. fasciatus liver from the perspective of the upstream regulation pathway of lipid metabolism. Additionally, this study provides a new perspective for identifying the cause of the formation of fatty liver in T. fasciatus and provides a theoretical basis for its treatment, and supplements the ecological safety data of CuNPs.

Section snippets

CuNPs preparation and particle characterization

The CuNPs employed in this study were obtained from Shanghai Aladdin Co., Ltd. China, which particle diameter is 10-30 nm on average and purity is 99.9%. Nanopowders were dispersed in ultrapure water and aggregates were decomposed by ultrasound at 600 W and 40 kHz for 30 min, then diluted to exposure concentrations to prepare stock solutions. A transmission electron microscope (TEM) (HITACHI-7650, Japan) was supplied to measure the morphology and size of the CuNPs. The particle size range was

Results and discussion

Many studies have reported that heavy metal exposure can induce hepatic lipid deposition (Yoon et al. 2007; Zheng et al. 2013; Pan et al. 2018; Song et al. 2018). No previous studies have found that Cu in a nano form can induce lipid deposition in fish. In this study, a fatty liver model was successfully established by CuNPs exposure in T. fastiatus, which can be used as a typical species for the study of fatty liver formation mechanisms after CuNPs exposure. The results of this study identify

Conclusions

This study revealed that exposure to CuNPs under environmental related concentration has been shown to alter ER morphologies and induce ERS and its mediated UPR signaling pathway in the livers of T. fasciatus. SREBPs were up-regulated by the UPR pathway to promote fatty liver formation. This study confirmed that the CuNPs pollutant induces lipid deposition in the livers of T. fasciatus triggered by the PERK-EIF2α-SREBP-1c pathway.

Ethics approval and consent to participate

All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in China. The protocols of this study were approved by the Institutional Animal Care and Use Committee of Nanjing Normal University (grant No. SYXK 2015–0028, Jiangsu).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

CRediT authorship contribution statement

Shaowu Yin and Tao Wang conceived this study, and designed and supervised the experiments. Xiaozhen Wei, Yadong Hu, Yiru Sun, Jie Li, and Xinyu Zhang performed the experiments. Yongxiang Zhu and Yonghai Shi conducted data analysis and prepared figures and tables. Tao Wang wrote the manuscript. Shaowu Yin reviewed and revised the manuscript. All authors reviewed and approved the manuscript.

Declaration of Competing Interest

The authors declare no competing financial interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (31800436); Natural Science Foundation (NSF) of Jiangsu Province of China (BK20180728); National Key R & D Program of China (2018YFD0900301).

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