Oxidative Stimuli Induce Antioxidant Responses by Affecting Nrf2/are Signal Pathway in Common Carp (Cyprinus Carpio)

doi:10.21203/rs.3.rs-946590/v1

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Oxidative Stimuli Induce Antioxidant Responses by Affecting Nrf2/are Signal Pathway in Common Carp (Cyprinus Carpio)

https://doi.org/10.21203/rs.3.rs-946590/v1

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The Nrf2/ARE signal pathway is a key pathway for antioxidant stress responses and resistance to external stimuli in most organisms. It plays an important role in anti-inflammatory, immune and antioxidant responses. In this paper, the expression of Nrf2/ARE signal pathway related genes in the larval and adult zebrafish were analyzed, including the degree of conservation of the key sites of Nrf2 and Keap1 in carp. And the stress status of the Nrf2/ARE signal pathway was investigated in response to external environment change. The result showed that nrf2 and keap1 binding sites were highly conserved in carp. The expression of nrf2a and keap1a was highest at 48 hpf, keap1b at 8 hpf and varied in adults, suggesting that these genes play different functional roles in different stages of zebrafish. Stimulation by oxidant can induce upregulation of genes related to the Nrf2/ARE signalling pathway, playing an important role in the protection of the organism. This paper not only lays the foundation for studying the role of Nrf2/ARE signaling pathway in the embryonic and adult stages of carp, but also provides a direction for the development of antioxidant drugs and feeds for carp.

Environmental Policy

Carp

Nrf2

Keap1

Antioxidant

Nrf2/ARE

As the main species of freshwater fish in China, carp are reared in large quantities and the demand is increasing. With economic and social development, the problem of water pollution is becoming more and more serious, exposing fish to more external environmental stress (Zheng et al. 2016), which in turn affects the growth and production of carp. Changes in the water environment often cause oxidative stress in the fish organism, and in order to cope with the damage caused by harmful substances such as organic and electrophilic substances on fish growth and development (Jia et al. 2021, Qiu et al. 2019), the antioxidant system plays an important protective role, and the Nrf2/ARE signaling pathway is one of them (Jiang et al. 2014, Takaya et al. 2012).

The Nrf2/ARE signal pathway is widely distributed in organisms and is important to protect against external environmental stress (Han et al. 2019, Sun et al. 2020). The transcription factor nuclear factor E2-related factor 2 (Nrf2) is a key factor which is regulated by Kelch-like ECH-associated protein 1 (Keap1) in the signal pathway response system (Jiang et al. 2015b, Yamamoto et al. 2018). Under normal conditions, Nrf2 is held in the cytoplasm and is inhibited by Keap1. When the organism is stimulated by the external environment or when homeostasis is disrupted or metabolism is disturbed(Wu et al. 2014), Nrf2 enters the nucleus and then Nrf2 binds to Maf (Katsuoka et al. 2005, Katsuoka &Yamamoto 2016), and induces the expression of downstream related antioxidant response elements to regulate the transcriptional activity of antioxidant proteins and phase II detoxification enzyme (Vomhof-Dekrey &Picklo 2012), including HO-1 (heme oxygenase-1), SOD (superoxide dismutase), Nqo1 (NAD(P)H:quinone oxidoreductase 1), GSH (glutathione-S Glutathione), EH (epoxide hydrolase) and others. These enzymes play a defensive and protective role in the body which can promote the metabolism in vitro and detoxification of harmful substances, ensuring that organisms maintain normal growth and development in the complex environment conditions (Wang et al. 2013).

Recent studies have found that the Nrf2/ARE signal pathway not only responded to external environmental stimuli, but also responded to intracellular physiologically active substances (Kobayashi et al. 2009). Kobayashi found that the Nrf2/ARE signal pathway was activated by intrinsic physiologically active substances differently from exogenous environmental stimuli (Hosoya et al. 2005, Komatsu et al. 2007). The Nrf2/ARE signaling pathway has become a focal point and is being studied by an increasing number of researchers both nationally and internationally as more studies have shown that disruption of the Nrf2/ARE signaling pathway is associated with various disease models (Chapple et al. 2012).

The Nrf2/ARE signal pathway has been extensively studied in mice and zebrafish (Li et al. 2008, Yu et al. 2019). In carp, a common freshwater fish, the Nrf2/ARE signal pathway has been little studied. When the water contains organic matter and electrophilic substances, the oxidative stress signal of carp can not only be used as an indicator for water quality monitoring, but also as a guide for the captive breeding of freshwater fishes. In this paper, we analysed the homology and conservation of nrf2 and keap1 between carp and other species. The expression of genes related to the Nrf2/ARE signaling pathway in embryonic development and adult fish was investigated. The stress state of the signal pathway was analyzed in response to environmental conditions. This paper lays the foundation for studying the role of Nrf2/ARE signaling pathway in the embryonic development and adult fish tissues of common carp. It also provides a new direction for the study of the Nrf2/ARE signaling pathway in carp in different water environments.

Materials and Instruments

The carp (Cyprinus carpio) was purchased from Heilongjiang Institute of Fisheries, Chinese Academy of Fisheries Sciences. Artemia was purchased from Xueshan Xiongying, China. H₂O₂ were purchased from Tian in Fuyu Fine Chemical Co., Ltd. Trizol reagent was purchased from Invitrogen, USA. DEPC was purchased from Biotopped, USA. DEM was purchased from Japan Wako Pure Chemical Industries, Ltd. Trizol, PrimeScript RT reagent Kit with gDNA Eraser Kit, SYBR GREEN Real-time PCR Master Mix kit were bought from Takara, Japan. Animal Genomic DNA Quick Extraction Kit was obtained from Beyotime Biotechnology Co., Ltd. PCR primers were synthesized by BGI Co., Ltd.

The automatic light-controlled circulating water system for carp feeding was purchased from Taiwan Chuanfu Science and Technology Development Co., Ltd. The SPX-250BIII biochemical incubator of Tester was used for embryo culture. Sterilization operation used GI54DWS type autoclave. Real-time PCR was ABI 7500, Applied Biosystems. DNA and RNA purity analysis used NanoDrop 2000, Thermo Fisher.

Carp Maintenance

The carp was maintained in the automatic light-controlled circulating water system. The temperature was constant at 28℃ and the carp was fed live Artemia twice daily, with taking the 14 h light/10 h dark photoperiod.

The sample acquiring

In order to obtain the embryos, we used wild type carp to spawn and hatch embryos for 120 hours. Then the embryos were randomly divided into control group, 100 mmol/L H₂O₂ and 100 µmol/L DEM treatment groups, and were treated with corresponding reagents for 6 hours. Finally, randomly collect 20 embryos or larvae at 8 hpf, 12 hpf, 24 hpf, 48 hpf, 96 hpf, 120 hpf and 168 hpf time points from the control group, these were used as samples for testing expression of the Nrf2/ARE signal pathway related genes and antioxidant target genes in different developmental stages. The expression of Nrf2 and Keap1 factors in skin, brain, gill, heart, intestine, liver were also measured.

Quantitative real-time PCR for gene expression

Firstly, total RNA of larvae were isolated using TRIZOL Reagent according to the instructions. And then, RNA reversed transcription into DNA with the PrimeScript RT reagent Kit with gDNA Eraser Kit. Real-time PCR primers were detected from NCBI, as sequences were shown in Table 1. Real-time PCR was performed with SYBR GREEN Real-time PCR Master Mix kit. PCR conditions for 95℃ 5min and 40 cycles of 95℃ (15s), 60℃ (30s) and then 72℃ (30s). All the qPCR primers used showing in Table 1. All the experiments were repeated three times, and β-actin was as the housekeeping gene. The real-time PCR results were calculated with 2^−△△Ct method.

Statistical analysis

Statistical analysis was done with GraphPad Prism 7 software with one-way ANOVA followed by Tukey's multiple comparison test. It is regarded as statistically significant at P < 0.05.

Gene tree and sequence analysis

Nrf2 and Keap1 exist in vertebrates and invertebrates (Kaspar et al. 2009). The amino acid sequences of Nrf2 and Keap1 in carp, zebrafish, mouse and human species were downloaded by NCBI/GenBank and compared with DNAMAN software to analyze the homology between them.

As shown in Fig. 1, the homology of Nrf2a and Keap1a (part the amino acid sequence) for both carp and zebrafish were the highest. The binding sites of DLG and ETGE were highly conservative (Fig. 2A). The binding region of Nrf2 and antioxidant response element^{[错误!未定义书签。]} was also highly conservative in the carp (Fig. 2B).

The expression of Nrf2/ARE signal pathway related genes in different tissue

In order to know what roles did the Nrf2a, Keap1a and Keap1b play in adult carp, we used Real-time PCR to detect the expression of Nrf2a, Keap1a and Keap1b in different tissues, including skin, brain, gill, heart, intestine and liver. As shown in Fig. 3, the expression of Nrf2a, Keap1a and Keap1b was different in different tissues. Nrf2a was the highest in the heart, the lowest was gill. Keap1a was highest in the skin and the lowest in the heart. Keap1b was highest in the skin and the lowest in the brain. However, the gene expression trends in each tissue were similar, Keap1a was a little higher than keap1b, and Nrf2a had the lowest relative expression.

In order to know the expression of Nrf2a, Keap1a and Keap1b in different developmental stages, we detected the expression of the related genes in different developmental stages (from 8 h to 168 h) with β-actin as housekeeping gene (Fig. 4). Nrf2a and Keap1a were highest at 48hpf and lowest at 96 hpf. Keap1b which showed a decreasing trend throughout the growth period. It can be seen that Nrf2a and Keap1a play an important role at 48 hpf, and Keap1b plays an important role at the early embryonic fertilization stage.

The influence of H₂O₂ and DEM on Nrf2/ARE signal pathway

The carp treated with 100 mmol/L H₂O₂ and 100 µmol/L DEM (diethyl malonate) caused an increase in Nrf2a, Keap1a and Keap1b (Fig. 5). It was shown that genes related to the Nrf2/ARE signaling pathway were upregulated in response to stimulation by the oxidants H₂O₂ and DEM. It is concluded that the Nrf2/ARE signaling pathway is activated by oxidative stress.

H₂O₂ increased the expression of the antioxidant target gene Gpx1a (Fig. 6A). The addition of DEM significantly increased the expression of the antioxidant genes Gpx1a, CuZnSOD and MnSOD (Fig. 6A-C). It suggests that external oxidative stimuli can cause up-regulation of antioxidant target gene expression, thus reducing oxidative damage and providing protection to carp.

Nrf2 and Keap1 factors are great important in most organisms. In order to understand how does Nrf2 and Keap1 factors work, this paper constructs a phylogenetic tree (Chen et al. 2016) of Nrf2 and Keap1 factors amino acid sequences of carp, zebrafish, mice and human. We find that Nrf2 is highly conserved in organisms, especially in the ETGE and DLG site. This illustrates the importance of the Nrf2/ARE signaling pathway for the study of important functional sites in the structural domain of Nrf2 in carp.

To understand how Nrf2a, Keap1a and Keap1b work, we use Real-time PCR to detect the expression of them. We find that Nrf2a, Keap1a and Keap1b play different roles in different stages and tissues. It can be seen that Nrf2a and Keap1a play an important role in the embryonic stage and Keap1b played an important role in the early embryo hatching stage (Hahn et al. 2015). We performed preliminary studies on genes related to the Nrf2/ARE signaling pathway at different time points to better understand the alterations of Nrf2 in response to external environmental stimuli from a molecular perspective (Wang et al. 2015, Zhao et al. 2014). This has important implications for the study of the molecular mechanisms of the Nrf2/ARE signaling pathway in different oxidative stress responses, and we can also better exploit the Nrf2/ARE signaling pathway to address related problems and diseases.

In the process of daily nutrient metabolism in fish, due to changes in external environmental conditions, the metabolism of free radicals generated in the body becomes abnormal, causing lipid peroxidation in fish, resulting in cellular tissue damage (Nguyen et al. 2005). Under normal conditions, the oxidative and antioxidant systems of the carp body maintain a dynamic balance, and a complete antioxidant system can maintain the metabolic balance of free radicals to ensure the normal growth of carp. Among them, SOD (Superoxide dismutase) (McMahon et al. 2004) and GSH-Px (glutathione peroxidase) (An et al. 2008) play an important role in antioxidation of the organism and are one of the main indicators to evaluate the antioxidant capacity of the organism (Jiang et al. 2015a). Our results showed that the antioxidant genes Gpx1a, CuZnSOD and MnSOD were significantly increased in carp under the induction of oxidants. The reason for this is that the Nrf2/ARE signalling pathway was activated by the induction of H₂O₂ and DEM, as indicated by the alteration of Nrf2a, Keap1a and Keap1b. This leads to the expression of Nrf2/ARE downstream antioxidant genes such as gpx1a, CuZnSOD and MnSOD. It suggests that the Nrf2/ARE signalling pathway can against oxidative damage and play a protective role when carp are exposed to external oxidative stimuli (Wang et al. 2016). Our findings further explain the mechanism by enhancing the antioxidant capacity of carp with drugs or feeds. It provides new ideas for the development of prevention or treatment of carp diseases.

Ethics declarations

Ethics approval--Not applicable

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Conflict of interest/competing interests--The authors declare no competing interests.

Data availability

All data generated or analysed during this study are included in this published article.

Acknowledgements

This work was supported by the National Key Technologies R&D Program of China under Grant Number: 2019YFA0705202; the National Natural Science Foundation of China (31701296 and 11474076).

Authors' contributions

All authors contributed to the study conception and design. Conceptualization: [Li Li]; Methodology: [Mingyue He]; Formal analysis and investigation: [Lidong Yu]; Writing - original draft preparation: [Yi Liu]; Writing - review and editing: [Xueting Li]; Resources: [Lei Cheng]; Supervision: [Li Li], [Ning Gu], [Bingsheng Li]. All authors read and approved the final manuscript.

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Due to technical limitations, table 1 is only available as a download in the Supplemental Files section.

Table1.xlsx

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Version 1

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You are reading this latest preprint version

Oxidative Stimuli Induce Antioxidant Responses by Affecting Nrf2/are Signal Pathway in Common Carp (Cyprinus Carpio)

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials and Instruments

Carp Maintenance

The sample acquiring

Quantitative real-time PCR for gene expression

Statistical analysis

Results

Gene tree and sequence analysis

The expression of Nrf2/ARE signal pathway related genes in different tissue

The influence of H₂O₂ and DEM on Nrf2/ARE signal pathway

Discussion

Declarations

Ethics declarations

Data availability

References

Tables

Supplementary Files

Status:

Version 1

Oxidative Stimuli Induce Antioxidant Responses by Affecting Nrf2/are Signal Pathway in Common Carp (Cyprinus Carpio)

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Materials and Instruments

Carp Maintenance

The sample acquiring

Quantitative real-time PCR for gene expression

Statistical analysis

Results

Gene tree and sequence analysis

The expression of Nrf2/ARE signal pathway related genes in different tissue

The influence of H2O2 and DEM on Nrf2/ARE signal pathway

Discussion

Declarations

Ethics declarations

Data availability

References

Tables

Supplementary Files

Status:

Version 1

The influence of H₂O₂ and DEM on Nrf2/ARE signal pathway