Elsevier

Aquaculture

Volume 544, 15 November 2021, 737040
Aquaculture

Pomelo polysaccharide extract inhibits oxidative stress, inflammation, and mitochondrial apoptosis of Epinephelus coioides

https://doi.org/10.1016/j.aquaculture.2021.737040Get rights and content

Highlights

  • The polysaccharide (YZW-A) was extracted wasted pomelo fruitlet.

  • YZW-A showed great potential as a feed additive for fisheries.

  • YZW-A mitigated oxidative stress induced by H2O2 in vivo and in vitro.

  • YZW-A inhibited the NF-κB signaling pathway to modulate inflammation.

  • YZW-A ameliorated the H2O2-induced extrinsic and intrinsic apoptosis.

Abstract

Although the production of pomelo, a citrus fruit, has recently increased considerably in China, the therapeutic potential of the fruitlets on aquatic animals has not been widely studied. Thus, in this study, we aimed to evaluate the protective effects of the crude polysaccharide extracted and purified from pomelo fruitlet (YZW-A), on oxidative stress, inflammation, and mitochondrial dysfunction-mediated apoptosis. For this, we added YZW-A (0, 100, 200 and 400 μg/mL) to Epinephelus coioides spleen (GS) cells before incubation with 0.8 mM of H2O2. Morphological indices showed significant deterioration when exposed to H2O2 (P < 0.05), whereas this was did not observed in the cells exposed to YZW-A. Administration of YZW-A (100 and 200 μg/mL) substantially mitigated the H2O2-induced oxidative stress, as shown by the elevated activities of the mitochondrial respiratory complexes and antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) (P < 0.05). NF-κB derived inflammation was also significantly attenuated by the diminished gene expression of the NF-κB regulated proinflammatory cytokines (TNF-α, IL-β and IL-6) (P < 0.05). In addition, apoptosis-related indicators (Bax, Bcl-2, and caspase-3, −8, and − 9) and the cell apoptosis rate showed that mitochondrial dysfunction-mediated apoptosis was alleviated due to the decreased oxidative stress. Pretreatment with the NF-κB inhibitor, BAY11–7082 (20 μM), dramatically reduced the expression of the downstream genes of NF-κB, including the proinflammatory genes, IL-β and IL-6 (P < 0.05), and the proapoptotic genes, Bax, caspase 3, and caspase 9 (P < 0.05), which confirmed that the NF-κB signaling pathway is involved in the mechanisms of inflammation and apoptosis. In support of the results in vitro study, we further added YZW-A at the doses of 0, 200, 400, 800 and 1600 mg/kg in the diet for 56 days prior to the treatment of H2O2 or saline. The results demonstrated that administration of YZW-A significantly ameliorated H2O2-induced liver injury as evident by the decline of Serum albumin (ALB), alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), and cholinesterase (CHE) content in the groupers. Furthermore, the changes on the activities of the antioxidative enzymes and the mRNA expression of the apoptosis-related indicators and the proinflammatory cytokines corresponded well with that of in GS cells. In addition, YZW-A treatment repressed the enhancement of enzymic activities of the proinflammatory cytokines induced by H2O2. In summary, these findings showed the therapeutic potential of YZW-A on H2O2-induced oxidative stress, and further mitigated the NF-κB regulated inflammation and apoptosis.

Introduction

A disequilibrium between reactive oxygen species (ROS) generation and consumption causes oxidative damage in aquatic animals, and this phenomenon has attracted increasing attention worldwide. This impaired equilibrium can be affected by multiple factors, such as metal ions (Eyckmans et al., 2011), temperature (Lushchak and Bagnyukova, 2007; Madeira et al., 2013; Vinagre et al., 2012), salinity (Kim et al., 2017), and oil and related pollutants (Lushchak, 2011). Changes in the levels of these factors in aquatic life can be accompanied by excessive ROS production that induces metabolic disorder, mitochondrial dysfunction, and DNA damage in biological systems (Kim et al., 2020). This may be a mechanism causing toxicity in aquatic lives (Livingstone, 2003), turning them into unmarketable products (in the form of emaciation and low reproduction efficacy), thus causing potential economic losses in the fishery industry.

There are numerous sites within cells that generate ROS, such as the xanthine oxidase and abundant cytochrome P450 enzymes, as well as the mitochondrial respiratory complexes (Su et al., 2011). Among them, the respiratory complexes, especially complex III, are highly involved in the increased formation and release of ROS in mitochondria (Suomalainen and Kaukonen, 2001; Morgan and Liu, 2011). The toxic buildup of ROS subsequently inhibits the antioxidation defenses, by using up the enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-Px), as well as antioxidant-associated enzymes (Davies, 2001).

An increasing number of researches have reported that ROS are involved in modulating various metabolic cytokines, such as the nuclear factor-kappa B (NF-κB), a common intracellular messenger that instigates various diseases (Schreck et al., 1991; Xie et al., 2020). It has been demonstrated that the common intracellular messenger and ROS, H2O2, causes inflammation by causing the release of NF-κB and enhancing the transcriptional level of cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF- α) (Kalousová et al., 2002; Yuk and Jo, 2011). Some studies have also reported that NF-κB activation induces apoptosis by upregulating the activity of Bcl-XS and Bax (Bcl-2 family protein) which are associated with pro-apoptotic mechanisms (Shou et al., 2002; Xu et al., 2019).

Recent literature has delineated the interaction between ROS and apoptosis (Wang et al., 2020a; Simon et al., 2000; Giorgio et al., 2005). The increased formation and release of ROS alters the expression of Bcl-2 and Bax, decreases the mitochondrial membrane potential (ΔΨm) and the content of ATP, induces the translocation of mitochondrial intermembrane space cytokines, such as cytochrome c into the cytoplasm, thus triggering apoptosis via downstream apoptotic executors (Oh and Lim, 2006; Petit et al., 1995; Azzolin et al., 2010). Taken together, cell inflammation and apoptosis are instrumental in the process of oxidative stress, via the pathways of NF-κB and ROS-mediated mitochondrial apoptosis.

The plant size of pomelo, a citrus fruit, has recently increased tremendously in China. It has been found to have antioxidant (Lan-Phi and Vy, 2015), antimicrobial (Aichayawanich and Ngaouwthong, 2012) and anti-inflammatory (Zhao et al., 2019) properties. Studies have reported that naringin (from citrus fruits) can mitigate the symptoms of oxidative stress, inflammation, and mitochondrial apoptosis in vitro, via NF-κB-mediated signaling cascades, that are associated with type 2 diabetes mellitus-induced steatohepatitis (Syed et al., 2020). During pomelo cultivation, pomelo fruitlet is commonly discarded in the orchard. It is unmarketable because of its unappealing pulp quality and causes significant economic losses to the farmers. Contemporary research has indicated that pomelo fruitlets possess many pharmacological functions. Wang et al. (2019) extracted flavonoids from young grapefruit using ultrasonic microwave assisted extraction, and found that all three flavone components inhibited 2,20-azobis (2-amidinopropane) dihydrochloride free radicals and reduced red blood cell reactive oxygen levels, thereby inhibiting hemolysis. Another study conducted by Lin and Xu You-rui (2008), demonstrated that the essential oil from pomelo fruitlets contains 43 different chemicals and certainly has medicinal value. Nevertheless, the literature concerning the therapeutic potential of pomelo fruitlets on aquatic animals is lack. Therefore, this study firstly explored the protective effect of the pomelo-fruitlet-derived crude polysaccharide (YZW-A) on the grouper, Epinephelus coioides. A chain of experiments was performed involving treatments of isolated spleen cells (GS) and liver tissue with YZW-A and H2O2 alone, or in different combinations, to further decipher the possible mechanism of ROS and NF-κB on inflammation and mitochondrial apoptosis.

Section snippets

Isolation and purification of YZW-A

The crude polysaccharides of pomelo fruitlets were extracted from the powder of dried pomelo fruitlets using ultrasonic-assisted extraction method, and then separated successively on DEAE-52 cellulose column. Subsequently, the main fraction of this separation, YZW was distilled and obtained after the elution of NaCl solution. We finally obtained YZW-A by dialyzing YZW with distilled water through DEAE-Sepharose column for further experiment.

Cell culture and treatment

GS cells were propagated in Leibovitz's L-15 medium

Selection of the appropriate concentrations of YZW-A

To establish the appropriate concentrations of YZW-A in the GS cells, we incubated the cells with YZW-A at concentrations of 0, 50, 100, 200, 400, and 800 μg/mL for 24 h. The viability results demonstrated a dose and time dependent trend. As exhibited in Fig. 1A(a), the viabilities for the GS cells treated with 50, 100, 200, 400, and 800 μg/mL YZW-A were 99.74 ± 1.12%, 108.91 ± 0.48%, 111.13 ± 0.48%, 106.39 ± 0.48%, and 95.01 ± 1.12%, respectively. The cell viabilities for the YZW-A 100, 200,

Discussion

H2O2, is easy to across through membranes, can be converted to the highly reactive hydroxyl radical which finally lead more generation of unpaired electrons and radical species by extracting electrons and protons form from macromolecules, such as nucleic acids, proteins, lipids and carbohydrates (Birnie-gauvin et al., 2017). Oxidative damage inflicted by H2O2 is a well-established chemical model to observe the mechanisms of oxidative stress in various cells and species (Kalpana et al., 2009).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Guangdong Provincial Natural Science Foundation Project [2019A1515011283].

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