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Bioactive Constituents, Metabolites, and Functions
Fish skin gelatin hydrolysate produced by ginger powder induces glutathione synthesis to prevent hydrogen peroxideinduced intestinal oxidative stress via Pept1-p62-Nrf2 cascade Liufeng Zheng, Hongkui Wei, Huichao Yu, Qian Xing, Yi Zou, Yuanfei Zhou, and Jiang Peng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02840 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 19, 2018
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Journal of Agricultural and Food Chemistry
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Fish skin gelatin hydrolysate produced by ginger powder induces glutathione
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synthesis to prevent hydrogen peroxide-induced intestinal oxidative stress via
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Pept1-p62-Nrf2 cascade
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Liufeng Zheng,†,‡ Hongkui Wei,†,§ Huichao Yu,† Qian Xing,‡ Yi Zou,† Yuanfei Zhou,†,§ and
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Jian Peng*,†,§
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†Department
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Agricultural University, Wuhan 430070, P. R. China
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‡State
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China
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§The
of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong
Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, P. R.
Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, P. R. China
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ABSTRACT
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Gelatin hydrolysate was reported to contain large amounts of biologically active peptides with
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excellent antioxidant properties. However, its inducement of antioxidant response within cells and
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the underlying molecular mechanism are far from clear. Here, gelatin from Nile tilapia skin was
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hydrolyzed by ginger protease to produce antioxidant hydrolysate, and three fish skin gelatin
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hydrolysate fractions (FSGHFs) were obtained by ultrafiltration. Fractionation of the hydrolysate
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increased the free radical scavenging capacity of the FSGHFs, particularly FSGHF3, which showed
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the lowest molecular weight (below 1000 Da). Furthermore, FSGHF3 treatment prior to H2O2
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exposure increased cell viability and membrane integrity in IPEC-J2 cells. H2O2-induced ROS
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production and epithelial barrier damage were suppressed by FSGHF3 pretreatment. FSGHF3
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stimulated the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), along with
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the increases in the mRNA and protein expression of catalytic and modulatory subunits of
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γ-glutamylcysteine ligase, as well as in the level of glutathione. Silencing of Nrf2 or p62 (an
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upstream regulator of Nrf2) suppressed FSGHF3-induced Nrf2 activation and its protection against
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H2O2-induced oxidative stress. Moreover, oligopeptides in FSGHF3 may mediate the cytoprotective
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effect against oxidative stress, which was confirmed by the result that FSGHF3 failed to inhibit the
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ROS production in H2O2-exposed cells with the knockdown of Pept1 (an oligopeptide transporter).
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Therefore, FSGHF3 can induce glutathione synthesis and prevent oxidative stress through
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Pept1-p62-Nrf2 cascade, and thus may be a functional food for gastrointestinal dysfunction.
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KEYWORDS: fish skin gelatin hydrolysate, small intestinal epithelial cells, Pept1-p62-Nrf2
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cascade, oxidative stress, glutathione synthesis 2
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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Reactive oxygen species (ROS) adversely affect proteins, lipids and DNA,1 and its overproduction
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can cause oxidative stress, promoting the development of numerous diseases such as cardiovascular
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disorders, obesity, and diabetes.2,3 Notably, the small intestine, an important organ for mucosal
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barrier, nutrition absorption and innate immunity,4 is a key source of ROS.5 Various gastrointestinal
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mucosal diseases and dysfunction were attributed to the excess production of ROS.6 Thus, the
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inhibition of oxidative stress is critical to prevent gastrointestinal diseases.
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Normally produced intracellular ROS is scavenged by endogenous antioxidants and antioxidant
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enzymes that are abundant in intestinal epithelial cells.7,8 Accordingly, the approaches to directly
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scavenge ROS and activate antioxidant enzymes may be a viable strategy for protecting intestine
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from oxidative injury. In this regard, induction of glutathione (GSH) synthesis is considered as the
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main mechanism for preventing oxidative stress.9 GSH is the most abundant endogenous antioxidant
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within mammalian cells, and its synthesis is mainly determined by the rate-limiting enzyme
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γ-glutamylcysteine ligase (GLC), which is composed of a catalytic (GCLC) and a modifier (GCLM)
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subunit.10 Therefore, induction of GLC expression may play a vital role in protecting intestine from
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oxidative stress.
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Numerous studies have demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2) is a
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redox-sensitive master regulatory transcription factor that mediates expression of a variety of
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antioxidant enzymes regulated by antioxidant response element (ARE).11 Generally, Nrf2 is located
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in cytoplasm and remains unactivated by binding with its inhibitor kelch-like ECH-associated
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protein-1 (Keap1), which facilitates the degradation of Nrf2. Upon stimulation, Nrf2 is dissociated 3
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from Keap1 and translocated to nucleus, resulting in the transcription of antioxidant enzymes.12
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Interestingly, a recent report demonstrated that the transcriptional activation of Nrf2-mediated
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antioxidant genes depends on p62, which is also called sequestosome 1.13 Meanwhile, p62 serves as
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a stress/nutrient sensor to regulate diverse cellular functions through its multi-domain structure.13,14
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Therefore, it is particularly important to find strategies to activate p62-Keap1-Nrf2 cascade and
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subsequently increase intracellular level of GSH to prevent oxidative damage of the intestine.
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It has been well demonstrated that gelatin hydrolysate (GH) derived from fish skin serves as a
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potential source of bioactive peptides with excellent antioxidative activities in vitro (such as
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radical-scavenging ability).15 Notably, research in humans and animals has shown that only
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oligopeptides can be absorbed in intact forms via oligopeptide transporter 1 (Pept1), and thereby
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effectively exerting their bioactivities in vivo.16-18 Furthermore, GH-derived oligopeptides were
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recently identified, and it was found high concentrations of these oligopeptides in blood after
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ingestion of GH.17 Therefore, increasing the production of oligopeptides derived from GH could be
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an effective way to prevent oxidative stress and cell damage.
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More recently, ginger protease was used to hydrolyze gelatin for producing a novel hydrolysate
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with a large amount of glycine-containing oligopeptides.17 However, little is known about the
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antioxidant activities in vitro of ginger-degraded GH and its inducement of antioxidant response
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within cells. Particularly, the precise molecular mechanism and the signal cascade by which
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ginger-degraded GH regulates cellular antioxidant capacity have not been reported so far.
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Therefore, in this study, fish skin gelatin hydrolysate (FSGH) was produced using ginger protease,
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and three hydrolysate fractions, FSGHF1 (>10 kDa), FSGHF2 (1–10 kDa) and FSGHF3 (
