**1. Introduction**

Infertility is a universal and serious human health problem, reportedly affecting 8–12% of couples of childbearing age worldwide [1]. Male-related sterility accounts for around 50% of all infertility cases, with approximately 1 in 20 men of reproductive age suffering from infertility [2]. Infertility could be caused by a number of factors, including reproductive system injuries, endocrine disruption, environmental pollution, modern lifestyles, and drug side effects [3–7]. Spermatogenesis occurs in the seminiferous tubules and is strictly dependent on the structure of the testis. Therefore, the integrity of testicular morphological structure and the maintenance of physiological function play important roles in spermatogenesis [8]. Abnormal testicular tissue structure, changes in reproductive hormones, oxidative damage, and cell apoptosis may be the mechanisms of male reproductive dysfunction [9].

**Citation:** Zhang, X.; Peng, Z.; Zheng, H.; Zhang, C.; Lin, H.; Qin, X. The Potential Protective Effect and Possible Mechanism of Peptides from Oyster (*Crassostrea hongkongensis*) Hydrolysate on Triptolide-Induced Testis Injury in Male Mice. *Mar. Drugs* **2021**, *19*, 566. https://doi.org/ 10.3390/md19100566

Academic Editors: Donatella Degl'Innocenti, Marzia Vasarri and Marialuisa Menna

Received: 28 August 2021 Accepted: 6 October 2021 Published: 9 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Triptolide (TP, C20H24O6), as the main active ingredient of the pharmacological and toxic effects of *Tripterygium wilfordii multiglycoside* (GTW), has long been used in the treatment of inflammatory and immune diseases [10–12]. Among the toxic and side effects of TP, its reproductive toxicity leads to the highest incidence of reproductive dysfunction. Excessive triptolide intake interferes with testicular energy metabolism and normal reproductive function, resulting in reduced sperm quality and testicular atrophy, thereby leading to male reproductive dysfunction. Therefore, TP was used as a model drug in the animal models of male sterility to explore the pathogenesis of male sterility and the improvement effect of related drugs on this disease [13].

Oysters are the largest farmed shellfish in the world, rich in protein, glycogen, taurine, and trace elements [14]. In addition to rich nutrition, oyster meat can also affect a variety of physiological functions and has certain health care effects, thus it has been included in the medicine food homology list published by China's Ministry of Health [15]. As an aquatic product with high protein content, oysters are a good source of polypeptides. Several studies have focused on the health benefits of peptides from oyster hydrolysate (OPs) and have demonstrated their antioxidant [16], immunity-improving [17], antimicrobial [18], antitumor [19], anti-fatigue [20], and liver-protecting [21] properties. In previous studies, Li et al. reported that oyster polysaccharide administration could improve sperm quality and protect reproductive damage in cyclophosphamide-induced male mice [22]. However, the protective effects of OPs on reproductive damage have not been systematically reported. In addition, the study found that OPs may protect the ovary from D-galactose-induced female reproductive dysfunction by reducing oxidative stress, thereby preventing ovarian cell apoptosis [23]. Therefore, OPs have the potential to protect reproductive function against drug damage and deserve further study.

This study aimed to investigate the protective effect of peptides from the oyster hydrolysates on sperm parameters, testicular histopathology, sex hormone levels, activities of testicular marker enzymes, antioxidant level, and cell apoptosis in TP-induced ICR male mice. Furthermore, the activation of OPs on the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway and inhibition of OPs on the activation of the c-Jun N-terminal kinase (JNK) phosphorylation and Bcl-2/Bax-mediated apoptosis pathway was examined. Eventually, the correlation among the sperm analysis, morphological, biochemical indicators, cell apoptosis, and oxidative stress signaling pathway in the testis was established via the combination of experimental determination and statistical analysis. These findings not only deepen the understanding of OPs against TP-induced testis injury but also provide an experimental basis for the development of OPs as a functional agent.

#### **2. Results**

## *2.1. Molecular Weight and Main Peptide Sequences of OPs*

The function and biological activity of peptides depends on their amino acid composition, sequence, and molecular mass. In this study, 89 peptides with molecular weight ranging from 662.41 to 1590.81 Da were identified by LC-MS/MS, and the peaks of OPs were mainly in the range of 300–800 m/z. As shown in Table 1, the scores for identifying peptide sequences were obtained, and 15 peptide sequences with higher scores were listed.


**Table 1.** Main peptide sequences of OPs.

#### *2.2. Amino Acid Composition of OPs*

Amino acid composition and content of OPs are presented in Table 2. The contents of total amino acids (TAA) in OPs were 54.75 g/100 g. The essential amino acid (EAA) cannot be synthesized by the body itself and must be obtained through the diet [24]. EAA content of OPs is 22.19 g per 100 g, accounted for 40.53% of TAA, which was highly sufficient to meet the minimum dietary intake of 35% recommended by the World Health Organization [25].



<sup>a</sup> TAA: Total amino acids. <sup>b</sup> Essential amino acid (EAA): Thr, Val, Met, Ile, Leu, Phe, Lys. <sup>c</sup> Hydrophobic amino acids (HAA): Ala, Leu, Ile, Met, Phe, Pro, Tyr, and Val. <sup>d</sup> Branched-chain amino acids (BCAA) = Leu, Ile, and Val. <sup>e</sup> ND: not detected.

The composition, concentration, and sequence of amino acids have a great influence on the biological activity of proteolytic compounds [20]. The amino acid composition of spermatozoa was significantly changed by amino acid supplementation in the internal environment, which affected sperm motility [26]. Of the 17 amino acids contained in OPs, glutamic acid (8.41 g/100 g), aspartic acid (5.56 g/100 g), arginine (4.23 g/100 g), lysine (4.78 g/100 g), leucine (4.56 g/100 g), and valine (3.34 g/100 g) accounted for a higher proportion. Among them, a diet supplemented with amino acids (mainly lysine, valine, and threonine) improved sperm quality, changed amino acid composition in seminal plasma, and improved sperm motility [27]. Lysine, aspartic acid, and glutamic acid have the ability to chelate metal ions because of the amino or carboxyl groups in their side chains. Hydrophobic amino acids (HAA) accounted for 31.58% of TAA, and its high amounts in OPs offer properties that are able to promote lipid interactions, which enhance entry of the peptides into target organs via hydrophobic associations [28]. BCAAs accounted for 19.95% of TAA, which plays a strong role in maintaining energy supply, and Bahadorani et al. found that appropriate supplementation of BCAAs may have synergistic effects on sperm function and testosterone secretion [24].
