**3. Discussion**

As one of the main target organs of Cd, acute hepatic injury was observed in Cdexposed mice. Fortunately, the OPs treatment clearly ameliorated the Cd-induced hepatic injury in this study. In particular, oxidative damage, inflammation, and cell apoptosis, as crucial triggers and contributors to the development of Cd-induced hepatic injury [30,34,35,37], were improved after OPs application in Cd-exposed mice.

Extensive literature indicates that the health benefits of protein hydrolysates may be partly attributed to their rich in free amino acids and peptides [38,39]. Extracts rich in free amino acids can be used in pharmaceutical applications [40]. A recent study found that free amino acids were related to the antioxidant property of protein hydrolysates of mackerel [38]. The protein hydrolysates with higher contents of free amino acids exhibited better antioxidant properties [41] and metal-chelating ability [42]. In the present study, a high level of free amino acids (15.8%) was detected in OPs. According to a previous report, the royal jelly hydrolysates with 8.389% of free amino acids had a stronger antioxidant activity than those of royal jelly with 0.572% of free amino acids [43]. A similar study also found the anchovy sprat hydrolysates with higher contents of free amino acids (16.28–27.53%) exhibited stronger ferrous-chelating activity and radical-scavenging activity compared to those with lower contents of free amino acids (9.05%) [44]. These data indicated that OPs

were rich in free amino acids, which may contribute to the potential health benefits of OPs against Cd toxicity.

Serum ALT and AST are leaked from damaged hepatocytes [45,46]. Cd intoxication led to a significant elevation in the levels of ALT and AST [47]. In the present study, significant improvements were observed in the hepatic injury and dysfunction biomarkers (serum AST, ALT, ALP, and LDH) in Cd-exposed mice after the OPs treatment. Compared to the Cd group, OPs significantly decreased hepatic dysfunction biomarkers in a dosedependent manner. This result is in line with an earlier report, in which oyster protein hydrolysate could reduce hepatic dysfunction biomarkers (serum AST, ALT, and ALP) and inflammatory response in alcoholic liver disease mice [48]. Likewise, Shi, Sun [49] reported that *ganoderma lucidum* peptides have an alleviative effect on D-GalN-induced hepatocellular injury via reversing AST and ALT levels in the liver. Moreover, Mumtaz, Ali [50] found that elevated level of LDH, AST, and ALT in the Cd-exposed batch was improved by ascorbic acid. Early evidence indicates that hepatic injury and cirrhosis usually lead to metabolic disturbances of amino acids [51]. The bioactive properties of protein hydrolysates mainly depend on free amino acids and peptides [38,39,52]. The present study showed that OPs are rich in hydrophobic free amino acids (i.e., Pro) and proline-rich peptides. Among these amino acids, Pro plays a beneficial role in plants under changing environments, including Cd stress [53]. Exogenous Pro could increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells [22]. Pro has shown tissue-protective effects against D-galactosamine-induced hepatic injury [54]. Dietary Pro could effectively decrease AST and ALT levels of shrimp under NH3 stress [55]. The derivatives of Pro, N-acetyl-seryl-aspartyl-lysyl-proline, were found to attenuate bile duct ligation-induced liver fibrosis by restoring hepatic dysfunction (serum AST and ALT) in mice [56]. Pro and proline-rich proteins are often implicated in stress tolerance in plants [57–59]. Salivary proline-rich proteins possess good antioxidant properties [60]. Hypothalamic proline-rich polypeptides were found to protect brain neurons in aluminum neurotoxicosis [61]. These data support the idea that OPs could ameliorate hepatic injury and improve hepatic dysfunction in Cd-exposed mice.

Oxidative stress is often implicated in the induction of multi-organ injury under Cd exposure [62]. Lipid peroxidation is a major consequence of Cd-induced oxidative stress [63]. The consequences of the peroxidative of membrane lipids have been considered in relation to the tissue aspects of liver injury, and these peroxidative reactions play a critical role in the pathogenesis of acute liver necrosis [64]. According to a previous report, the liver, kidneys, and heart were most susceptible to Cd-induced oxidative stress in mice [65]. Some amino acid derivatives, such as N-Acetylcysteine, showed ameliorative effects on cisplatininduced multiple organ toxicity in rats [66–68]. Betulinic acid was found to alleviate the kidney and liver damage induced by Cd [69]. In this study, Cd exposure induced serious hepatic toxicity and oxidative stress, which were significantly improved after the OPs supplement. These data suggest that amelioration of hepatic oxidative injury may be the key to the treatment of Cd toxicity by OPs in mice.

Oxidative stress plays a crucial role in Cd-induced hepatic toxicity [70]. The development of liver injury usually involves the lipid peroxidation of hepatic cell membranes in Cd-exposed mice [71]. According to a recent report, Cd-induced hepatic injury is tightly coupled with enhanced lipid peroxidation (MDA) and the significant depletion of antioxidants (CAT and SOD) [72]. In this study, the OPs supplement clearly reduced the formation of MDA and significantly restored the activity of antioxidant enzymes (SOD, CAT, and GPH-Px) in the liver of the Cd-exposed mice. OPs displayed a strong antioxidant activity, which might also be attributed to their abundance of hydrophobic amino acids. Commonly, protein hydrolysates with higher content of hydrophobic amino acids possess better antioxidant properties due to their more effective interaction with lipid-soluble free radicals and the prevention of lipid peroxidation [17,73–75]. Thus, this evidence clearly indicated that OPs possess good antioxidant properties to delay hepatic oxidative injury

via retaining hepatic antioxidant enzymes and preventing MDA production in Cd-exposed mice.

Hepatic histopathological damage in Cd-exposed mice is characterized by apoptotic bodies, hemorrhage, lymphocyte accumulation, and inflammatory cell infiltration in liver tissue. Increasing evidence demonstrates that hepatic injury and fibrosis are accompanied by the elevation of the inflammatory response [76]. As well known, TNF-α and IL-6 are two key inflammatory mediators of tissue injury-induced inflammatory response [77]. IL-1β and IL-6 are two stimulators of the hepatic synthesis of acute-phase proteins in the inflammatory response to stress [25,26]. Cd exposure will trigger an acute inflammatory response in mice [78]. The present study showed that hepatic apoptotic cells in Cd-exposed mice were significantly minimized, and histopathological appearance was obviously improved after OPs treatment. In addition, the Cd-triggered inflammatory responses (IL-1β, IL-6, and TNF-α) were significantly inhibited, as expected. This result is consistent with the findings in an earlier report, in which peptides from oyster soft tissue hydrolysates selectively repressed TNF-α, IL-1β, and IL-6 [79]. To go even further, we found that as important activators and regulators of inflammatory responses, MIP-2 [29], COX-2 [28], NF-κB, and the ERK signal pathway [80], were significantly stimulated in Cd-exposed mice. The results are in agreement with a previous study, in which Cd activated the ERK signal pathway, then subsequently up-regulated TNF-α, COX-2, IL-1β, IL-6, and NF-κB in swine [31]. Likewise, Huang, Xia [30] reported that Cd exposure led to an increase in MIP-2 and COX-2. Actually, ROS production could activate MAPK signaling to induce inflammation and skin aging by promoting the phosphorylation of ERK [32]. Peng, Chen [81] also found that the upregulated ERK phosphorylation in ultraviolet B-exposed mice was significantly inhibited by the application of oyster protein hydrolysates. A recent study found that seahorse protein hydrolysates could significantly inhibit p-ERK levels in ethanol-exposed cells. [82]. In this study, Western blotting assays showed that OPs significantly decreased the levels of p-ERK and NF-κB proteins, as well as the MIP-2 and COX-2 in Cd-exposed mice. Therefore, the ameliorative effect of OPs in Cd-caused liver injury may be related to its anti-inflammation properties via suppressing the production of inflammatory mediators and inhibiting the inflammatory response associated with NF-κB and the ERK signal pathway.

In addition to inflammatory responses, hepatic injury is also accompanied by the development of apoptosis in Cd-exposed mice. Reducing Cd-induced apoptosis is also considered to be one of the feasible ways to prevent Cd-induced hepatic injury [69]. In the process, the NF-κB inflammation pathway indirectly activated the apoptosis-related factors Bcl-2, Bax, and Caspase-3 [31]. The present study revealed that the OPs supplement strongly up-regulated the expression of the anti-apoptotic factor Bcl-2 while significantly down-regulated the expression of the pro-apoptotic factors (caspase-3 and Bax), eventually restoring the balance of pro-/anti-apoptotic proteins in Cd-exposed mice. Moreover, the PI3K/AKT pathway is an important signaling pathway associated with apoptosis [83]. Actually, Cd selectively induces MIP-2 and COX-2 through the activation of the PI3K/AKT [30]. A previous study showed that curcumin alleviated lipopolysaccharideinduced hepatic injury and apoptosis via inhibiting the PI3K/AKT and NF-κB pathways [84]. MiR-130a alleviated neuronal apoptosis and changes in the expression of Bcl-2/Bax and caspase-3 in cerebral infarction rats through the PI3K/AKT signaling pathway [85]. In the present study, we found that the OPs supplement significantly inhibited the expression of PI3K and p-AKT proteins. These results are in agreement with an earlier report, in which Selenomethionine ameliorated Cd-induced hepatocyte apoptosis by suppressing the PI3K/AKT pathway [86]. Therefore, we may conclude that OPs possess the ability to ameliorate hepatocyte apoptosis, possibly by restoring the balance of pro-/anti-apoptotic proteins via suppressing the PI3K/ AKT pathway in Cd-exposed mice.

In conclusion, from our study, we found that OPs could effectively ameliorate Cdinduced hepatic injury through their antioxidative and anti-inflammatory properties. In addition, OPs displayed an important role in restoring the balance between pro-apoptotic and anti-apoptotic proteins by suppressing the activation of the PI3K/AKT pathway, contributing to the development of hepatocyte apoptosis in Cd-exposed mice (Figure 8). These results may provide a new insight for a better understanding of the ameliorative function of OPs to Cd toxicity and provide a theoretical basis for the use of OPs to prevent or treat Cd-induced hepatic injury.

**Figure 8.** Schematic representation of OPs improved mechanisms of cadmium-induced oxidative stress, inflammation, and apoptosis in mice.
