Protective Effect of Lycium ruthenicum Polyphenols on Oxidative Stress against Acrylamide Induced Liver Injury in Rats

Acrylamide (ACR) is formed during tobacco and carbohydrate-rich food heating and is widely applied in many industries, with a range of toxic effects. The antioxidant properties of Lycium ruthenicum polyphenols (LRP) have been established before. This study aimed to research the protective effect of LRP against ACR-induced liver injury in SD rats. Rats were divided into six groups: Control, ACR (40 mg/kg/day, i.g.), LRP (50, 100, and 200 mg/kg/day, i.g.) plus ACR, and LRP groups. After 19 days, we evaluated oxidative status and mitochondrial functions in the rat’s liver. The results showed that glutathione (GSH) and superoxide dismutase (SOD) levels increased after LRP pretreatment. In contrast, each intervention group reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels compared to the ACR group. Meanwhile, alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver mitochondrial ATPase activity, mRNA expression of mitochondrial complex I, III, and expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and its downstream proteins were all increased. This study suggested that LRP could reduce ACR-induced liver injury through potent antioxidant activity. LRP is recommended as oxidative stress reliever against hepatotoxicity.


Introduction
Acrylamide (ACR) is widely used to synthesize industrial chemicals such as polyacrylamide [1]. Previous studies have found that ACR has severe harmful effects on mammals' genetic material, nervous system, and immune function [2][3][4][5][6]. Furthermore, ACR can be exposed to humans in several ways and threaten human health [1,7,8]. In 2002, Swedish researchers found high-concentration ACR in starchy foods after heat treatment [9]. Since then, ACR has become a foodborne hazard, attracting the attention of researchers all over the world [7]. With the changing dietary habits and the increasing consumption frequency of baked and fried foods, people can intake a certain amount of ACR through their daily diet, resulting in higher exposure and increasingly serious hazards for their health [10]. In addition, children and young people are especially easily exposed to foods containing ACR, such as French fries, bread, cookies, and so on [1,11,12].
As the primary metabolic target organ of ACR, the liver is the first to be affected [13]. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are closely related to liver function [14]. Previous studies have shown that ACR can increase the serum levels of Note: Data are expressed as the mean ± SD. ** p < 0.01 versus the CON group; # p < 0.05 and ## p < 0.01 versus the ACR group. ACR, acrylamide; ALT, alanine transferase; AST, aspartate transferase; LRP-L, Lycium ruthenicum polyphenols in low dosage; LRP-M, Lycium ruthenicum polyphenols in medium dosage; LRP-H, Lycium ruthenicum polyphenols in high dosage; LRP, Lycium ruthenicum polyphenols control group.

Effect of LRP on the ACR-Induced Histopathological Changes
Hepatic tissue from the CON and LRP groups were with typical histological structures under light microscopy. The liver from the ACR group showed localized inflammatory cell infiltration and some damage to hepatocytes. Three doses of LRP pretreated with LRP-L, LRP-M, and LRP-H slightly improved the histopathological morphology induced by ACR ( Figure 1). Note: Data are expressed as the mean ± SD. ** p < 0.01 versus the CON group; # p < 0.05 and ## p < 0.01 versus the ACR group. ACR, acrylamide; ALT, alanine transferase; AST, aspartate transferase; LRP-L, Lycium ruthenicum polyphenols in low dosage; LRP-M, Lycium ruthenicum polyphenols in medium dosage; LRP-H, Lycium ruthenicum polyphenols in high dosage; LRP, Lycium ruthenicum polyphenols control group.

Effect of LRP on the ACR-Induced Histopathological Changes
Hepatic tissue from the CON and LRP groups were with typical histological structures under light microscopy. The liver from the ACR group showed localized inflammatory cell infiltration and some damage to hepatocytes. Three doses of LRP pretreated with LRP-L, LRP-M, and LRP-H slightly improved the histopathological morphology induced by ACR (Figure 1).
Transmission electron microscopy showed that the hepatocytes in the CON and LRP groups had regular morphology with clear borders, mitochondria were abundant, and the endoplasmic reticulum's surface was rough; the cristae of mitochondria had a clear structure. Compared to the CON group, hepatocytes in the ACR group suffered from organelle destruction, mitochondrial swelling, mitochondrial cristae loss, and rough endoplasmic reticulum. After the intervention of three doses of LRP, the nuclear structure of hepatocytes gradually improved, and the mitochondrial membrane structure and rough endoplasmic reticulum were restored with does dependant (Figure 2).  Transmission electron microscopy showed that the hepatocytes in the CON and LRP groups had regular morphology with clear borders, mitochondria were abundant, and the endoplasmic reticulum's surface was rough; the cristae of mitochondria had a clear structure. Compared to the CON group, hepatocytes in the ACR group suffered from organelle destruction, mitochondrial swelling, mitochondrial cristae loss, and rough endoplasmic reticulum. After the intervention of three doses of LRP, the nuclear structure of hepatocytes gradually improved, and the mitochondrial membrane structure and rough endoplasmic reticulum were restored with does dependant (Figure 2).

Effects of LRP on ROS in the Liver
As one can see in Figure 3A, ACR (40 mg/kg) significantly increased the level of hepatic ROS after 12 days of continuous intervention (p < 0.01). In addition, the ROS were decreased in the LRP (50, 100, and 200 mg/kg) groups compared to the ACR group (p < 0.01).

Effects of LRP on ROS in the Liver
As one can see in Figure 3A, ACR (40 mg/kg) significantly increased the level of hepatic ROS after 12 days of continuous intervention (p < 0.01). In addition, the ROS were decreased in the LRP (50, 100, and 200 mg/kg) groups compared to the ACR group (p < 0.01).

Effects of LRP on ROS in the Liver
As one can see in Figure 3A, ACR (40 mg/kg) significantly increased the level of hepatic ROS after 12 days of continuous intervention (p < 0.01). In addition, the ROS were decreased in the LRP (50, 100, and 200 mg/kg) groups compared to the ACR group (p < 0.01).

Effects of LRP on SOD, GSH, and MDA in the Liver
As can be see in Figure 3, administration of ACR (40 mg/kg) for 12 consecutive days significantly increased the MDA level compared to the control group in the liver (p < 0.01). ACR-treated rats showed lower levels of SOD and GSH than the CON group (p < 0.01). In contrast, different doses of LRP inhibited oxidative damage in liver tissue. In addition, the levels of MDA were significantly decreased in the LRP (50, 100, 200 mg/kg) group compared to the ACR group rats (p < 0.01). Compared with the ACR group, administration with LRP at 50, 100, and 200 mg/kg dose increased the levels of SOD and GSH (p < 0.05).

Effect of LRP on ATPase Activities Induced by ACR in the Liver Mitochondrion
As shown in Table 2, liver mitochondrial ATPase activity was significantly decreased in ACR-treated rats compared with the CON group (p < 0.01). In addition, Na + /K + -ATPase activity and Mg 2+ -ATPase activity in the ACR group were approximately 50% lower than those in the CON group. LRP pretreatment significantly restored the activity of these ATPases, and at the high dose (200 mg/kg) of LRP essentially restored the Ca 2+ -ATPase activity to control levels (p < 0.05). Note: Data are expressed as the mean ± SD. ** p < 0.01 versus the CON group; # p < 0.05 versus the ACR group. ACR, acrylamide; LRP-L, Lycium ruthenicum polyphenols in low dosage; LRP-M, Lycium ruthenicum polyphenols in medium dosage; LRP-H, Lycium ruthenicum polyphenols in high dosage; LRP, Lycium ruthenicum polyphenols control group.

Effect of LRP on the mRNA Expression of Mitochondrial Complexes I-III Induced by ACR in Liver Tissue
Compared with the CON group, we did not find any significant change in complex II in the ACR group, but a significant decline was observed in mRNA levels of complexes I and III (p < 0.01). Furthermore, administration of LRP at the doses of 50, 100, and 200 mg/kg in ACR-induced toxicity rats, significantly increased mRNA levels of complexes I and III compared with the ACR group (p < 0.01) ( Figure 4).

Effect of LRP on the Nrf2 Pathway Induced by ACR in Liver Tissue
Compared with the CON group, Nrf2, NQO1, GCLC, GCLM, and HO-1 proteins expression was reduced significantly in the ACR group (p < 0.01). ACR can reduce intracellular Nrf2-regulated downstream antioxidant proteins Nrf2, NQO1, GCLC, GCLM, and HO-1. In contrast, the expression levels of antioxidant proteins in the Nrf2 pathway, including Nrf2, NQO1, GCLC, GCLM, and HO-1 were increased after LRP pretreatment with a dose-response effect. These results suggest that LRP may exert antioxidant effects by regulating the expression of Nrf2 pathway antioxidant proteins (Figures 5 and6).

Effect of LRP on the Nrf2 Pathway Induced by ACR in Liver Tissue
Compared with the CON group, Nrf2, NQO1, GCLC, GCLM, and HO-1 prot pression was reduced significantly in the ACR group (p < 0.01). ACR can reduce i lular Nrf2-regulated downstream antioxidant proteins Nrf2, NQO1, GCLC, GCL HO-1. In contrast, the expression levels of antioxidant proteins in the Nrf2 pathw cluding Nrf2, NQO1, GCLC, GCLM, and HO-1 were increased after LRP pretre with a dose-response effect. These results suggest that LRP may exert antioxidan by regulating the expression of Nrf2 pathway antioxidant proteins (Figures 5 and

Effect of LRP on the Nrf2 Pathway Induced by ACR in Liver Tissue
Compared with the CON group, Nrf2, NQO1, GCLC, GCLM, and HO-1 proteins expression was reduced significantly in the ACR group (p < 0.01). ACR can reduce intracellular Nrf2-regulated downstream antioxidant proteins Nrf2, NQO1, GCLC, GCLM, and HO-1. In contrast, the expression levels of antioxidant proteins in the Nrf2 pathway, including Nrf2, NQO1, GCLC, GCLM, and HO-1 were increased after LRP pretreatment with a dose-response effect. These results suggest that LRP may exert antioxidant effects by regulating the expression of Nrf2 pathway antioxidant proteins (Figures 5 and 6).

Discussion
The aim of this study was to investigate the protective effect of LRP against ACRinduced hepatotoxicity. ACR, a water-soluble toxic substance with solid permeability, can quickly go into the bloodstream and may trigger liver damage [29]. Recently, a study by Elhelaly et al. indicated that ACR-induced toxicity caused damage to liver tissue by increasing DNA oxidative damage and reducing antioxidant enzyme activity [30]. Previously, we reported that LRP had a protective impact on oxidative damage in vitro. In this study, we further investigated the protective effect of LRP against ACR-induced toxicity in rat liver and its related mechanism.
The liver is the target organ for exogenous toxicants, which is considerably more sensitive to these toxicants and plays a vital role in the detoxification process [4]. One previous study showed ACR-induced oxidative stress in the liver by allicin compared with the CON group. The levels of ALT and AST were increased in the ACR group mice, indicating the ACR staining has caused acute damage to liver function. The vitality of ALT and AST in all groups of allicin has decreased with the increase of allicin concentration, and the liver function was also improved [31]. The results we obtained were consistent with those that have been reported. The activity of ALT and AST in the serum of rats in the ACR group was significantly higher, indicating that the liver function was damaged by the rupture of hepatocytes and even mitochondria. There were significant differences in the ALT and AST viability in each dose group of LRP compared with the ACR group. The LRP improved the recovery of membrane structure and normalized the permeability and function of hepatocytes under ACR-induced liver injury. Therefore, LRP has a protective effect on ACR-induced liver function injury in rats. It has been shown that the central

Discussion
The aim of this study was to investigate the protective effect of LRP against ACRinduced hepatotoxicity. ACR, a water-soluble toxic substance with solid permeability, can quickly go into the bloodstream and may trigger liver damage [29]. Recently, a study by Elhelaly et al. indicated that ACR-induced toxicity caused damage to liver tissue by increasing DNA oxidative damage and reducing antioxidant enzyme activity [30]. Previously, we reported that LRP had a protective impact on oxidative damage in vitro. In this study, we further investigated the protective effect of LRP against ACR-induced toxicity in rat liver and its related mechanism.
The liver is the target organ for exogenous toxicants, which is considerably more sensitive to these toxicants and plays a vital role in the detoxification process [4]. One previous study showed ACR-induced oxidative stress in the liver by allicin compared with the CON group. The levels of ALT and AST were increased in the ACR group mice, indicating the ACR staining has caused acute damage to liver function. The vitality of ALT and AST in all groups of allicin has decreased with the increase of allicin concentration, and the liver function was also improved [31]. The results we obtained were consistent with those that have been reported. The activity of ALT and AST in the serum of rats in the ACR group was significantly higher, indicating that the liver function was damaged by the rupture of hepatocytes and even mitochondria. There were significant differences in the ALT and AST viability in each dose group of LRP compared with the ACR group. The LRP improved the recovery of membrane structure and normalized the permeability and function of hepatocytes under ACR-induced liver injury. Therefore, LRP has a protective effect on ACR-induced liver function injury in rats. It has been shown that the central veins, sinus venosus, and vascular structures in the portal area of the liver profile are congested after ACR administration. Inflammatory cells infiltrate the portal connective tissue and periportal area. Areas of hepatocyte necrosis and hemorrhage of different sizes were seen in the liver parenchyma [17,30]. From the results of this experiment, it was observed that there was local inflammatory cell infiltration in the liver of the ACR group, and the degree of the damage was not significant. In order to observe some changes that occurred in the liver, we also observed the liver ultrastructure with transmission electron microscopy. The results showed that the nuclear structure of hepatocytes was damaged after the action of ACR, the mitochondrial bilayer structure was not clear, and the rough endoplasmic reticulum was proliferated. The above changes in the ultrastructure of hepatocytes mean that the liver is damaged [32]. From this study, we can know that LRP can promote the recovery of hepatocyte membrane structure after ACR-induced liver injury and restore its permeability and function to normal in rats. It indicates that LRP has a protective effect on ACR-induced liver injury in rats with dose-dependence.
Oxidative stress occurs in an imbalance between the production and elimination of oxidative products in the body when stimulated by various external harmful substances. A previous study showed that ACR could disrupt the balance between oxidation and antioxidants. It is linked with the overproduction of ROS and thus leads to oxidative damage [19,33]. Our current study suggested ACR-induced oxidative stress in rat tissues. SOD and GSH activities were significantly reduced. Conversely, the level of MDA and ROS increased during ACR treatment. Our results were consistent with others that ACR could increase lipid peroxidation and cause damage to the antioxidant enzyme systems [29]. Different studies suggested the antioxidant effects of LR [34,35]. Tian et al. found that LRP normalizes high lipid peroxidation levels in mice [36]. In addition, the GSH content of the rodent liver was significantly increased after gavage LRP. In the present study, all three doses of LRP (50, 100, and 200 mg/kg) reduced MDA levels, while different amounts of LRP were effective in increasing the levels of GSH in the liver tissue.
Mitochondria are the main sites of endogenous ROS generation and easily occur oxidative stress. The results showed that ACR reduced the activity of ATP hydrolase, which affected mitochondrial function and resulted in insufficient energy supply in vivo. It is consistent with Er et al. [37]. In addition, we observed mitochondrial structural damage in ACR-induced hepatocytes by transmission electron microscopy. We found that LRP can directly scavenge the free radicals generated during mitochondrial damage. Polyphenols restore mitochondrial ATPase activity by maintaining the structural and functional integrity of the inner and outer mitochondrial membranes. Zhao et al. [38] found that blueberry anthocyanins restored ACR-induced Na + , K + -ATPase, and Mg 2+ -ATPase activities in rats with simultaneous dose-response effects. Our results indicate that LRP increases mitochondrial ATPase activity in a dose-dependent manner. Compared with the ACR group, the activity of ATPase in the LRP group was significantly increased. Complexes I, II, III, and IV have electron transport functions, and complexes I and III are necessary sites for ROS production. The change in its activity can reflect the change in mitochondrial respiratory function. Er et al. observed that the mitochondrial respiratory enzyme activity (complexes I-V) and the phospholipid level in the center of the mitochondrial membrane decreased after ACR induction in mouse liver [37]. We found LRP could significantly reduce ACR-induced mRNA expression of mitochondrial complexes I and III genes in rat liver. No differences were found for complex II mRNA. ACR inhibits the function of the mitochondrial respiratory chain complex and reduces its activity, leading to a decrease in ATP synthesis [39]. The antioxidant properties of LRP can slow down the damage of ACR to the mitochondrial complex, thus maintaining the normal electron transport function in the mitochondrial respiratory transport chain. The results showed that LRP medium and high doses could inhibit the decrease of complexes I and III mRNA expression. According to previous studies [38], blueberry anthocyanin extract can inhibit electron leakage of complexes I and III induced by ACR, thus improving mitochondrial function in the liver.
Nrf2/ antioxidant response element (ARE) is an important antioxidant signaling pathway. In addition to Nrf2 nuclear translocation, the Nrf2 pathway is mainly responsible for ARE activation. Through Nrf2 activation, downstream targets such as HO-1, GCLM, GCLC, and NQO1 are highly expressed [40]. Previous experiments have shown that the Nrf2 signaling pathway is a crucial compensatory protective mechanism that attenuates ACR-induced oxidative damage [41,42]. Our results showed that ACR induced a decrease in Nrf2, HO-1, GCLC, GCLM, and NQO1 in the liver of rats compared to the CON group. Liu et al. found that chlorogenic acid could be protective in rats with cerebral ischemia/reperfusion injury by activating the Nrf2 pathway [43]. Tan et al. suggested that ACR-treated cells activated the Nrf2/NQO-1 pathway and increased the expression of mitochondrial respiratory complexes by resveratrol in ACR-treated cells [44]. This study also confirmed that LRP has a potent antioxidant activity to increase Nrf2, HO-1, GCLC, GCLM, and NQO1 protein expression in a dose-dependent manner. Based on the above results, it is further demonstrated that LRP can alleviate the damage of mitochondrial structure and function and inhibit apoptosis through its good antioxidant effect, thus playing a role in protecting ACR-induced hepatotoxicity. This study provides a new perspective to improve the hepatotoxicity induced by ACR intake with LRP.

Animals and Experimental Design
Forty-eight healthy male Spraque Dawley rats (7-week old and weighing 225-275 g) were obtained from the Experimental Animal Center of Ningxia Medical University (Yinchuan, China). The study was carried out in agreement with the Ethical Committee Acts and Guidelines of Ningxia Medical University of Medical Sciences (ethical number: IACUC-NYLAC-2020-119). Rats were randomly divided into six groups, including the control (CON) group, ACR group, three LRP intervention groups in low, medium, and high dosages (LRP-L, LRP-M, LRP-H), and LRP control group (LRP group), each group consisting of 8 animals. These groups are shown in Table 3. After one week of adaptive feeding, rats in the LRP intervention group were gavaged with the corresponding dose of LRP for 7 days, while rats in the control and ACR groups were gavaged with normal saline. On the eighth day of the intervention, rats were gavaged with ACR in each group. Meanwhile, the corresponding dose of LRP or regular saline for 1 h before manipulation for 12 days. The dose settings of ACR and LRP were obtained from pre-experiment and related literature [19,45].

Sample Collection
By the end of the experimental time, isoflurane was used for rat anesthesia. After the corneal reflex, flip reflex, and pain reflex disappeared, the liver tissue was removed by exposing the abdomen. The blood on the surface of the tissue was washed with saline and placed in a lyophilization tube. After that, the treated tissues were quickly cryopreserved in liquid nitrogen and stored at −80 • C.

Isolation of Liver Mitochondria
We extracted rat liver mitochondria with reference to the method of Zhang et al. [38]. Briefly, about 0.1 g of rat liver tissue was taken, washed, homogenated, and centrifuged at 600× g for 10 min. Prepared the supernatant for further use was taken, centrifuged at 11,000× g for 15 min, and precipitated as mitochondria. The prepared mitochondria were stored at −80 • C for the future.

Histopathological Studies
Liver tissue was fixed in 10% formalin, processed in wax blocks, sliced, dyed, closed, etc., and examined under a microscope [15].

Measurement of the AST, ALT Indexes in Serum
The enzymes AST and ALT activities were determined with the commercial assay kits referring to the manufacturers' instructions [47].
The liver single-cell suspension was prepared, and DCFH-DA was added at a recommended concentration of 10 µM. The cells were incubated at 37 • C for 30 min. The probe labeled single-cell suspension was collected, washed once or twice with PBS, centrifuged, and collected the precipitate. The fluorescence intensity was measured at the optimal excitation wavelength of 485 nm and the optimal emission wavelength of 525 nm. The measurement results were expressed as fluorescence intensity/mg protein.

Measurement of the Liver Mitochondrial ATPase
The activities of Na + -K + -ATPase, Ca 2+ -ATPase, and Mg 2+ -ATPase were detected using a commercial kit [49]. The tissue RNA was extracted by the Trizol method, the RNA concentration was measured, and the extracted total RNA was reverse transcribed to cDNA according to the reverse transcription kit instructions. Quantitative real-time PCR analysis was performed on the PCR system using TB Green Premix Ex Taq™ II. Then relative gene expression was normalized to GAPDH and calculated using the ∆∆ CT method [50]. The primer sequences utilized in the research are listed in Table 4.

Statistical Analysis
Results were expressed as mean ± SD. All statistical comparisons were performed by a one-way ANOVA test followed by Tukey's post hoc analysis. SPSS (version 22.0, IBM, Armonk, NY, USA) and GraphPad Prism 6.0 were used for drawing and data analysis. p < 0.05 was considered significant.

Conclusions
The present study showed that ACR could induce liver injury through oxidative stress and mitochondrial dysfunction. Moreover, oral LRP administration at 50, 100, and 200 mg/kg/day could protect the rat's liver tissue from ACR-induced oxidative stress in a dose-dependent manner, improving mitochondrial structure and function by activating Nrf2 signaling pathway. It is suggested that LRP could be a promising liver protective agent against ACR toxicity. LRP is recommended as oxidative stress relievers against hepatotoxicity.