Oligosaccharide attenuates aging‐related liver dysfunction by activating Nrf2 antioxidant signaling

Abstract Chitosan oligosaccharide (COS) is the depolymerized product of chitosan possessing various biological activities and protective effects against inflammation and oxidative injury. The aim of the present study was to investigate the antioxidant effects of COS supplements on aging‐related liver dysfunction. We found that COS treatment significantly attenuated elevated liver function biomarkers and oxidative stress biomarkers and decreased antioxidative enzyme activities in liver tissues in D‐galactose (D‐gal)‐treated mice. Furthermore, COS treatment significantly upregulated the expression of Nrf2 and its downstream target genes HO‐1, NQO1, and CAT. Moreover, in vitro experiments showed that COS treatment played a vital role in protecting H2O2‐exposed L02 cells against oxidative stress by activating Nrf2 antioxidant signaling. These data indicate that COS could protect against D‐gal‐induced hepatic aging by activating Nrf2 antioxidant signaling, which may provide novel applications for the prevention and treatment of aging‐related hepatic dysfunction.


| INTRODUC TI ON
Aging is a biological process characterized by progressive degeneration of physiological functions that results in an increase in the prevalence of morbidity and mortality. Hepatic cells are rich in mitochondria and prone to aging-related injury and metabolic abnormalities due to mitochondrial reactive oxygen species (ROS) production (Luceri et al., 2018;Yang et al., 2019). Herein, effective antioxidants against ROS stress may be applied to protect against ROS-induced liver dysfunction during the aging process.
NF-E2-related factor 2 (Nrf2) is a key transcription factor that controls many aspects of cell homoeostasis in response to oxidative and toxic insults (Wardyn, Ponsford, & Sanderson, 2015;Zhang, Yao, et al., 2019). The Nrf2 pathway regulates the expression of several antioxidant and detoxification enzymes including the catalytic subunits of glutamate-cysteine ligase (GCLC), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase 1 (NQO1) by binding to the antioxidant response element (ARE) in their promoter regions (Kubben et al., 2016). Previous studies have shown that Nrf2 is an essential regulator of longevity (Bruns et al., 2015). Current studies suggest that chitosan oligosaccharide (COS) activates numerous antioxidant genes and promotes Nrf2 translocation (Hyung, Ahn, Il Kim, Kim, & Je, 2016;Zhang, Ahmad, et al., 2019). However, whether COS can regulate the oxidative and antioxidant balance in aging cells by regulating the expression of Nrf2 pathway remains unclear.
Chitosan oligosaccharide is prepared from the degradation of chitosan and is a mixture of β-1,4-linked D-glucosamine residue oligomers and abundant in insect cell walls and crustacean exoskeletons. Studies have shown that COS possesses a wide range of biological effects including anti-inflammation, antioxidation, antitumor, anti-Alzheimer's disease, antihypertension, and anti-obesity (Azuma, Osaki, Minami, & Okamoto, 2015;Muanprasat & Chatsudthipong, 2017). Furthermore, there is also evidence showing that COS has an anti-aging role via inhibition of cellular senescence and maintaining a favorable redox balance in mice triggered by D-galactose (D-gal) (Kong et al., 2018).
Although previous studies have already demonstrated that COS possesses hepatoprotective and renoprotective effects in D-galinduced subacute aging mice to realize its anti-aging activity (Kong et al., 2018), the effects of COS on aging-related liver injury triggered by D-gal and its potential molecular mechanism remain to be explored. Herein, we investigated the effects of COS on liver injury at two dose levels using a D-gal-induced animal aging model.
All other chemicals and reagents used in the study were purchased from Sigma-Aldrich.

| Animals
Male C57BL/6 mice at 8 weeks of age were obtained from the animal center of Binzhou Medical University. Animals were fed according to the national standard rodent feed. All mice were kept in the condition that relative humidity is 45%-55%, room temperature is

| Detection of liver index
The mice and their livers were weighed 30 min after the last administration, and the liver index was calculated according to the following equation: Liver index = weight liver wet weight/body weight × 100%.

| Detection of liver function biomarkers
Serum were separated for the assess of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels using commercially available colorimetric assay kits. Serum total bilirubin (TBIL) and direct bilirubin (DBIL) were assessed using Bilirubin (Total and Direct) colorimetric assay Kit.

| Detection of liver malondialdehyde (MDA)
Malondialdehyde was quantified as thiobarbituric acid reactive substances (TBARS). Briefly, the weighed samples were homogenized in 1 ml 5% trichloroacetic acid. The samples were centrifuged (10,000 g), and 250 ml of the supernatant was reacted with the same volume of 20 mM thiobarbituric acid for 35 min at 95°C, followed by 10 min at 4°C. Sample fluorescence was read using a spectrophotometric plate reader (Victor3 1420-050; Perkin Elmer) with an excitation wavelength of 515 nm and an emission wavelength of 553 nm.

| Detection of 8-Hydroxy-2′-deoxyguanosine (8-OH-dG)
The liver pellets were resuspended, and the DNA was isolated using the method recommended by ESCODD. The 8-OH-dG in the DNA was detected using an ESA Coulochem II electrochemical detector in line with a UV detector as previously described (Lodovici et al., 2007).

| Detection of advanced glycation end-products (AGEs)
The supernatant was collected as described above, and the levels of AGEs in the liver in each group were measured by ELISA kits according to the manufacturer's protocol.

| Detection of proinflammatory cytokines
The liver tissues were homogenized in PBS, centrifuged at 10,000 g for 15 min, and collected supernatants for the measurement of cytokines, including monocyte chemoattractant protein-1(MCP1), tumor necrosis factor alpha (TNF-α), and IL-6 (interleukin-6) by ELISA kits, and the detection methods were performed according to the manufactures' instruction.

| Detection of liver antioxidant markers biomarkers
An equal amount of liver was homogenized, and the supernatants were used for the detection. The supernatant was collected after centrifugation at 12,000 rpm and 4°C for 15 min. GSH-Px, CAT, and SOD activity were detected by colorimetric analysis according to the manufacturer's protocol (Xiao et al., 2018).

| Histopathological analysis
Liver tissues were fixed in 4% paraformaldehyde for 12-24 hr, dehydrated in absolute ethanol, transparentized in dimethylbenzene, and embedded in paraffin. Sections of 4 µm were cut, mounted on glass slides, deparaffinized, dehydrated with gradient ethanol, and routinely stained with hematoxylin-eosin (HE), and sealed with optical resin. The stained sections were observed by light microscopy (Nikon).

| Western blot analysis
Total cell extracts were prepared in 1 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. Cell fractions were extracted with nuclear and cytoplasm protein extraction kit (Wanleibio). Cell proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were incubated with primary antibodies overnight at 4°C and appropriate HRP-secondary antibodies for 1 hr at room temperature. Detection was performed using a Thermo Scientific Pierce enhanced chemiluminescence Western blotting substrate (Thermo Scientific) (Xiong et al., 2016).

| RNA extraction and quantitative realtime PCR
Total RNA was isolated using TRIzol reagent as manufacturer's instructions. We performed real-time PCR assay by using SYBR green dye on Step One sequence detection system (ABI). Using β-actin as internal control, we calculated relative abundance of genes using 2 −∆∆CT formula. Primers attached in the Table 1.

| Detection of ROS
The intracellular ROS levels were measured using the ROS assay kit according to the manufacturer's instructions. Briefly, The L02 cells were plated in a 96-well plate (5 × 10 4 cells/well). After the COS and H 2 O 2 treatment, the cells were washed with Hanks balanced salt solution (HBSS) and incubated with 500 μM of the luminol derivative L02 in HBSS at 37°C for 15 min. ROS-induced chemiluminescence was determined every 10 min for a total of 60 min using a Microplate Luminometer (Tropix).

Name Accession No
Primer sequences (Forward/Reverse primer)

| Cell viability assay
Cell viability was determined by MTT assay. After the COS and H 2 O 2 treatment, MTT was added and incubated for 4 hr at 37°C.
Subsequently, the plate was centrifuged at a speed of 800 g for 5 min and the supernatant was discarded. Then, the formazan crystals formed in each well were dissolved using 100 μl DMSO and the absorbance was measured at a wavelength of 540 nm. The relative cell viability was calculated by comparison with the absorbance of untreated control group.

| Statistical analysis
Results are expressed as mean ± SEM of three independent experiments in triplicate. Data were conducted by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Significance was defined as p < .05.

| COS alleviated D-gal triggered liver dysfunction in mice
As shown in Figure 1a

| Effect of COS on liver oxidative stress biomarkers and inflammatory cytokines of D-galtreated mice
There was a markedly increase in the oxidative stress biomarkers ( Figure 2d-f). In addition, the levels of MCP1, TNF-α, and IL-6 levels decreased significantly by 37.9%, 33.0%, and 32.3% in COS-H group compared with D-gal group, respectively.

| Effect of COS on the expressions of Nrf2 pathway in the liver tissue of D-gal-treated mice
We further investigated whether COS initiated the activation of

| Effects of COS on viability and ROS levels of H 2 O 2 -treated L02 cells
We constructed an H 2 O 2 oxidation model in vitro to simulate oxidative stress microenvironment in aging organisms and to explore the molecular mechanism by which COS alleviating dysfunction of liver cells. As shown in Figure 4a, with increasing concentrations of

| Effect of COS on the expressions of Nrf2 pathway of H 2 O 2 -treated L02 cells
The mRNA and protein levels of Nrf2 were increased significantly after incubated with 200 μM H 2 O 2 for 12 hr. In addition, pretreated with 100 or 300 μg/ml COS can further promote the expression of Nrf2 mRNA and protein compared with H 2 O 2 group (Figure 4d,e).
In turn, COS treatment significantly increased the mRNA expression levels of NQO1, HO-1, and CAT (p < .05 for all) (Figure 4f-h).

| D ISCUSS I ON
Oxidative damage has long been implicated in the aging process.
Dietary antioxidant supplementation may be an effective treatment for the correction of impaired plasma membrane redox systems by reducing the incidence of aging-related diseases in the elderly (Chen, Chen, & Zhou, 2018;Xiao et al., 2018;Xiong et al., 2017). In this study, D-gal-induced subacute aging in mice was chosen to investigate the possible anti-aging effects of COS and explore the underlying mechanism. Our study demonstrates that COS treatment could induce Nrf2 pathway activation and subsequently upregulate the expression of the downstream target genes NQO1, HO-1, and CAT. These antioxidant genes further play vital roles in scavenging free radicals in liver tissues of D-gal-treated mice. Additionally, in vitro experiments showed that COS treatment protected H 2 O 2 -exposed L02 cells against oxidative stress by activating Nrf2 antioxidant signaling. These findings uncover some novel molecular events in COS anti-aging biological effects.
Aging is associated with the gradual alteration of hepatic structure and function as well as various changes in liver cells including hepatic sinusoidal endothelial cells . In the present study, we observed a significant elevation in liver enzyme bio- and DNA oxidation that are commonly used as oxidative stress biomarkers (Mizoue et al., 2007;Xiao et al., 2018). AGEs are nonenzymatic glycosylation reaction end-products and considered to be associated with aging-related inflammation and oxidation in liver tissue (Hollenbach, 2017). Our current study showed that continuous administration of COS significantly downregulated MDA, AGE, and 8-OH-dG contents, while significantly upregulating the activities of the antioxidative enzymes SOD, CAT, and GSH-Px in liver tissues in D-gal-treated mice. SOD, CAT, and GSH-Px are important enzymes that participate in the removal of ROS from the cellular environment (Kong et al., 2018). These results indicated that COS may play an anti-aging role by enhancing the activity of endogenous antioxidative enzymes in oxidized cells and reducing the levels of peroxidation products.
Nrf2 is a transcription factor that regulates various antioxidation and detoxification enzymes (Ahmed, Luo, Namani, Wang, & Tang, 2017;Ambrozewicz et al., 2018;Zhang, Yao, et al., 2019) induces an significant decrease in the expression of Nrf2 and its downstream target genes, accompanied by a significant increase in ROS levels and significant decrease in viability of H 2 O 2 -exposed L02 cells. This reveals the therapeutic effects of COS on aging-related oxidative stress injure of liver via activating the Nrf2 antioxidant pathway. These results are in agreement with previous results that COS prevents apoptosis and oxidative stress in mice and H9C2 cells by activating the Nrf2/ARE pathway (Zhang, Ahmad, et al., 2019).

| CON CLUS IONS
In summary, our study demonstrated that COS provides protection against D-gal-induced inflammatory, oxidative stress, and liver dysfunction. Furthermore, COS initiates protective effects by activating Nrf2 and its downstream target genes. These findings provide novel applications of COS to prevent and treat aging-related hepatic dysfunction.

ACK N OWLED G M ENTS
This study was supported by the Shandong Provincial Natural

CO N FLI C T O F I NTE R E S T
The authors have declared that there is no conflict of interest.

E TH I C A L A PPROVA L
Animal experiments were performed in accordance with the National