Testosterone propionate activated the Nrf2-ARE pathway in ageing rats and ameliorated the age-related changes in liver

The present study aimed to evaluate the protective efficacy of testosterone propionate (TP) on age-related liver changes via activation of the nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway in aged rats. Aged rats received subcutaneous injections of TP (2 mg/kg/d, 84 days). Oxidative stress parameters and the expression levels of signal transducer and activator of transcription 5b (STAT5b), Kelch-like ECH associating protein-1 (Keap1), Nrf2, haem oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO1) in liver tissues were examined to check whether the Nrf2-ARE pathway was involved in the age-related changes in liver. Our results showed that TP supplementation alleviated liver morphology, liver function and liver fibrosis; improved oxidative stress parameters; and increased the expression of STAT5b, Nrf2, HO-1 and NQO-1 and decreased the expression of Keap1 in the liver tissues of aged rats. These results suggested that TP increased the expression of STAT5b, and then activated the Nrf2-ARE pathway and promoted antioxidant mechanisms in aged rats. These findings may provide new therapeutic uses for TP in patients with age-related liver changes.

Ageing is characterized by a process of physiological degeneration and is accompanied by pathophysiological age-related changes, including impaired functioning and increased vulnerability to death [1][2][3] . Age-related changes in the liver are commonly associated with structural and physiological changes 4,5 . The mechanisms of hepatic injury caused by ageing are complicated and involve apoptosis, inflammation, hepatic clearance function and toxicity [6][7][8] . Recent studies have shown that oxidative stress is one of the mechanisms implicated in the progression of age-related changes in the liver 9,10 . In this way, antioxidants could be important therapeutic strategies for controlling such changes in the liver.
Nuclear factor erythroid-related factor 2 (Nrf2), an important antioxidant transcription factor, regulates the expression of many antioxidant and phase II detoxifying enzyme genes, such as haem oxygenase-1 (HO-1) and NADP(H): quinine oxidoreductase-1 (NQO1), through the antioxidant response element (ARE). Nrf2 is repressed in the cytoplasm by the Kelch-like ECH associating protein-1 (Keap1) under normal conditions. However, Nrf2 dissociates from Keap1 and translocates to the nucleus to bind to ARE upon oxidative stress 11 . Activation of the Nrf2-ARE pathway has a protective effect against various diseases via antioxidative mechanisms [12][13][14][15] .
Testosterone is an important hormone that participates in a variety of physiological functions, including embryonic development 16 , stimulation of genital growth and secondary sexual characteristics 17 , maintenance of spermatogenesis 18 , improved sexual function 19 and promotion of erythropoiesis 20 . It has been reported that concentrations of testosterone decrease during the ageing process 21,22 . The level of growth hormone (GH) show the age-related reduction, and testosterone increases GH secretion in older men 23 . Signal transducer and activator of transcription 5b (STAT5b), responds to a variety of extracellular cytokine and growth factor signals, including The effect of TP on liver function in aged rats. The levels of alanine transaminase (ALT, Fig. 2a), aspartate transaminase (AST, Fig. 2b), total bilirubin (TBIL, Fig. 2c), direct bilirubin (DBIL, Fig. 2d) and indirect bilirubin (IBIL, Fig. 2e) in 24 Mon rats were significantly increased compared to those in 6 Mon rats (P < 0.01). The levels of total protein (TOP, Fig. 2f), albumin (ALB, Fig. 2g) and globulin (GLB, Fig. 2h) in 24 Mon rats were significantly decreased compared to those in 6 Mon rats (P < 0.01). The levels of ALT, AST, TBIL, DBIL and IBIL of 24 Mon-TP rats were decreased, and the levels of TOP, ALB and GLB of 24 Mon-TP rats were increased after TP treatment compared with those of untreated 24 Mon rats (P < 0.01). However, the serum levels of ALT, AST, TBIL, DBIL, IBIL, TOP, ALB and GLB of 24 Mon-TP rats were not restored to the corresponding levels of 6 Mon rats (P < 0.01) (Fig. 2).
The effect of TP on liver fibrosis in aged rats. The levels of hyaluronidase (HA, Fig. 3a), laminin (LN, Fig. 3b), type III precollagen (PC-III, Fig. 3c) and type IV collagen (IV-C, Fig. 3d) in 24 Mon rats were significantly increased compared to those in 6 Mon rats (P < 0.01). The levels of HA, LN, PC-III and IV-C in 24 Mon-TP rats were significantly decreased after TP treatment compared with those in 24 Mon rats (P < 0.01) and were not restored to the levels in 6 Mon rats (P < 0.01) (Fig. 3).
The effect of TP on serum growth hormone (GH) in aged rats. The level of serum GH in 24 Mon rats (0.4 ± 0.1 μg/L) was significantly decreased compared to that in 6 Mon rats (1.8 ± 0.2 μg/L, P < 0.01). The level of serum GH in 24 Mon-TP rats (1.4 ± 0.2 μg/L) was significantly increased after TP treatment compared with that in 24 Mon rats (P < 0.01) and was not restored to the level in 6 Mon rats (P < 0.01).
The effect of TP on oxidative stress parameters in aged rats. The levels of malondialdehyde (MDA, Fig. 4a) and lipid peroxidation (LPO, Fig. 4b) in 24 Mon rats were significantly increased compared to those in 6 Mon rats (P < 0.01), and the levels of reduced glutathione (GSH, Fig. 4c), glutathione peroxidase (GSH-px, Fig. 4d), catalase (CAT, Fig. 4e) and superoxide dismutase (SOD, Fig. 4f) in 24 Mon rats were significantly decreased compared to those in 6 Mon rats (P < 0.01). Notably, the levels of MDA and LPO in 24 Mon-TP rats were decreased, and the levels of GSH, GSH-px, CAT and SOD in 24 Mon-TP rats were increased after TP treatment compared to those in 24 Mon rats (P < 0.01). The serum levels of MDA, LPO, GSH, GSH-px, CAT and SOD in 24 Mon-TP rats were not restored to the corresponding levels in 6 Mon rats (P < 0.01) (Fig. 4).

Discussion
In the present study, TP supplementation improved age-related liver morphological changes. The serum levels of ALT, AST, TBIL, DBIL, IBIL, TOP, ALB and GLB were used to explore liver function. Our data showed that TP treatment decreased ALT, AST, TBIL, DBIL and IBIL levels and increased TOP, ALB and GLB levels. The serum levels of HA, LN, PC-III and IV-C were used to explore liver fibrosis. These data showed that TP treatment resulted in a significant decrease in HA, LN, PC-III and IV-C levels. These results confirmed that TP supplementation alleviated changes in liver morphology, liver function and liver fibrosis in aged rats.
Accumulating evidence indicates that reactive oxygen species (ROS) regulate longevity in many organisms 27,28 . This free radical theory suggests that ageing is caused by an accumulation of molecular damage resulting from ROS, such as superoxide anions, hydroxyl radicals and hydrogen peroxide 29 . In the antioxidant defence system, Nrf2 is the most important transcription factor in regulating multiple antioxidants 30 . Activation of the Nrf2/ HO-1 pathway might play a potential protective role against doxorubicin-induced hepatotoxicity 31 . Moreover, the preventive effect against CCl 4 -induced hepatotoxicity and fibrosis was partly dependent on modulation of the Nrf2-ARE signalling pathway 32 . The mechanism of the cytoprotective effect of rutaecarpine against tert-butyl hydroperoxide occurs via upregulating antioxidant enzymes in part via the Nrf2-ARE pathways 33 . Ethyl acetate treatment has been shown to significantly increase the levels of Nrf2 and inhibit CCl 4 -induced liver fibrosis in rats 34 . In this way, activation of the Nrf2-ARE signalling pathway has a protective effect on the liver. In our previous studies, intranasal testosterone propionate supplementation increased the levels of Nrf2, HO-1 and NQO1 and enhanced dopaminergic functional activity in the substantia nigra and ventral tegmental area 22 . However, the role of TP in peripheral liver tissues has not been reported.
STAT5b is downstream of GH signalling. With the pulsatile secretion of GH, this leads to the formation of STAT5b homodimers 24 . Whether STAT5b regulates the Keap1 and Nrf2-ARE pathway in the liver is unclear. Our data shows that the level of serum GH and the expression level of STAT5b were increased after TP treatment in aged rats. The levels of MDA, LPO, GSH, GSH-px, CAT and SOD in hepatic tissues represent the state of oxidative stress 35 . In our study, the levels of MDA and LPO were decreased, and the levels of GSH, GSH-px, CAT and SOD were increased in aged rats after TP treatment. Nrf2 is a key transcription factor that plays a crucial role in defending against oxidative stress through modulation of its downstream antioxidant and detoxifying enzymes 36 . The cytoplasmic genes HO-1 and NQO1 could be induced by Nrf2 nuclear translocation 37,38 . The expression levels of Nrf2 (nuclear), HO-1 and NQO1 (cytoplasm) were increased and the expression level of Keap1 (cytoplasm) was decreased after TP supplementation in aged rats. These results suggested that TP promotes GH secretion, increases the expression of STAT5b, induces the separation of Keap1 and Nrf2 in the cytoplasm and promotes Nrf2 transfer to the nucleus and bind to ARE, subsequently activates the Nrf2-ARE pathway and improves oxidative stress in aged hepatic tissues. Therefore, the protective effects of TP on aged liver tissues may partly depend on activation of the Nrf2-ARE signalling pathway followed by a decrease in ROS. The molecular mechanism of www.nature.com/scientificreports www.nature.com/scientificreports/ testosterone on age-related changes in the liver is not fully understood, and more detailed studies are needed to accurately identify the mechanism. Our results of TP on the activation of Nrf2 in the liver show some disparity with a previous study 25 . In male mice, Nrf2 was activated by ethinyl estradiol and was suppressed by dihydrotestosterone, whereas in female mice, Nrf2 was suppressed by testosterone and was activated by ethinyl estradiol 25 . Testosterone can be converted to dihydrotestosterone and estradiol by 5α-reductase and aromatase respectively in male rats 39 . Thus, we presume that the difference of our results with the previous study is the sum effects of dihydrotestosterone and estradiol. In the liver of aged male rats, testosterone might show efficacy of estradiol on Nrf2. This requires further investigation in future research. In addition, different species, different time of administration and dose and the status of oxidative stress in the liver might contribute to the difference.
In conclusion, the results of the present study suggested that TP increased the expression of STAT5b, and then activated Nrf2-ARE signalling and promoted antioxidant mechanisms in aged rats. TP treatment appeared to play the role of an Nrf2 activator and alleviate age-related changes in the liver. This finding may provide new therapeutic uses for TP in patients with age-related liver disease.

Animals and testosterone propionate supplement. Forty-eight male Wistar rats (Experimental
Animal Center of Hebei Medical University) were divided into three groups consisting of a 6-month-old group (6 Mon), a TP-supplemented group (24 Mon-TP) and an aged vehicle control group (24 Mon). Each group included sixteen rats. The 24 Mon-TP group rats received a daily subcutaneous TP injection (2 mg/kg/d at 5:00 PM to 6:00 PM) beginning at the age of 21 months. Supplementation with TP was continued for 12 weeks (84 days). The 6 Mon group rats and 24 Mon group rats received a sesame oil injection (vehicle) 40 . All rats were housed in a room maintained at a constant temperature (22 ± 2 °C). All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Local Animal Use Committee of Hebei Medical University. www.nature.com/scientificreports www.nature.com/scientificreports/ Histopathologic evaluation. The eight rats in each group were anaesthetized by intraperitoneal injection of 4% chloral hydrate (300 mg/kg body weight) and transcardially perfused with saline. Then, the rats underwent perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Livers were removed, and tissue sections were post-fixed in 4% paraformaldehyde for 4 h (4 °C). The liver tissue sections of the rats were washed in two changes of fresh phosphate buffer, dehydrated in titrated ethanol and cleared in xylene before being embedded in paraffin wax. The blocks were sliced into sections at a 5 μm thickness on a sliding microtome (Leica-RM2145, Germany) and the sections were stained with H&E. The preparations were evaluated by light microscopy and photographed (Olympus, BX 61, Japan) 40 .

Measurement of liver fibrosis indexes and GH in serum.
Levels of serum HA, LN and GH were measured using an ELISA kit. Levels of serum PC-III and IV-C were analysed by radioimmunoassay (RIA) and determined from a standard curve. Kits for HA (FA02052B) and LN (FA01730B) were provided by the Shanghai Yantuo Biological Technology Co., LTD. Kits for PC-III (HY-10089) and IV-C (HY-10086) were purchased from Shanghai Huzhen Industrial Co., Ltd. Kit for GH (ARB12139) was purchased from Beijing Baiao Laibo Technology Co., Ltd. All steps were performed according to the manufacturers' protocols.

Measurement of oxidative stress parameters in liver.
The liver tissue was homogenized separately with 10 times (w/v) ice-cold 0.1 M phosphate buffer (PB) at pH 7.4. The homogenates were used to assess oxidative stress parameters. MDA, LPO, GSH, GSH-px, CAT and SOD levels were measured spectrophotometrically using detection kits, following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China) 40 .
Western blot analysis. The liver tissue was homogenized in ice-cold lysis buffer (10 mmol/L HEPES pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.1 mmol/L EGTA) for 15 min. After adding NP-40, the homogenate was centrifuged at 10,000 rpm at 4 °C for 3 min, and the supernatant was collected as cytoplasmic protein for STAT5b, Keap1, HO-1 and NQO1. The pellets were homogenized in ice-cold lysis buffer (20 mmol/L HEPES, pH 7.9, 400 mmol/L NaCl, 1 mmol/L EDTA, 0.1 mmol/L EGTA) for 15 min. Then, the pellets were centrifuged at 12,000 rpm at 4 °C for 10 min, and the supernatant was collected. Phenylmethanesulfonyl fluoride was added to the supernatant at a final concentration of 1 mmol/L as the nuclear protein for Nrf2 40 .
Immunohistochemistry and densitometric analysis. Liver tissue sections from histopathologic evaluation were also used for immunohistochemical analysis. Liver tissue sections from each of the three groups were placed on individual slides. After deparaffinization and hydration, sections were subjected to antigen retrieval (in 0.01 M citrate buffer, pH 6.0) by microwave for 30 min and immersed in 3% hydrogen peroxide in methanol for 30 min to abolish endogenous peroxidase activity. The sections were incubated with 5% normal goat serum to block non-specific binding. This procedure was followed by an overnight incubation with polyclonal rabbit anti-Nrf2 antibody (1:100, Abcam, ab31163), polyclonal rabbit anti-HO-1 antibody (1:100, Abcam, ab13243) and mouse anti-NQO1 monoclonal antibody (1:500, Abcam, ab28947) at 4 °C. After washing, the sections were incubated with biotinylated goat anti-mouse IgG (Jackson ImmunoResearch, 1:300) or goat anti-rabbit IgG www.nature.com/scientificreports www.nature.com/scientificreports/ (Jackson ImmunoResearch, 1:300) for 2 h at room temperature. Following the incubation of sections for 1 h at RT in horseradish peroxidase-conjugated streptavidin (1:300), the sections were stained for 5 min in a solution containing 0.05% diaminobenzidine (Sigma) and 0.03% H 2 O 2 in 0.05 M Tris-HCl buffer (pH 7.6) to reveal immunoreactions 40 .
A computer-assisted image analysis system (Olympus, BX 61, Japan; Image-Pro Plus 6.0) was used for the average optical density (AOD) measurements of Nrf2, HO-1 and NQO1 immunoreactive intensity. Images were acquired after calibration of the system to eliminate saturation of grey levels for an accurate determination of optical density. The sections processed without primary antibody were used to determine the level of nonspecific staining for the entire experiment. After subtraction of the nonspecific staining, the AOD of Nrf2, HO-1 and NQO1 was measured. To prevent differences arising from variations in the conditions of tissue processing and densitometric analysis, all sections were simultaneously processed. All microscopic and computer parameters were kept constant throughout the study. Ten sections were selected from each rat. The averaged value of the AOD of Nrf2, HO-1 and NQO1 was presented for each rat 40 . Statistical analyses. We applied tests of normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene's test) to all data. If both normal distribution (P > 0.1) and homogeneity of variance (P > 0.1) were found, then a parametric test was performed by one-way analysis of variance (one-way ANOVA) followed by a Student-Newman-Keuls (SNK) post hoc test for multiple comparisons. Otherwise, we used non-parametric statistics, namely the Kruskal-Wallis test, and if P < 0.05, post hoc analysis between groups were performed using the Mann-Whitney U test. Differences were considered to be significant when P values were less than 0.05. All of the data are presented as the mean ± SD 40 .