Abstract
NaCl has beneficial effects in preventing fish disease. However, the effects of NaCl in regulating fish growth and survival at different food supply levels under hypoxic conditions are unknown. Herein, the effects of NaCl on body weight, survival rate (SR), H2O2 content, lipid peroxidation, protein carbonylation, antioxidant (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxidase [GPX]) enzyme activities, and the glutathione (GSH) content were investigated in goldfish (Carassius auratus) livers at three food supply levels (50, 200, and 400 mg day−1 fish−1) under hypoxic conditions. The highest and lowest SRs were detected in the 200 and 400 mg food groups, respectively. Interestingly, 50 mM NaCl markedly elevated survival, but not the body weight of goldfish. Enhanced H2O2 content; SOD, CAT, and GPX activities; GSH content; and reduced lipid peroxidation and protein carbonylation were detected in goldfish livers after NaCl treatment compared with those in the control. However, these effects of NaCl were dramatically attenuated by 50 µM hydroxychloroquine, an inhibitor of autophagy. This showed that nutrition stress reduced goldfish survival, which could be improved by NaCl via regulation of the antioxidant system and autophagy under hypoxic conditions.
Graphical abstract
1 Introduction
Abiotic stress such as salinity, starvation, and hypoxia induces reactive oxygen species (ROS) and oxidative stress in aquatic animals [1,2]. Oxidative stress leads to severe oxidative damage, such as lipid peroxidation and protein carbonylation. The antioxidant system (enzymatic and non-enzymatic antioxidants) can alleviate oxidative damage [3]. The three key antioxidant enzymes are the superoxide dismutase (SOD, EC 1.15.1.1) that converts O2˙− into H2O2, which can be detoxified by either catalase (CAT, EC 1.11.1.16) or peroxidases. A major peroxidase is glutathione peroxidase (GPX, EC 1.11.1.9), which catalyzes the reduction of H2O2 or lipid peroxides via glutathione (GSH). Moreover, ROS also acts as signaling molecules during programmed cell death, redox signaling, and autophagy [4,5].
Salinity is one of the abiotic variables of estuarine ecosystems (3–4% NaCl); however, few freshwater fish species live in estuaries because of their inability to cope with the large salinity ranges that occur in most estuaries and their altered food resources [6,7]. For example, starvation could attenuate salinity tolerance [8], but enhance cold resistance in fish [9]. Thus, the health of aquatic animals is dramatically affected by salinity. Their health can be assessed by monitoring the SOD and CAT activities under salinity [10]. Interestingly, the activities of the two enzymes could be regulated by dietary nutrients and environmental stress [11]. Moreover, exogenous stimuli, such as salinity and nutrient deficiency or excess, can trigger autophagy by inducing autolysosome formation [4,12,13].
Natural or cultured fish often experience periods of poor food supply (e.g., during reproduction) and are well adapted to starvation [14,15]. Starvation activates the antioxidant system and autophagy in fish [12,15]. In contrast, high fat or starch diets also trigger the antioxidant system and autophagy in aquatic animals [16,17]. Moreover, eutrophication of freshwater ecosystems induces hypoxia in many aquatic environments. Hypoxia in pond water causes severe damage to fish when the dissolved O2 levels remain below 2 mg L−1 for several hours, promoting fish death [18,19]. Interestingly, hypoxia also elicits the antioxidant system and autophagy in largemouth bass [20]. Goldfish (Carassius auratus) is a popular freshwater fish with a high market value in the ornamental fish trade [21]. However, goldfish frequently experience intermittent and severe hypoxia in their cultured habitat.
Research has shown that nutrition stress, salinity, and hypoxia can all trigger the antioxidant system and autophagy [4,13,20], ultimately affecting fish growth and survival. Interestingly, beneficial effects of salinity have been reported in the prevention of some diseases in fish [21,22]. This begs the question as to whether NaCl also improves fish growth and survival at different food supply levels under hypoxic conditions? Moreover, what are the possible mechanisms underlying it? In the present study, we aimed the determine effects of NaCl, nutrition stress, and hydroxychloroquine (an inhibitor of autophagy) on goldfish and their body weight, survival rate (SR), and antioxidant response under hypoxic conditions. The results of the present study would help to better understand the beneficial roles of NaCl in regulating goldfish growth and survival under multiple stress conditions from antioxidant defense and autophagy perspectives.
2 Materials and methods
All methods were performed in accordance with the relevant guidelines and regulations.
2.1 Reagents
All reagents, including nitroblue tetrazolium (NBT), hydroxychloroquine (HCQ), 2,4-dinitrophenylhydrazine (DNTP), reduced GSH, and 5,5′-dithiobis-2 nitrobenzoic acid (DTNB) used in the present study were of analytical grade and were obtained from Macklin Biochemistry & Technique Company (Shanghai, China).
2.2 Fish management
One-month-old goldfish were obtained from the Luoyang Xilan fish farm (Henan, China). The fish were adapted to the experimental environment (25 ± 0.5°C; 12:12 dark/light cycle; the salinity of the tap water used here was about 0.6 ppt or approximately equal to 50 mM NaCl) for 7 days. A total of 250 goldfish (20.1 ± 2.1 g and 6.6 ± 0.4 cm) were randomly assigned to five concrete tanks (1.2 m water depth and 2 m diameter; the tap water was placed in the tank for 3 days before being used as the acclimation water for goldfish), resulting in 50 fish per tank. All fish in each tank were fed once a day (09:00 h). Each tank was individually aerated (the dissolved O2 level was maintained at 10.2 ± 0.8 mg L−1).
During the trial, goldfish were distributed in 40 L glass aquaria (50 cm × 20 cm × 40 cm), filled with 20 L tap water and the tap water was changed each week. The goldfish were acclimatized to the new environment for 7 days and dead fish were removed. Subsequently, the experiment was divided into three groups: low nutrition group (50 mg fish−1 day−1), medium nutrition group (200 mg fish−1 day−1), and high nutrition group (400 mg fish−1 day−1). For each nutrition group, they were further divided into four subgroups: water group (control), NaCl group (50 mM NaCl treatment), HCQ group (50 µM HCQ treatment) [23], and NaCl + HCQ group (50 mM NaCl and 50 µM HCQ treatment).
Ten fish were placed in every aquarium giving a rearing density of 10 g L−1. Each aquarium was equipped with an oxygen pump with a sponge biological filter. The dissolved O2 level in the aquaria was maintained at 5.4 ± 0.8 for upper layer of tap water and 3.5 ± 0.5 mg L−1 for the lower layer of tap water. During the periods of acclimation and the 30-day experiment, the goldfish were hand-fed a commercial feed (Brand name: Yubaohong; purchased from Yubaohong Agricultural Development Co., Ltd, Guangzhou, China) containing 42% crude protein, 17% crude lipid, 9% crude ash, and 6% crude carbohydrates with total energy of 2,320 kcal kg−1. The goldfish were fed once daily at 09:00 h with 50, 200, or 400 mg per fish per day. For the NaCl addition, each aquarium filled with 20 L tap water was added with 2 g NaCl (0.1‰) every day (08:30 h) during the first 6 days. Fish were weighed individually, sacrificed, and their livers were excised and frozen immediately in liquid N2. Samples were stored at −80°C for further analysis.
2.3 H2O2 content measurement
The H2O2 content was measured using the method of Velikova et al. [24]. The liver tissue sample was homogenized at 4°C in trichloroacetic acid (TCA; 0.1%) before being centrifuged for 10 min at 10,000×g. Then, 1 mL of the supernatant was mixed with 1 mL of 10 mM Ki-phosphate-buffered saline (PBS) solution (pH 7.0). One milliliter of KI was added to the mixture before it was incubated under dim light for 10 min. The absorbance was then measured spectrophotometrically at 390 nm.
2.4 Oxidative damage measurement
Thiobarbituric acid reactive substance (TBARS) was used to evaluate the lipid damage and was determined using the method of Buege and Aust [25]. The goldfish liver samples were ground in 20% TCA containing 0.25% thiobarbituric acid, and the supernatant was collected after centrifugation at 10,000×g for 10 min. The supernatant was heated at 98°C for 15 min, and the absorbance of TBARS was measured at 532 and 600 nm.
The carbonyl content was used to evaluate protein damage and was determined using the method of Reznick and Packer [26]. Liver samples were incubated at 37°C every 10 min for 1 h with DNTP (10 mM). Proteins were precipitated using TCA (20%) and centrifuged for 20 min at 3,000×g. The precipitated protein pellet was washed three times with ethanol:ethylacetate (1:1). The proteins were re-pelleted by centrifugation at 13,000×g for 5 min. The precipitates were dissolved in 6 M guanidine hydrochloride and kept at room temperature for 30 min. The carbonyl content was then determined spectrophotometrically at 370 nm.
2.5 Antioxidant enzyme activity measurement
Crude enzymes were extracted from fresh livers of control and treated samples that were ground in liquid nitrogen and suspended in 50 mM Ki-PBS buffer (pH 7.5). The homogenate was centrifuged at 12,000×g for 10 min at 4°C, and the supernatant was immediately collected for SOD, CAT, and GPX activity assays. The protein content of the goldfish livers was determined using the Bradford method [27] with bovine serum albumin as the standard.
SOD activity was determined based on the ability of the enzyme to inhibit the reduction of NBT [28]. One unit of SOD was defined as the amount of protein required to decrease the reference rate to 50% of maximum inhibition at 560 nm.
CAT activity was assayed spectrophotometrically using the method of Aebi [29], by monitoring the H2O2 decomposition from the decrease of absorbance at 240 nm.
GPX activity was assayed spectrophotometrically by measuring the decrease in NADPH absorbance measured at 340 nm, via a coupled reaction with glutathione reductase [30].
2.6 GSH content measurement
Fresh goldfish livers were ground in liquid nitrogen and suspended in TCA (5%). After centrifugation at 12,000×g for 15 min at 4°C, the supernatant was immediately collected and used to measure the GSH content. The GSH content was measured using the spectrophotometric method of Sedlak and Lindsay [31]. DTNB turns dark yellow via reduction with sulfhydryl compounds. The absorbance was read at 412 nm against a GSH standard curve.
2.7 Data analysis
All experiments were conducted using a completely randomized design, with three replicates per treatment. All data were analyzed using Duncan’s multiple range test using SPSS 13.0 software (IBM Cop., Armonk, NY, USA) at p < 0.05.
3 Results
3.1 Effects of NaCl on body weight and SR
As shown in Table 1, the effects of three food supply levels on body weight were investigated in goldfish during 4 weeks under hypoxic conditions. Feeding with 50 mg per fish per day for 4 weeks decreased the body weight by approximately 18.7 and 22.1% in the goldfish in the water control and NaCl supplemented groups, respectively (Table 1; p < 0.05). In contrast, feeding with 400 mg per fish per day for 4 weeks increased the body weight by approximately 35 and 33% in goldfish in the water control and NaCl supplemented groups, respectively, under hypoxia conditions (Table 1; p < 0.05). However, no significant differences were detected for the NaCl-treated goldfish compared with the water control at the medium nutrition level.
Effects of NaCl on goldfish body weight
| Week 1 | Week 2 | Week 3 | Week 4 | |
|---|---|---|---|---|
| Control-50 | 25.6 ± 0.9a | 23.9 ± 1.2cd | 22.9 ± 0.8c | 20.8 ± 1.1c |
| NaCl-50 | 25.3 ± 0.7a | 23.4 ± 0.7d | 21.8 ± 0.5c | 19.7 ± 0.7c |
| Control-200 | 25.3 ± 1.2a | 25.1 ± 0.9c | 24.8 ± 1.1b | 24.6 ± 0.8b |
| NaCl-200 | 25.2 ± 0.9a | 24.9 ± 1.1cd | 24.8 ± 0.7b | 24.5 ± 0.8b |
| Control-400 | 25.4 ± 1.1a | 28.8 ± 0.7a | 31.8 ± 0.9a | 34.3 ± 1.1a |
| NaCl-400 | 25.2 ± 0.8a | 27.2 ± 0.9b | 30.4 ± 1.1a | 33.5 ± 0.9a |
Means associated with the same letter are not significantly different within each time-point (n = 3; p > 0.05).
Effects of 50 mM NaCl on the body weight of goldfish at three food supply levels (50, 200, and 400 mg day−1 fish−1) after treatment for 1, 2, 3, and 4 weeks under hypoxic conditions.
Compared with the water control, NaCl enhanced the SR by approximately 38, 19, and 83% in goldfish at the low, medium, and high nutrition levels, respectively, after 4 weeks (Table 2; p < 0.05). Without NaCl treatment, feeding with 200 mg per fish per day enhanced the SR by approximately 29 and 100% compared with the 50 and 400 mg food groups, respectively, after 4 weeks under hypoxic conditions (Table 2; p < 0.05). After supplementation with NaCl, feeding with 200 mg per fish per day enhanced the SR by approximately 11 and 34% compared with the 50 and 400 mg food groups, respectively, after 4 weeks under hypoxic conditions (Table 2; p < 0.05).
Effects of NaCl on the goldfish SR
| Week 1 | Week 2 | Week 3 | Week 4 | |
|---|---|---|---|---|
| Control-50 | 95.3 ± 2.6a | 89.4 ± 2.8b | 82.3 ± 1.6c | 61.6 ± 2.3e |
| NaCl-50 | 96.6 ± 1.6a | 94.6 ± 2.3a | 88.6 ± 2.3b | 85.3 ± 1.6b |
| Control-200 | 96.3 ± 1.6a | 90.3 ± 1.3b | 84.6 ± 2.6bc | 79.3 ± 1.6c |
| NaCl-200 | 97.3 ± 1.3a | 95.6 ± 1.6a | 95.3 ± 1.3a | 94.6 ± 2.3a |
| Control-400 | 93.6 ± 2.6a | 80.3 ± 2.6d | 56.6 ± 2.3e | 38.6 ± 3.3f |
| NaCl-400 | 94.6 ± 2.3a | 87.6 ± 2.3c | 78.3 ± 1.6d | 70.6 ± 1.6d |
Means associated with the same letter are not significantly different within each time-point (n = 3; p > 0.05).
Effects of 50 mM NaCl on the SR of goldfish at three food supply levels (50, 200, and 400 mg day−1 fish−1) after treatment for 1, 2, 3, and 4 weeks under hypoxic conditions.
3.2 Effects of NaCl on oxidative damage and antioxidant capacity
The effects of NaCl on lipid peroxidation (evaluated by the TBARS content) and protein carbonylation (evaluated by the carbonyl content) were investigated in goldfish at three food supply levels under hypoxic conditions (Table 3). The results showed that applying 50 mM NaCl reduced the TBARS content by approximately 44, 36, and 25% in goldfish livers from the high, medium, and low nutrition groups, respectively, under hypoxic conditions (p < 0.05). Similar change patterns were also observed for protein carbonylation (Table 3). Treatment with 50 mM NaCl decreased the carbonyl content by 25, 18, and 27% in goldfish livers from the high, medium, and low nutrition groups, respectively, under hypoxic conditions (Table 3; p < 0.05).
Effects of NaCl on oxidative damage and the antioxidant capacity
| 50 mg group | 200 mg group | 400 mg group | |
|---|---|---|---|
| Lipid damage | 0.295 ± 0.012b | 0.263 ± 0.013c | 0.364 ± 0.015a |
| + NaCl | 0.222 ± 0.011d | 0.168 ± 0.008e | 0.203 ± 0.014d |
| Protein damage | 1.76 ± 0.12b | 1.47 ± 0.08c | 2.07 ± 0.11a |
| + NaCl | 1.28 ± 0.11d | 1.21 ± 0.09d | 1.56 ± 0.13bc |
| SOD activities | 14.1 ± 1.1b | 12.4 ± 0.5c | 8.5 ± 1.3d |
| + NaCl | 17.5 ± 0.9a | 17 ± 0.7a | 15 ± 0.8b |
| CAT activities | 7.84 ± 0.24c | 7.31 ± 0.22d | 5.62 ± 0.44e |
| + NaCl | 9.12 ± 0.16a | 8.62 ± 0.31b | 7.71 ± 0.25cd |
| GPX activities | 18.5 ± 1.3b | 15.5 ± 0.8c | 14.9 ± 1.1c |
| + NaCl | 21.1 ± 1.2a | 19.5 ± 0.7ab | 19.1 ± 0.6b |
| GSH content | 16.42 ± 0.88bc | 13.71 ± 0.69d | 11.38 ± 0.62e |
| + NaCl | 19.52 ± 0.84a | 17.51 ± 0.72b | 15.42 ± 0.74c |
Means associated with the same letter are not significantly different for each parameter (n = 3; p > 0.05). SOD, superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase; GSH, glutathione.
Effects of NaCl treatment on oxidative damage (lipid peroxidation and protein carbonylation) and the antioxidant capacity (SOD, CAT, and GPX activities, and GSH content) of goldfish at three food supply levels (50, 200, and 400 mg day−1 fish−1) under hypoxic conditions.
The results also showed that NaCl treatment markedly enhanced SOD, CAT, and GPX activities and the GSH content in goldfish livers at the three food supply levels under hypoxic conditions (Table 3). NaCl increased SOD activities by approximately 76, 36, and 25% in goldfish in the high, medium, and low nutrition groups, respectively, under hypoxic conditions (Table 3; p < 0.05). Similarly, NaCl enhanced CAT and GPX activities by approximately 38 and 27%, respectively, in goldfish in the high nutrition group (Table 3; p < 0.05). Treatment with NaCl enhanced the GSH content by approximately 35, 28, and 19% in goldfish livers from the high, medium, and low nutrition groups, respectively, under hypoxic conditions (Table 3; p < 0.05).
3.3 Effects of NaCl on the H2O2 content
Treatment with NaCl markedly affected the H2O2 content in goldfish livers under hypoxic conditions. Compared with that at 0 day, in goldfish fed with the 400, 200, and 50 mg diet for 7 days, their H2O2 contents increased by approximately 18, 5, and 8%, respectively, without NaCl treatment under hypoxic conditions (Figure 1; p < 0.05). Compared with that at 0 h, applying 50 mM NaCl induced an acute increase (65–89%) in H2O2 during the first day, which then declined to lower levels in the goldfish livers under hypoxic conditions (Figure 1; p < 0.05). Compared with that at 0 day, NaCl treatment decreased the H2O2 content by approximately 30, 28, and 30% in livers of goldfish in the high, medium, and low nutrition groups, respectively, after 7 days under hypoxic conditions (Figure 1; p < 0.05).
Effects of NaCl on H2O2 content. Effects of NaCl addition on the H2O2 content in the livers of goldfish at high, medium, and low nutrition levels within the first 7 days under hypoxic conditions. Bars represent the standard deviation of the mean (n = 3); means associated with the same letter are not significantly different (p > 0.05).
3.4 Effect of autophagy inhibition on the SR and oxidative damage
As shown in Figure 2a, inhibition of autophagy using 50 µM HCQ drastically reduced the SR of the goldfish at three nutrition levels under hypoxic conditions. Treatment with HCQ reduced the SR by approximately 50, 44, and 75% in goldfish without NaCl treatment at high, medium, and low nutrition levels, respectively, after 4 weeks under hypoxic conditions (Figure 2a; p < 0.05). However, applying 50 mM NaCl enhanced the SR of the HCQ-treated goldfish by approximately 50, 44, and 33% at high, medium, and low nutrition levels, respectively, after 4 weeks under hypoxic conditions (Figure 2a; p < 0.05).
Effects of inhibition of autophagy on the SR and oxidative damage. Effects of inhibition of autophagy by HCQ on the SR (a) after treatment for 4 weeks, lipid damage (b), and protein damage (c) in the livers of goldfish at high, medium, and low nutrition levels after treatment for 7 days under hypoxic conditions. Bars represent standard deviation of the mean (n = 3); means associated with the same letter are not significantly different (p > 0.05). HCQ, hydroxychloroquine.
Moreover, the results showed that HCQ treatment enhanced the TBARS content by approximately 93, 73, and 166% in livers of goldfish without NaCl treatment at high, medium, and low nutrition levels, respectively, under hypoxic conditions (Figure 2b; p < 0.05). In addition, NaCl treatment reduced the TBARS content by approximately 31, 17, and 26% in the livers of HCQ-treated goldfish at high, medium, and low nutrition levels, respectively, under hypoxic conditions (Figure 2b; p < 0.05). Similar change patterns were also observed for protein carbonylation (Figure 2c). The application of NaCl reduced the carbonyl content in the livers of HCQ-treated goldfish by approximately 21, 26, and 17% at high, medium, and low nutrition levels, respectively, under hypoxic conditions (Figure 2c; p < 0.05).
3.5 Effects of autophagy inhibition on enzyme activities and the GSH content
Application of HCQ dramatically enhanced the SOD, CAT, and GPX activities and the GSH content in the livers of goldfish under hypoxic conditions (Figure 3). Treatment with HCQ increased the SOD, CAT, and GPX activities and GSH content by approximately 171, 157, 100, and 168%, respectively, in the livers of goldfish from the high nutrition level group under hypoxic conditions (Figure 3; p < 0.05). However, the HCQ-induced increase in enzyme activities and the GSH content were attenuated by NaCl (Figure 3; p < 0.05). Applying NaCl reduced the SOD, CAT, and GPX activities by approximately 11, 14, and 13%, respectively, in the livers of HCQ-treated goldfish in the high nutrition level group under hypoxic conditions (Figure 3a–c; p < 0.05). Similarly, NaCl decreased the GSH content by approximately 12, 15, and 14% in the livers of HCQ-treated goldfish at high, medium, and low nutrition levels, respectively, under hypoxic conditions (Figure 3d; p < 0.05).
Effect of inhibition of autophagy on antioxidant capacity. Effects of the inhibition of autophagy by HCQ on the activities of SOD (a), CAT (b) and GPX (c), and content of GSH (d) in the livers of goldfish in the high, medium, and low nutrition level group after treatment for 7 days under hypoxic conditions. Bars represent the standard deviation of the mean (n = 3); means associated with the same letter are not significantly different (p > 0.05). HCQ, hydroxychloroquine; SOD, superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase; GSH, glutathione.
4 Discussion
In addition to nutrition stress, hypoxia is another important threat to fish growth and survival [32–34]. In the present study, all the experiments were conducted under hypoxic conditions. However, effects of nutrition stress on fish growth and survival under hypoxia conditions are not well studied.
Herein, feeding goldfish with 200 mg food did not drastically alter their body weight under hypoxic conditions. This indicated that 200 mg per fish per day could be considered as a half-starving state (defined as the “medium nutrition group”) for goldfish. However, application of 400 and 50 mg food profoundly enhanced (29–35%) or reduced (19–23%) the fish body weight, respectively (Table 1; p < 0.05). This showed that 400 mg per fish per day represented a satiated state (defined as the “high nutrition group”) and 50 mg food per fish per day represented a starvation state (defined as the “low nutrition group”) under hypoxic conditions. Interestingly, food supply levels also drastically affected the SR of goldfish under hypoxic conditions, in the order medium nutrition group (∼80%) > low nutrition group (∼60%) > high nutrition group (∼40%) (Table 2). The high death rate (∼20%) observed in the medium nutrition group could be partly attributed to the low dissolved O2 (3.5–5.4 mg L−1) in tap water under hypoxic conditions. In general, the normoxic level should be maintained at 10 mg L−1 for goldfish [20]. Compared with the medium and low nutrition groups, supplying high nutrition levels (400 mg per fish per day) resulted in the lowest SR (∼40%) under hypoxic conditions (Table 2; p < 0.05). This phenomenon could be partly explained by the free radical theory of aging [35]. The theory considers that high caloric diets can produce more free radicals/ROS and reduce life expectancy. Here, the satiated goldfish (high nutrition group) might generate increased free radicals/ROS, which would impair the health and reduce the lifespan/SR of the goldfish. Accordingly, severe oxidative damage (e.g., lipid peroxidation and protein carbonylation) was recorded in liver tissues of goldfish in the order high nutrition group > low nutrition group > medium nutrition groups (Table 3; p < 0.05). This was partly consistent with previous reports, which showed that a high fat diet induced high accumulation of TBARS and carbonyl in turtles compared with those in the normal feeding group [16]. It also showed that oxidative damage (high nutrition group > low nutrition group > medium nutrition group) has a close negative correlation with the SR (medium nutrition group > low nutrition group > high nutrition group) in goldfish under hypoxia conditions (Tables 2 and 3).
Interestingly, beneficial effects of sea salinity were reported to prevent some fish diseases [21,22]. Here, we wanted to know whether NaCl improves the growth and survival of goldfish at different food supply levels under hypoxic conditions. A previous report showed that only high salinity (>2‰ NaCl) could inhibit goldfish growth [21]. In this study, 50 mM NaCl (0.06% or 0.6 ppt, which is close to the salinity of tap water) was applied to goldfish and no significant difference was detected in the body weight of the goldfish at the same nutrition level (Table 1). However, applying NaCl dramatically enhanced the SR of goldfish, especially in the high nutrition level group, under hypoxic conditions (Table 2). Moreover, this nutritional stress-induced oxidative damage, such as lipid peroxidation and protein carbonylation, could be alleviated by NaCl (Table 3; p < 0.05). We then investigated the possible underlying mechanisms.
Hypoxia, as well as nutrition stress, could trigger the antioxidant system in aquatic animals [12,15–17,20]. Moreover, published data showed that high and low salinity could decrease or increase antioxidant enzyme activities, respectively, in freshwater fish [36,37]. This suggested that the NaCl-enhanced SR might be associated with activation of the antioxidant system in goldfish under hypoxic conditions. Research showed that salinity-mediated redox signaling could activate the antioxidant system in aquatic animals [38]. Here, we speculated that the exogenous NaCl might also trigger ROS signaling in goldfish at different food supply levels under hypoxic conditions. To test this speculation, the effect of 50 mM NaCl on the H2O2 content was measured in goldfish livers. Without NaCl treatment, nutrition stress (e.g., starvation) increased the H2O2 content in goldfish livers to different extents after 2 days (Figure 1, p < 0.05). However, NaCl addition induced an acute increase (∼100%) of H2O2 within 24 h, which then declined to a relative low level in goldfish livers (Figure 1, p < 0.05). Previous research showed that an increase in salinity could cause a decrease in O2 consumption, but an increase of H2O2 levels in crabs [2,39]. This showed that exogenous NaCl could induce a ROS burst, which might act as a signal [38,40] in goldfish livers during the early stage (within the first 24 h). To determine whether this ROS burst could activate antioxidant system in goldfish livers, the antioxidant enzyme (SOD, CAT, and GPX) activities and the antioxidant (GSH) content in goldfish livers were measured (Table 3).
Compared with the high nutrition group, starvation (50 mg group) markedly enhanced SOD, CAT, and GPX activities, and the GSH content in goldfish livers (Table 3; p < 0.05). This was consistent with published data, which showed that glucose restriction enhanced antioxidant gene expression and improved cell survival of the protozoan parasite Trichomonas vaginalis [41]. Similarly, starvation-induced SOD, CAT, and GPX activities were also reported in Chinese perch [42]. Furthermore, NaCl also markedly increased antioxidant enzyme activities and the GSH content in goldfish livers under hypoxic conditions (Table 3; p < 0.05). This was partly in agreement with previous report, which showed that a slight enhancement of salinity could increase SOD and GPX activities for scavenging ROS to maintain the health of red strain of the oyster Pinctada fucata [43]. Moreover, these enzyme activities were increased to a greater extent in the high nutrition group compared with the starved group after NaCl treatment (Table 3; p < 0.05). This was partly consistent with a previous report, which showed that sufficient food supply induced higher antioxidant enzyme activities compared with fasted fish under salinity stress [44]. Thus, the results indicated that the NaCl-improved SR could be partly attributed to the enhanced antioxidant capacity in the livers of goldfish via triggering the ROS signaling at different food supply levels under hypoxic conditions.
Moreover, reports showed that a high fat diet induced lipid peroxidation and triggered autophagy in the liver of Chinese softshell turtle [16]. In contrast, starvation induced oxidative damage and elicited autophagy in Chinese perch [42,45]. Similarly, glucose restriction triggered autophagy and enhanced cell survival of the protozoan parasite T. vaginalis [41]. In the present study, nutrition stress (50 and 400 mg group) enhanced oxidative damage and reduced the SR in goldfish compared with the water control group under hypoxic conditions (Tables 2 and 3). We speculated that this phenomenon might also be associated with autophagy. To test this, HCQ, an autophagy inhibitor [46], was applied to the goldfish. Research showed that 55–110 µM chloroquine caused high mortality in large yellow croakers [23]. Here, 50 µM HCQ was applied to goldfish and a reduced SR was detected, especially in the low and high nutrition level groups, under hypoxic conditions (Figure 2a; p < 0.05). This showed that autophagy might play an important role in elevating the SR of the goldfish at three food supply levels under hypoxic stress. Interestingly, oxidative stress induction of autophagy and reduction in cell survival was also reported [5]. Herein, the oxidative damage (lipid peroxidation and protein carbonylation) was aggravated by HCQ, especially in the starved goldfish (Figure 2b and c; p < 0.05). Similarly, published data showed that inhibition of autophagy using 3-methyladenine induced ROS and TBARS accumulation in HepG2 cells [47]. This indicated that nutrition stress-induced autophagy is important to alleviate oxidative damage and improve the SR of goldfish under hypoxic conditions.
How does autophagy inhibition affect the antioxidant capacity goldfish livers at different food supply levels under hypoxic stress? Further study showed that inhibition of autophagy by HCQ profoundly enhanced SOD, CAT, and GPX activities, and the GSH content in goldfish livers (Figure 3; p < 0.05). Similarly, a report showed that inhibition of autophagy dramatically increased SOD, CAT, and GPX activities in rats [48]. It showed that nutrition stress triggered autophagy, as well as the elevated antioxidant capacity, playing a synergistic role in alleviating the oxidative damage to enhance the goldfish SR under hypoxia conditions. This raised an interesting question: why did the HCQ-increased antioxidant capacity not enhance the SR of the goldfish under hypoxic conditions? One plausible explanation could be that autophagy inhibition led to the over-production and accumulation of TBARS and carbonyl groups (Figure 2b and c). To alleviate oxidative stress, more resources (e.g., ATP and building block) would be allocated to biosynthesize the antioxidant metabolites in fish cells [4]. However, inhibition of autophagy also impairs the recycling of damaged macromolecules and organelles for biosynthesis of substrates to maintain primary metabolism, which is indispensable for cell survival [4]. Then, the catabolic and anabolic processes would become unbalanced, especially for starving goldfish, after inhibition of autophagy [13]. It also confirmed that autophagy is the last defense mechanism [4], rather than the antioxidant system, to alleviate the effect of nutritional stress on goldfish survival.
However, treatment with NaCl drastically improved the SR, reduced oxidative damage and antioxidant capacity in HCQ-treated goldfish, especially at low nutrition levels, under hypoxic conditions (Figures 2 and 3; p < 0.05). One possible explanation for this phenomenon could be that the exogenous NaCl triggered H2O2 signaling, which could activate the antioxidant system in goldfish livers (Figure 1; Table 3). The increase in antioxidant capacity meant that the HCQ-aggravated oxidative damage could be alleviated and goldfish survival would be improved under hypoxia conditions. Moreover, a previous report showed that the rapid change of salinity concentration could also induce autophagy in fish cells [13]. Herein, the inhibitory effects of HCQ might have been attenuated by the rapid addition of NaCl in tap water. Thus, the HCQ-reduced survival could also be partly reversed by NaCl (Figure 2a). Based on the above-mentioned data, we suggested that the NaCl-triggered antioxidant system, coupled with autophagy, played a synergistic effect in elevating goldfish survival at different food supply levels under hypoxic conditions.
To better interpret our results, we constructed a hypothetical model (Figure 4). Nutritional stress slowly increased the accumulation of ROS, which induced severe oxidative damage and impaired goldfish health. NaCl addition induced an acute increase in ROS and triggered the antioxidant system. However, this NaCl-enhanced goldfish SR could be markedly attenuated by inhibiting autophagy.
Hypothetical model. A hypothetical model based on ROS signaling was proposed to illustrate how NaCl addition enhanced the antioxidant defense ability and SR of goldfish at starvation or satiated levels under hypoxic conditions. The sharp and blunt arrows represent the positive and negative effects, respectively. For details, see the text. ROS, reactive oxygen species; HCQ, hydroxychloroquine; SR, survival rate.
Some challenges remain. For example, further exploration of the detailed mechanism by which NaCl regulates autophagy in the livers of goldfish under hypoxia and nutrition stress conditions is required.
5 Conclusions
In this study, hypoxia severely reduced the goldfish SR, even at the medium nutrition level. However, supplementation with NaCl markedly enhanced the goldfish SR at different nutrition levels under hypoxic conditions. Our results allowed us to draw interesting conclusions. First, high nutrition drastically increased the body weight, but decreased the SR of goldfish compared with those in the control groups under hypoxic conditions. Second, starvation profoundly reduced the body weight, but enhanced the antioxidant capacity in goldfish, compared with those in the satiation group. Third, NaCl addition markedly promoted the SR and inhibited oxidative damage in goldfish at the three food supply levels under hypoxic conditions. Fourth, the application of NaCl induced an acute increase in H2O2, coupled with enhanced antioxidant capacity, in goldfish at the three food supply levels under hypoxic conditions. Finally, inhibition of autophagy by HCQ enhanced the antioxidant capacity and reduced the SR, which could be dramatically attenuated by NaCl. These findings provide insight into the roles of NaCl in regulating goldfish survival at different food supply levels under hypoxic stress from the antioxidant defense and autophagy perspectives.
Acknowledgements
The authors would like to thank Benliang Deng (PhD) for his advices in this work.
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Funding information: This study was supported by a grant from the Key Project of Henan High Education (grant number 15A180008 to Xueyi Yang), the National Natural Science Foundation of China (grant number U1504323 to Mingyan Shi).
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Author contributions: Xueyi Yang and Mingyan Shi: conceptualization, funding, and supervision; Xueyi Yang, Jing Zhao, and Yumeng Zhang: investigation, writing, and editing the original draft; Zhiyong Pan, Xiaowen Xu, Yingye Weng, and Xinyu Su: investigation, data curation, and methodology.
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Conflict of interest: The authors declare no competing interest.
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Ethical approval: This study was conducted according to the Guidelines for the Laboratory Animal Use and Care Committee of the Ministry of Health, China and the Welfare Committee of Luoyang Normal University (No. 12009158). The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
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Data availability statement: The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
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