Introduction

Oxidative stress is the process of generating and accumulating reactive oxygen intermediates, which results in a burst in the production of reactive oxygen species (ROS), leading to steady-state alterations in the body as the result of oxidative damage (Lesser 2006; Lushchak 2011). ROS has oxidative properties for many macromolecular substances, such as proteins, lipids, and DNA, and it can directly cause oxidative stress damage to the body (Snezhkina et al. 2019; Tanaka et al. 2006). Temperature is an important environmental factor with significant effects on organisms, and extreme temperature conditions can result in harmful effects, such as lipid peroxidation (LPO). Acute shifts in temperature can also result in failure to maintain the steady levels of ROS required for metabolism, which leads to aggravated stress levels (Lushchak 2011; Tseng et al. 2011).

Aquatic animals have evolved an efficient antioxidant defense system (ADS) to remove ROS and resist oxidative stress (Guo et al. 2018). The ADS consists of antioxidant enzymes including glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), and non-enzymatic antioxidant molecules such as glutathione (GSH) (Dadras et al. 2016; Lin et al. 2018; Wilhelm Filho 1996). Hence, the activity of antioxidant enzymes and the content of non-enzymatic antioxidant molecules can reveal the oxidative status in cells (Dadras et al. 2016).

As poikilothermic animals living in water, fish will produce a stress response when the ambient temperature exceeds the optimal range. If the limit of self-regulation is exceeded, the body's immune system will be destroyed, leading to increased morbidity and mortality. Studies have shown that long-term exposure to increased temperature induces the expression of genes such as heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) in adult zebrafish and the phosphorylation of stress-activated protein kinases in gilthead seabream Sparus aurata (Feidantsis et al. 2009; Malek et al. 2004). Variation in temperature was found to affect the physiological status of both fish cells such as RTH-149 cells and organisms such as green sturgeon Acipenser medirostris and killifish (Babich et al. 1993; Lankford et al. 2003; Leggatt et al. 2007). It has been reported that in hepatic cells of grass carp, heat stress can increase the level of lipid peroxide and destroy antioxidant homeostasis (Cui et al. 2013). Moreover, when fish are exposed to adverse environmental conditions, for survival they will adapt to the new environment, and in that process the biosome produced accommodation; however the thermal adaption process in fish is complex. In most of the world's oceans as well as in freshwater, living organisms are suffering deadly consequences of global warming (Rossati 2017). In catfish Heteropneustes fossilis, for example, it was reported that when the water temperature increased, regardless of the sex of the individual catfish, the level of lipid peroxide in the respiratory apparatus increased. However, after exposure to the higher temperature, the ascorbic acid content in the air sac of the fish was decreased (Parihar and Dubey 1995). Bagnyukova et al. (2007) also noted changes to several oxidative stress markers associated with glutathione-dependent enzymes following exposure of goldfish Carassius auratus auratus to warm temperature stress (a change from 3 to 23 °C), which included a rapid increase in lipid peroxide levels and a 20–40% reduction in protein carbonyl content in the liver. Furthermore, the effects of temperature reduction on gene expression in adult zebrafish skeletal muscle indicated that approximately 600 genes related to the oxidative stress response were upregulated by 1.7-fold or greater (Malek et al. 2004).

As mentioned, most organisms adapt to oxidative stress by increasing antioxidant potential, as has been reported in killifish, three-spined stickleback, green sturgeon, brown trout, and goldfish (Lankford et al. 2003; Leggatt et al. 2007; Lushchak and Bagnyukova 2006; Murphy et al. 2005; Kammer et al. 2011; Hansen et al. 2006).

For further details on the process of ADS in acute temperature stress response, here the process was examined using fish cells, considering that fish cells live in a wide range of temperatures, such as grass carp Ctenopharyngodon idellus kidney (CIK) cells, which can live in the range of 10–40 °C. In addition, the kidney has a powerful ADS, and is a suitable model for studying temperature and oxidative stress.

Therefore, this study explored the effects of temperature on cellular antioxidant enzymes and oxidative damage indicators in CIK cells, which is a more accurate approach to understand the ADS response of the fish to temperature stress.

Materials and methods

Cell culture

CIK cells were purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, and then cultured with medium M199 (Gibco, USA), supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 mg/ml streptomycin, and grown at 28 °C in 5% CO2. All experimental operations adhered to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Huazhong Agricultural University, Wuhan, China.

Temperature stress

After culturing at 28 °C for 2 weeks, the CIK cells (1 × 106 cells/well) were collected with 0.25% trypsin–EDTA and divided into five groups, with three replicate bottles in each group. A temperature of 28 °C was set as the control, and the incubator temperature was continuously increased or decreased relative to 28 °C, at a rate of 1 °C every 15 min. CIK cells were exposed to different temperatures (20, 24, 28, 32, 36 °C) for 24 h.

Reactive oxygen species (ROS) assay

The cells (1 × 106 cells/well) were seeded into a 25-mm2 culture dish. After temperature stress, the cells were incubated with medium containing 2′,7′-dichlorofluorescein diacetate (DCFH-DA; 10 μmol/l) (Sigma-Aldrich, USA) at 37 °C for 20 min, then washed twice with PBS and resuspended. The levels of ROS were tested using a fluorescence microplate reader (SpectraMax i3X, Molecular Devices), at an emission wavelength of 530 nm and excitation wavelength of 488 nm.

Mitochondrial membrane potential (\(\Delta \Psi {\text{m}}\)) assay

The cells (1 × 106 cells/well) were exposed to different temperatures for 24 h, then incubated with medium containing Rh123 (1 μg/ml) (Sigma, USA) at 37 °C for 15 min, washed three times with PBS, and then resuspended. The fluorescence activity of Rh123 was tested using a fluorescence microplate reader, at an emission wavelength of 534 nm and excitation wavelength of 504 nm.

Oxidative stress indicator analysis

The activity of SOD, CAT, GPx, glutathione reductase (GR), and xanthine oxidase (XOD), the content of GSH and malondialdehyde (MDA), and the total antioxidant capacity (TAC) were measured according to the instructions of commercially available kits (Jiancheng, Nanjing, China). Protein carbonyl was measured by reaction with 2,4-dinitrophenylhydrazine (DNPH) (Lushchak et al. 2005). SOD assay was based on the method described by Beyer and Fridovich (1987). CAT activity was determined by the rate of disappearance of H2O2 according to Aebi (1984), with slight modifications. GPx activity was measured using GSH as a substrate to measure the conjugation of GSH and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (Drotar et al. 1985). MDA content was used as an index of LPO and was determined according to Ohkawa et al. (1979). TAC was determined using the FRAP (ferric reducing/antioxidant power) assay described by Benzie and Strain (1999). Protein content was determined by the Coomassie blue method using bovine serum albumin (BSA) as a standard.

Total RNA extraction and quantitative real-time PCR analysis

RNAiso Plus (Takara Bio, Dalian, China) was used to extract total RNA from CIK cells according to the manufacturer’s instructions. The RNA samples were quantified spectrophotometrically with a NanoDrop® 2000 (Thermo Fisher Scientific), and 1 μg RNA was used to synthesize the cDNA.

The reaction mixture contained 12.5 μl SYBR Premix Ex Taq™II (Perfect Real Time) (Takara Bio, Dalian, China), 0.5 μl of each forward and reverse primer, and 9.5 μl ddH2O. The primers used in the experiment are shown in Table 1. The reaction procedure was as follows: pre-denaturation at 95 °C for 5 min, then 35 cycles of 95 °C for 30 s, 95 °C for 5 s, 60 °C for 30 s, and 72 °C 30 s. The transcript levels of related genes were quantified with the 28 °C group as a positive control, and eif1a was the reference gene, the data were calculated using \(2^{{ - \Delta \Delta C_{{\text{t}}} }}\) (Livak and Schmittgen, 2001).

Table 1 Primers used for quantitative real-time PCR
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Statistical analysis

Data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test; the statistical analysis was performed using SPSS 20.0 software. A value of P < 0.05 was considered statistically significant. All data were expressed as mean ± SD (standard deviation).

Results

Reactive oxygen species (ROS)

The ROS level at 28 °C (control) was the lowest. When the temperature was reduced from 28 to 24 °C, the level of ROS increased significantly (P < 0.05). No significant change was observed with a decrease from 24 to 20 °C. In the heat treatment groups, the level of ROS increased significantly when the temperature increased from 28 to 32 °C (P < 0.05), but it showed no significant change from 32 to 36 °C. Compared with the control group, all temperature treatment groups except the 20 °C group showed significant differences in ROS levels (P < 0.05; Fig. 1).

Fig. 1
figure 1

The intracellular fluorescence relative intensity of dichlorofluorescein (DCF) in CIK cells after exposure to different temperatures. Significant differences between different temperature are indicated with different case letters (P < 0.05). All values represent the mean ± SD (n = 9)

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Oxidative damage state indicator analysis

The \(\Delta \Psi {\text{m}}\) is represented by the relative fluorescence intensity ratio of Rh123. The \(\Delta \Psi {\text{m}}\) is stable in normal cells, and is decreased or lost when the membrane is damaged (Halestrap 2010). Compared with the control group, the Rh123 fluorescence ratio of cells increased significantly in the cold groups (P < 0.05). In the of heat treatment process, the ratio increased significantly when the temperature increased from 28 to 32 °C, while the ratio decreased significantly from 32 to 36 °C (P < 0.05; Fig. 2a).

Fig. 2
figure 2

The ratio of Rh123 fluorescence (a) and the content of MDA (b) and protein carbonyl (c) in CIK cells after exposure to different temperatures. Significant differences between temperatures are indicated with different case letters (P < 0.05). All values represent the mean ± SD (n = 9)

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The MDA content showed no significant difference between 28 and 24 °C. When the temperature decreased from 28 to 20 °C, the MDA content increased significantly (P < 0.05). In the heat treatment process, the MDA content increased significantly when the temperature increased from 28 to 32 °C (P < 0.05), but showed no difference with a temperature increase from 32 to 36 °C (Fig. 2b). The protein carbonyl content at 32 °C was 1.70 times higher than that at 28 °C (P < 0.05). Compared with the control group, the protein carbonyl content was significantly decreased in the 20 and 24 °C groups (P < 0.05; Fig. 2c).

Antioxidant relative enzyme indicator analysis

The SOD activity of CIK cells was lowest in the 28 °C group; when the temperature was increased to 32 °C, SOD activity increased significantly (P < 0.05). There was no significant difference when the temperature increased from 32 to 36 °C, whereas SOD activity showed a significant increase when the temperature decreased from 24 to 20 °C (P < 0.05; Fig. 3a).

Fig. 3
figure 3

The intracellular activity of SOD (a), GPx (b), CAT (c), GR (d), and XOD (e) in CIK cells after exposure to different temperatures. Significant differences between different temperatures are indicated with different case letters (P < 0.05). All values represent the mean ± SD (n = 9)

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The GPx activity of CIK cells was lowest in the 28 °C group. During the period of cold acclimatization, GPx activity showed no significant change from 28 to 24 °C, but it increased with an increase in temperature from 24 to 20 °C. However, the increase was lower than that in the warm acclimatization groups. The GPx activity of cells in the 32 °C group was 1.91 times that of the 28 °C group, and 1.24 times that in the 20 °C group (P < 0.05). The GPx activity in the 36 °C group was the highest (Fig. 3b).

The CAT activity of CIK cells was relatively low in the 28 and 32 °C groups, with no significant difference between them. In the heat treatment groups, when the temperature increased from 32 to 36 °C, the CAT activity increased significantly (P < 0.05). On the other hand, CAT activity also increased significantly when the temperature decreased from 28 to 24 °C (P < 0.05). The CAT activity of cells incubated at 20 °C increased by 2.12 times on average compared with that in the 28 °C group (P < 0.05; Fig. 3c).

The GR activity increased significantly when the incubation temperature increased from 28 to 32 °C (P < 0.05), and showed no significant difference between the 32 and 36 °C groups. In the cool treatment groups, the GR activity increased significantly when the temperature decreased from 28 to 24 °C, while it decreased significantly when the temperature decreased from 24 to 20 °C (P < 0.05; Fig. 3d).

There was no significant difference in XOD activity among the 24, 28, and 36 °C groups. Compared with the control group, when the temperature decreased to 20 °C, the XOD activity of cells increased significantly (P < 0.05). XOD activity in the 32 °C group was the greatest, and was significantly higher than that in other groups (P < 0.05; Fig. 3e).

Oxidative-reductive state indicator analysis

The two indicators in Fig. 4 represent the oxidative and reductive state in the CIK cells. TAC is the total antioxidant capacity of the cells, and there were no significant differences among the 24, 28, and 32 °C groups. When the temperature was increased from 32 to 36 °C, the TAC declined (P < 0.05). Interestingly, when the incubation temperature decreased from 28 to 20 °C, the TAC at 20 °C was significantly higher than that in other groups (P < 0.05), and was 3.40 times higher than the 28 °C group (Fig. 4a).

Fig. 4
figure 4

The levels of TAC (a) and GSH (b) in the CIK cells after exposure to different temperatures. Significant differences between different temperatures are indicated with different case letters (P < 0.05). All values represent the mean ± SD (n = 9)

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As shown in Fig. 4b, the GSH content was lowest in the 28 °C group. When the temperature was increased to 32 °C, the GSH content increased, but there was no significant difference between the 32 and 28 °C groups. A significant increase was seen when the temperature was increased to 36 °C (P < 0.05). In the cold stress process, the GSH content increased. The GSH content was significantly higher in the 20 °C group than in the 24, 28, and 32 °C groups (P < 0.05), but there was no significant difference between the 20 and 36 °C groups.

Quantitative real-time PCR assay for target gene expression

For the transcript levels of the cat gene, there was no significant difference among the 20, 24, and 32 °C groups. In the cold treatment, the transcript levels of cat in the 20 and 24 °C groups was significantly higher than that in the control group (P < 0.05). In the heat treatment groups, the expression level of the cat gene was significantly increased when the temperature increased from 28 to 32 °C (P < 0.05), and continued increasing when the temperature was increased from 32 to 36 °C (P < 0.05; Fig. 5a).

Fig. 5
figure 5

The relative transcript levels of Cu–Zn sod (a), gpx (b), and cat (c) in the CIK cells after exposure to different temperatures. Significant differences between different temperatures are indicated with different case letters (P < 0.05). All values represent the mean ± SD (n = 9)

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The expression level of gpx in the 28 and 32 °C groups was lower than that in other groups, and there was no significant difference between the two groups. When the temperature increased from 32 to 36 °C, the expression level of gpx was significantly upregulated (P < 0.05). With cool treatment, its expression was also significantly upregulated (P < 0.05). There was no significant difference in the expression level of gpx between the 20 and 36 °C groups (Fig. 5b).

Compared with the 20, 24, 32, and 36 °C groups, the Cu/Zn-sod transcript level of the 28 °C group was the lowest. When the temperature increased to 32 and 36 °C, the transcript level of Cu/Zn-sod was upregulated, and there was no significant difference between the 32 and 36 °C groups. In the cool treatment process, the transcript level of Cu/Zn-sod was upregulated when the temperature decreased from 28 to 24 °C (P < 0.05). When the temperature decreased from 24 to 20 °C, the transcript level of Cu/Zn-sod was further enhanced, and was significantly higher than that in the 24 °C group (P < 0.05; Fig. 5c).

Discussion

In this experiment, antioxidant enzymes and oxidation indicators were investigated as research markers to demonstrate the effects of temperature stress on CIK cells (Klein et al. 2017; Van der Oost et al. 2003).

Temperature had immediate stress effects on CIK cell antioxidant status, and one of the most important effects was reflected in the elevated ROS content. ROS content was at the lowest level when CIK cells were incubated at 28 °C, and increased significantly when the incubation temperature was increased and decreased. Similar to our study, Cheng et al. (2015) found that high-temperature exposure (34 °C) increased the ROS level in the blood cells of pufferfish (Takifugu obscurus). ROS levels in grass carp serum increased significantly at 30 and 33 °C compared with that at 28 °C (Luo et al. 2017). High temperature improved the oxygen requirement and accelerated the metabolism of cells to increase the ROS level (An and Choi, 2010; Scaion et al. 2008). Xu et al. (2016) found that cold temperatures of 18 and 10 °C induced the production of ROS in a zebrafish embryo fibroblast-like cell line (ZF4 cells). Decreased temperature weakened the ADS and led to the accumulation of ROS molecules (Malek et al. 2004; Marchant et al. 2008; Niyogi et al. 2001). Acute temperature changes disturb the steady state of cells and generate excessive ROS that cannot be promptly cleared by the ADS.

The increase in the ROS level caused biological macromolecules such as lipids and proteins to be attacked (Kültz 2005). Rh123 is a cationic fluorescent dye that can penetrate the cell membrane and is an indicator of \(\Delta \Psi {\text{m}}\). At 28 °C, the Rh123 fluorescence ratio remained at the normal level, and most of the cell mitochondrial membrane remained intact. When the temperature was increased and decreased, the Rh123 fluorescence ratio increased significantly compared with that in the 28 °C group. The increased ratio of Rh123 fluorescence indicated that the mitochondria were destroyed and \(\Delta \Psi {\text{m}}\) decreased (Halestrap 2010). When the temperature changed, some of the components of the mitochondrial membranes such as proteins and lipids were attacked by the elevated free radicals, leading to a loss of membrane integrity.

ROS molecules attack the lipid molecules, initiating a reaction that generates lipid peroxide, and MDA content accumulates as one of the reaction products (Kailasam et al. 2011). In the present experiment, the increased MDA content in CIK cells was associated with the elevated ROS level. Cui et al (2013) found that the MDA content in grass carp hepatic cells stressed at 32 and 34 °C was significantly higher than that in the control group, which is similar to the present result in CIK cells. Similar to in vitro experiments, the MDA content of catfish exposed to high temperature increased significantly, and cold stress increased the MDA content in the liver of juvenile rabbitfish Siganus guttatus (Qiang et al. 2019; Song et al. 2015; Yang et al. 2018). The variation in temperature induced lipid peroxide in fish both in vivo and in vitro.

Carbonyl protein is a product of proteins attacked by free radicals, whose content reflects the degree of protein damage and oxidative stress status in organisms. The increase in temperature led to elevated ROS concentrations, and the carbonization rate of proteins accelerated, so the protein carbonyl content in CIK cells was significantly higher than that in the control group when the temperature increased (Hansen et al. 2006). The decrease in temperature caused a significant decrease in protein carbonyl content, and this result was not fit with ROS. In contrast to our study, the protein carbonyl level increased during cold acclimation over a period of a few weeks in three-spined stickleback Gasterosteus aculeatus (Kammer et al. 2011). The possible reason for this phenomenon is that the cold stress time was not long enough, and as the material itself, the stress response in fish is more complex than that in CIK cells.

It is widely known that cells have a well-developed ADS to protect against oxidative stress. SOD and CAT are vital components of the ADS, and they can indirectly reflect the body’s ability to eliminate free radicals (Yang et al. 2018). SOD is a metalloenzyme that transforms superoxide anions (O2−) into molecular oxygen (O2) and H2O2, and the H2O2 that is formed is decomposed into H2O and O2 by CAT (Garcia et al. 2008). The present study found that SOD and CAT activity increased significantly with increasing temperature and decreasing CIK cells compared with that in the control group. Similar results were found in L8824 cells and grass carp serum, which suggests that the organisms improved the activity of antioxidant enzymes to protect against temperature stress (Cui et al. 2013; Luo et al. 2017).

The glutathione-related antioxidant system, mainly including glutathione (GSH), glutathione peroxidase (GPx), and glutathione reductase, is an important component of ADS (GR) (Li et al. 2010; Prokopenko et al. 2002). Similar to our temperature stress study, Leggatt et al. (2007) also found that increased temperature promoted GSH content in a rainbow trout Oncorhynchus mykiss hepatoma cell line. As an important enzyme in the GSH regeneration process, GR catalyzed oxidized GSH (GSSH) to GSH and functioned indirectly in the periods of free radical metabolism (Coelho et al. 2017). The activity of GR in CIK cells increased significantly when the incubation temperature was increased, a trend consistent with the discovery in killifish liver (Leggatt et al. 2007). GPx can use GSH or other reducing equivalents to reduce various hydroperoxides to corresponding hydroxylates (Rocha-Santos et al. 2018). The gradually increasing GPx activity with the changing temperature in CIK cells indicated that the glutathione-related antioxidant system was activated to resist oxidative stress.

TAC has been used as a comprehensive index for evaluating the functional status of cellular ADS, and reflects oxidation resistance capability in the body (Feng et al. 2012). In the present study with CIK cells, the highest level of TAC was seen in the 20 °C group and the lowest in the 36 °C group. With regard to this phenomenon, Feng et al. (2012) speculated that some constituent substances of ADS were reduced to inhibit oxidation of exogenous electrophilic groups and avoid LPO, which ultimately resulted in reduced TOC.

XOD is the main catalyzing enzyme for organs that generate free radicals when animals are under non-normal states. A previous study showed that changes in temperature affected XOD activity in juvenile Chinese sturgeon, with XOD activity highest in the 30 °C group (Feng et al. 2012). In the present study, the highest XOD activity of CIK cells appeared in the 32 °C group, which indicates that XOD was able to effectively scavenge free radicals under low-level stress to avoid the toxic effects of free radicals in the cell.

The gene expression levels of antioxidant enzymes can be used to assess cellular oxidative stress affected by temperature (Malek et al. 2004). SOD, CAT, and GPx are major antioxidant enzymes encoded by Cu–Zn sod, cat, and gpx. In the present study, the relative mRNA expression level of Cu–Zn sod increased 2.72-fold in the 36 °C group and 4.32-fold in the 20 °C group compared with the control group. Kammer et al (2011) similarly found that the transcription levels of Cu–Zn sod increased 1.8-fold in the liver of three-spined stickleback during cold acclimation. Moreover, the present study showed that the increased SOD activity was accompanied by increased Cu–Zn sod mRNA level, which both showed a trend of an increase followed by a decrease. The temperature variation upregulated the transcription levels of gpx and cat in CIK cells. In summary, genes that code for peroxisomal antioxidant enzymes were upregulated by temperature in CIK cells to resist oxidative stress (Malek et al. 2004).

In summary, changes in temperature induce oxidative stress in CIK cells, cause oxidative damage by producing ROS, and activate ADS to maintain cell stability. Temperature variation can also directly upregulate the gene expression levels of antioxidant enzymes.