Reagents source
The serum stress indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glucose (GLU), antioxidant components superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) determination kit and ATP content kit were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The serum cortisol (Cor) levels were measured by using the enzyme-linked immunoassay for fish species. The ROS kit was purchased from Beyotime (Shanghai, China). RNAiso Plus kit was purchased from TaKaRa Bio (Japan). Primers were synthesized by Chengdu Youkang Jianxing Bio-technology Co., Ltd. (China), the specific information of the primary antibody used in Western blot is shown in Table S1. β-Actin (Abclonal, China, 1:3,000) was chosen as the internal reference protein, and the secondary antibody was also purchased from Abclonal and diluted with 1:8,000. TUNEL Apoptosis Detection kit was purchased from Boster (Wuhan, China). Immunol Fluorence Staining kit with Alexa Fluor 488 and 555-Labeled Goat Anti-Rabbit IgG were purchased from Beyotime, and the specific information of primary antibodies used for immunofluorescence is shown in Table S2.
Diet design
The animal research protocol was approved by the Animal Care Advisory Committee of Sichuan Agricultural University (Chengdu, Sichuan, China).
The basal diet is shown in Table S3. Juvenile grass carp were purchased from Deyang City, Sichuan Province. After 2-week acclimatization period, 720 healthy fish with an initial weight of 11.07 ± 0.07 g were randomly divided into 4 groups of 3 replicates each and put into 12 net cages of 60 fish each. OTA and CUR were added to the diets to form four diets of 0 (no addition), 1.2 mg/kg OTA, 400 mg/kg CUR, and 1.2 mg/kg OTA + 400 mg/kg CUR. The amount of OTA and CUR added was obtained from our previous studies [21, 22]. And then conducted a 60-d feeding experiment, during which the water temperature was maintained at 28 ± 2 °C, pH was 7.5–8.0, and the dissolved oxygen was above 6.0 mg/L. The final average weight of the four groups were 152.59 ± 5.12 g, 93.94 ± 2.23 g, 200.97 ± 2.79 g, and 131.22 ± 3.69 g.
At the end of the feeding experiment, 32 fish were selected from each net cage and divided into normoxia (18 fish) and hypoxia (18 fish) groups for a 96-h hypoxia stress experiment. The normoxia group was kept at the normal oxygen level (above 6.0 mg/L) and the hypoxia group was controlled at 1 mg/L with a timer. The specific experimental design is shown in Fig. 1A. Furthermore, the level and duration of hypoxia according to Wang’s study [23].
Experimental design diagram (A) and liver serum biochemical indices (B–F). Data represent means of each group; error bars indicate SD. n = 6. Different letters denote significant differences between normoxia or hypoxia groups (P < 0.05), respectively, and * denotes significant differences between normoxia and hypoxia groups at the same level (P < 0.05). Cor: cortisol; GLU: glucose; ALT: alanine aminotransferase; AST: aspartate aminotransferase; LDH: lactate dehydrogenase
Sample collection
At the end of the 96-h hypoxia stress experiment, blood was taken from the tail vein and centrifuged at 3,000 × g for 10 min at 4 °C to obtain serum samples. The fish were then anaesthetized in a benzocaine bath (50 mg/L) and then executed as described by Dong et al. [24]. Meanwhile, the visceral mass was immediately dissected on an ice-cold plate to strip the liver tissue. Liver tissues were preserved in liquid nitrogen, 4% paraformaldehyde, and 2.5% glutaraldehyde, respectively.
Histological and ultrastructural observation
Histological observation
Histological observations refer to He’s study [25]. A small portion of the liver sample was removed from the fish and immediately rinsed with saline. It was then stored in 4% paraformaldehyde for fixation. The tissue samples were progressively dehydrated with ethanol (75%, 85%, 95%, 100%) and then paraffin-embedded. The wax blocks were then cut into thin slices using the Leica slicer, and after Hematoxylin and Eosin (H&E) staining, the tissue sections were observed at 200× using an OLYMPUS BX43 light microscope.
Ultrastructural observation
Ultrastructural observations refer to Liu’s study [26]. A small piece of fresh liver tissue with minor mechanical damage (e.g., contusion and crush) was selected and fixed in 2.5% glutaraldehyde. After further fixation in 1% osmium acid protected from light, it was dehydrated with ethanol and acetone in steps (ethanol: 30%-50%-70%-80%-95%-100%-100%; acetone: 100%-100%). They were then osmotically embedded with an epoxy resin embedding agent. Ultrathin sections were cut with a diamond knife and then stained with uranium acetate and lead citrate for 8 min. Finally, the sections were observed under a transmission electron microscope (HT7800, Servicebio, China), and images were collected for analysis.
Analysis of serum and liver tissue biochemical parameters
Serum biochemical indices of Cor, GLU, ALT, AST, and LDH were measured by kit using commercial methods. Liver samples were homogenized in saline at ice-cold temperature; then the tissue homogenate was centrifuged at 6,000 × g for 20 min at 4 °C. And the supernatant was separated to assay the relevant enzyme activities. Antioxidant-related enzyme indicators, such as SOD and CAT, non-enzymatic such as GSH, oxidative damage indicators ROS and ATP content were measured by kit using commercial methods.
TUNEL analyses
The TUNEL experiment was performed as follows: Paraffin sections were deparaffinized and tissues were repaired with proteinase K. The liver cells were then membrane-broken and buffer incubated at room temperature before labeling. Then, the nuclei were restained with DAPI to seal the sections with anti-fluorescence quenching sealer. Sections were observed under a DMI4000B inverted fluorescence microscope, keeping the settings of the inverted fluorescence microscope the same for all subsequent images. Three images were acquired at 200× objective for each treatment. Images were thresholded to exclude background fluorescence and were gated to contain only intensity measurements of positively stained cells. TUNEL relative fluorescence intensity was calculated using the formula: Integrated Density of TUNEL positively stained cells/Area of DAPI staining × 100. The resulting ratio was normalized to 1 in the control normoxia group.
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis
RT-qPCR analysis was performed as follows: Total RNA was isolated from the liver using an RNAiso Plus kit (Takara, Dalian, China). At the same time, the purity and content were detected by agarose gel electrophoresis (1.5%) and (A260/280) spectrophotometry respectively. The cDNA synthesis kit was used to reverse-transcribe RNA to form cDNA [27]. All primer sequences used in this study are shown in Table S4. The results are represented as matrix bubble plots.
Western blot analysis
Western blot analysis was performed as follows: The liver tissues were ground using a tissue grinder after the addition of RIPA Lysis Buffer (Beyotime, China). Using a BCA assay kit (Beyotime, China), we measured the protein concentration of the supernatant. Liver samples were separated by SDS-PAGE (10%) and transferred onto a PVDF membrane using a wet Trans-Blot system (Bio-Rad, USA). After sealing, the membrane was incubated with primary and secondary antibodies. Protein signals were visualised using the ECL kit (Beyotime, China) in a high-sensitivity chemiluminescent imaging system (Bio-Rad, USA). The grey scale values of each band were obtained using Image J, 1.54f (NIH Image J system, Bethesda, MD, USA). The relative expression levels of each protein were normalized using the internal reference protein (β-actin) and the resulting ratio was normalized to 1 in the control normoxia group.
Immunofluorescence staining
The immunofluorescence staining experiments were performed as follows: Paraffin sections were deparaffinized with dewaxing solution, and then the sections were placed in 3% hydrogen peroxide to remove catalase. Thermal antigen repair was then performed in a microwave oven. Primary antibodies were incubated overnight at 4 °C and washed thrice with PBS. Afterward, secondary antibodies were incubated and washed in PBS, followed by DAPI staining, PBS washing, and dropwise sealing of the sections with an anti-fluorescence quencher. The stained sections were observed using a DMI4000B inverted fluorescence microscope. The settings of the inverted fluorescence microscope were kept the same for all subsequent images. Six images per treatment were acquired at 200× objective. Images were thresholded to exclude background fluorescence and gated to contain only intensity measurements of positively stained cells. Relative fluorescence intensity was calculated using the formula: Integrated Density of positively stained cells/Area of DAPI staining × 100. The resulting ratio was normalized to 1 in the control normoxia group.
Statistical analysis and plotting
SPSS 19.0 was used for data analysis. Data from normoxia and hypoxia groups were analyzed separately by one-way ANOVA and compared using Duncan’s multiple comparison test. The student’s t-test was used to compare the normoxia and hypoxia groups. Differences were considered statistically significant at P < 0.05. Results were expressed as mean ± standard deviation (SD). After data analysis was completed, all figures were plotted using Origin 2021.
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