Introduction

Climate change and global warming are serious environmental problems. Environmental security is more prominent in the Caspian Sea. Due to its geopolitical and geoeconomic dimensions as well as its unique characteristics, this large lake is highly vulnerable to climate change. In fact, the natural isolation of the Caspian Sea has made its global warming conditions unique (Yazdanpanah Dero et al. 2020). From 1982 to 2019, the water surface temperature of the Caspian Sea in Iran increased between 5 and 8°C (Leroy et al. 2020; Zakerinejad et al. 2022).

In the aquatic environment, temperature is a crucial variable that directly affects marine organism survival. Temperature also influences the physiological stress response of fish (Cho et al. 2015). The stress response of fish depends on the duration of exposure and the temperature approaching the upper and lower limits (Islam et al. 2022). The adverse effects of temperature changes in fish have been fully proven to be related to oxidative stress (Birnie-Gauvin et al. 2019; Klein et al. 2017). Reactive oxygen species (ROS) may be produced by climate change. High levels of ROS can damage DNA, proteins, and lipids in cells as well as lysozymes, peroxidases, and complement factors in humoral immunity (Filho 2007; Kim et al. 2021; Mozanzadeh et al. 2021). Increased ROS production can lead to the production of proinflammatory cytokines, which further link inflammation to inflammatory diseases (Dominguez et al. 2005). A number of immune system processes are regulated by ROS, such as proliferation, differentiation, intracellular signaling, chemoattraction, and antigen cross-presentation (Birnie-Gauvin et al. 2019). Among the enzymes that protect against oxidative stress are glutathione S-transferases (GST), glutathione peroxidases (GPx), and total antioxidant capacity (TAC) (Ghafoori et al. 2017). Fish can maintain homeostasis under various stressors, including temperature and salinity changes, by scavenging ROS (Neamatallah et al. 2022). Cortisol, a corticosteroid, regulates water balance and is produced during stress (Lin et al. 2000). Stress triggers physiological mechanisms that maintain body homeostasis, affecting fish immunity (Lu et al. 2022). The fish innate immune system includes complement component 3 (C3), lysozyme (LYZ), and immunoglobulin M (IgM), which respond to pathogens and environmental stressors (Biller-Takahashi and Urbinati 2014). Successful aquaculture depends on the organism's environment. During the culture period, therefore, it is essential to maintain an optimum environment in order maximize productivity (Sharma et al. 2017).

Several factors contribute to the plight of Caspian sturgeon populations, including overfishing, increased water pollution, changes in water chemistry, and blocked migration routes. These factors put sturgeons at critical risk in approximately 85% of cases (Moghim et al. 2006; Prokopchuk et al. 2016). Acipenser ruthenus is one of the smallest sturgeon species (Stanic et al. 2006), while the sterlet sturgeon is a critically endangered species in the Caspian Sea (Lenhardt et al. 2006). Iran's Fisheries Organization maintains sturgeon stocks in the Caspian Sea by releasing up to three million fingerling fish into rivers every year (Baharloei et al. 2020). However, after release, fish fingerlings often suffer extensive economic losses and mass mortality due to environmental stresses (Khodabandeh et al. 2009). Stressors can cause fish to experience different stress reactions at the cellular level. In 1998, 24.5 million sturgeon fingerlings were released into the Caspian Sea, but in 2008, this number decreased to 10 million. In recent years, this number has reached one million (Afraei Bandpei et al. 2010; Hajirezaee et al. 2017). It is challenging for the Fisheries Organization to maintain sturgeon stocks.

As living organisms are subjected to constantly changing conditions, maintaining cellular protein homeostasis is critical for cell survival and integrity because protein misfolding and aggregation can cause protein malfunctions and a variety of diseases (Boshoff et al. 2004; Hu et al. 2022). Heat shock proteins (HSPs) are ubiquitous and conserved proteins that function to maintain proteostasis in both prokaryotes and eukaryotes (Pirali et al. 2020; Taheri et al. 2022). The stress-coping abilities of HSPs were long known to function. Environmental factors and oxidative stress increase HSP27, HSP70, and HSP90 protein levels (Mahanty et al. 2017; Yu et al. 2022). Aquaculture could benefit from natural compounds that induce HSP in animals without causing traumatic injury. A variety of natural and manufactured compounds can stimulate HSP expression, resulting in the activation of HSP and the induction of HSP. Pro-Tex® induces HSPs in Persian sturgeons and other organisms as a resistant precursor. Pro-Tex® is a soluble form of TEX-OE®, a plant-derived substance made from Opuntia ficus indica or Nopal cactus (NOP) (Otaka et al. 2007; Salmani et al. 2024; Vahdatiraad et al. 2023b). Another commonly used HSPi is amygdalin (AMG) (Ahmed et al. 2012). Numerous studies have shown that pyrazole-based compounds activate the HSF1 gene to upregulate HSP70 expression (Brough et al. 2005; Küçükgüzel and ŞenkardeŞ 2015).

Fish cell lines have been developed from a broad range of tissues, such as the ovary, fin, swim bladder, heart, spleen, liver, kidney, gill, eye muscle, vertebrae, brain, and skin (Lakra et al. 2011). A gill cell regulates acid‒base balance, whereas a liver cell regulates metabolism and detoxification (Stapp et al. 2015; Tresguerres et al. 2020). This study investigated whether HSPi can mitigate the effects of temperature changes on Acipenser ruthenus liver, gill, and kidney cells. Specifically, we will assess the effects of HSPis, including Tex-OE and amygdalin, together with newly synthesized pirano-piranazole-based inducers of HSPs called SZ, MZ, HN-P1, and HN-P2, on HSP27, HSP70, and HSP90 protein expression and antioxidant and immunological responses. Our hope is that this research will provide insights into the mechanisms underlying the protective effects of HSP induction and contribute to the development of effective strategies for mitigating the side effects of environmental changes on sturgeons.

Materials and methods

Synthesis of novel compounds based on pirano-piranazole

The synthesis of pyrazolone or 5-methyl-2,4-dihydro-3H-pyrazole-3-one is carried out via a condensation reaction between ethyl acetoacetate and hydrazine. Acetic acid is used as a catalyst for this reaction.

Synthesis of 4,4- (1,4-phenylene) bis(5-amino-3-methyl-1,4-dihydropyrano [2,3-c]pyrazole-6-carbonitrile: SZ)

This compound was synthesized from the reaction of 2 mmol of pyrazolone, 1.2 mmol of malonitrile, and 1 mmol of terephthalaldehyde in the presence of 5% sodium hydroxide solution (NaOH) as a catalyst in ethanol under reflux conditions. H-NMR, C-NMR, and FT-IR were used to confirm the structure of the synthesized compound (Supplementary information (SI): Fig. 1a, b, and c, respectively) (Shahriyari-Nejad et al. 2020; Zarei et al. 2024a).

Synthesis of 4,4- (1,3-phenylene) bis(5-amino-3-methyl-1,4-dihydropyrano [2,3-c]pyrazole-6-carbonitrile: MZ)

Under reflux conditions, 2 mmol of pyrazolone was reacted with 1.2 mmol of malonitrile and 1 mmol of isophthalaldehyde in ethanol with 5% sodium hydroxide (NaOH) as a catalyst. By using FT-IR measurements, the structure of the synthesized compound was confirmed (see SI Fig. 2) (Shahriyari-Nejad et al. 2020).

Synthesis of 6-amino-4-(2-methoxyphenyl)-3-methyl-1,4-dihydropyrano [2,3-c]pyrazole-6-carbonitrile (HN-P1)

Synthesis was carried out using 1 mmol of a pyrazolone base compound, 1.1 mmol of malonitrile, and 1 mmol of 2-methoxybenzaldehyde (a methoxyarylaldehyde). The structure of the synthesized compound was confirmed by FT-IR (SI Fig. 3) (Pourmousavi et al. 2022).

Synthesis of 6-amino-4-(3-methoxyphenyl)-3-methyl-1,4-dihydropyrano [2,3-c]pyrazole-5-carbonitrile (HN-P2)

1 mmol of pyrazolone base compound was reacted with 1.1 mmol of malonitrile and 1 mmol of 3-methoxybenzoaldehyde to yield this compound. FT-IR confirmed the structure of the synthesized compound (SI Fig. 4) (Pourmousavi et al. 2022).

Isolation of cells from liver, gill, and kidney tissue

In this study, a fingerling species, Acipenser ruthenus, which measured 18 cm in length and weighed 15.28 g, was used. The specimens were obtained from the International Sturgeon Research Institute. The liver, gill, and kidney tissues were dissected under completely sterile conditions and transferred to separate T-25 cm2 flasks. The culture was performed in a T-25 cm2 flask at 22°C and 95% humidity with essential medium supplemented with culture medium [80% Leibovitz's L-15 medium (L15), 20% fetal bovine serum (FBS), 100 U/ml streptomycin/penicillin (1%) and 100 U/ml amphotericin B (1%) prepared by Sigma‒Aldrich]. The complete medium was replaced every 24 h. Under a microscope, the cells were examined daily for contamination and growth. Monolayers of liver, gill, and kidney cells formed after 10–15 days. Two washes with phosphate-buffered saline (PBS, pH: 7.4) were conducted to remove any remaining culture medium from the cells adhering to the bottom of the flask. Trypsin–EDTA (0.25%; Sigma‒Aldrich) was then used to detach the cells from the flask. The monolayer cells were dispersed throughout the flask by using trypsin–EDTA. After 3 min, the cells were centrifuged for 5 min at 3000 rpm. For treatment, the supernatant was discarded, and the cell sediment was transferred to a flask (Butler 2004; Zarei et al. 2024a).

Determination of the optimal HSPi dose

Treatment with HSPis and MTT assay

Liver, kidney, and gill cells (5 × 105 cells/ml) were counted and plated in 96-well plates. At 22°C, 5% CO2, and 95% humidity, the cells were incubated in DMEM with 10% FBS, 100 U/ml streptomycin/penicillin (1%) and 100 U/ml amphotericin B (1%). After 24 h, the medium was replaced with DMEM containing 1% FBS. The HSPi concentrations used were as follows: NOP (Source Naturals, INC, Box 2118, Santa Cruz, CA 95062): 0, 50, 100, 200, 400, 800, and 1600 mM; AMG (Sigma‒Aldrich): 0, 1.25, 2.5, 5, 10, 20, 40, and 80 mM; and synthetic compounds: 0, 5, 10, 20, 40, 80, and 160 µM for 24 h. A group without treatment was considered the control group. To determine the toxicity of the HSPi compounds and cell viability, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test was conducted, with a final volume of 200 µl for each well. Each concentration was tested three times.

The MTT assay measures the production of purple‒blue color when tetrazolium salts are reduced to formazan forms, which can be measured spectrophotometrically. At the end of the treatment period, the medium without FBS was replaced with 10 µl (10% of final volume) of MTT reagent (5 mg/ml, Sigma‒Aldrich), and the plates were incubated at 22°C, 5% CO2, and 95% humidity for 3‒4 h. The final volume of each well was 100 µl. The supernatant was removed, and the formazan crystals formed were solubilized in 100 μl of dimethyl sulfoxide (DMSO; Sigma‒Aldrich) for 30 min. The absorbance was measured at 570 nm and is expressed as a percentage of the control (which is considered 100%) (Ahmadi et al. 2014). The viability was calculated using the following formula:

$$\mathrm{Cell viability }= [ \frac{\mathrm{OD }({\text{treatment}})}{\mathrm{OD }({\text{control}})}]\times 100\mathrm{\%}$$

The optimal doses of HSPi were determined based on results from the MTT assay (Fig. 1): 800 mM NOP (N800), 80 mM AMG (A80) and 80 µM SZ (SZ80) (Zarei et al. 2024a; Zarei et al. 2024b).

Fig. 1
figure 1figure 1figure 1figure 1figure 1figure 1

The optimal dose of each compound (HSPi) was determined (means ± SEM). Livers, gills, and kidneys of sterlet cells were treated with a) (0–160) µM SZ, b) (0–160) µM MZ, c) (0–160) µM HN-P1, d) (0–160) µM HN-P2, e) (0–1600) mM Nopal Endurance (NOP), and f) (0–80) mM Amygdalin (AMG). Each treatment group was repeated three times. Within each column, different letters indicate significantly different groups according to Duncan's test (a, b, c, and…; P < 0.0001)

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Grouping of treatments

A temperature of 22°C (T22) was considered the control temperature, and temperatures of 18 (T18) and 26°C (T26) were considered stress temperatures (Ndong et al. 2007; Wiles et al. 2020). The 5 × 105 cells/ml were counted and transferred to a 24-well plate with culture medium [90% DMEM, 10% FBS, 100 U/ml streptomycin/penicillin (1%) and 100 U/ml amphotericin B (1%)]. The plates were incubated at 22°C, 5% CO2, and 95% humidity. After 24 h, the culture medium was replaced with medium containing 1% FBS, and the cells were divided into four groups (Fig. 2): 1) the control group (without HSPi; T22, T18, and T26), 2) the group in which the cells received different concentrations of HSPi (N800, A80, and SZ80; at 22°C: HSPi + T22), 3) the group in which the cells were treated with HSPi for 24 h at 22°C and then placed at 18°C (HSPi + T18), and 4) the group in which the cells were treated with HSPi for 24 h at 22°C and then incubated at 26°C (HSPi + T26). Afterward, the cells were treated for 24 h and then centrifuged for 5 min at 4°C at 5000 rpm. Each group was repeated 3 times. The final volume of each well was 500 µl. Aseptic conditions were followed in all patients.

Fig. 2
figure 2

Grouping of treatments Twelve groups were considered: 1) Control cells without treatment at 22°C (T22), HSPi groups containing 22°C, 2) 80 mM amygdalin (A80 + T22), 3) 80 µM SZ (SZ80 + T22), 4) 800 mM Nopal Endurance (N800 + T22), 5) cells without treatment at 18 °C (T18), and HSPi groups incubated at 22°C. After 24 h, the cells were transferred to a temperature of 18°C, 6) 80 mM amygdalin (A80 + T18), 7) 80 µM SZ (SZ80 + T18), 8) 800 mM Nopal Endurance (N800 + T18), 9) cells without treatment at 26°C (T26), and HSPi groups incubated at 22°C. After 24 h, the cells were transferred to a temperature of 26°C: 10) 80 mM amygdalin (A80 + T26), 11) 80 µM SZ (SZ80 + T26), and 12) 800 mM Nopal Endurance (N800 + T26). A total of 5 × 105 cells/ml from each cell line (kidney, liver and gill) were counted and transferred to 24-well plates. Each group was repeated 3 times, and after 24 h, the cells were collected from the wells and used for subsequent tests

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The protein concentration in each treatment group (5 × 105 cells/ml) was determined using the Bradford assay (Bradford 1976), with bovine plasma albumin (Sigma‒Aldrich) used as a standard (SI section 1-3).

Western blotting analysis

A western blotting technique described by Werner et al. was used to determine the expression patterns of HSP27, HSP70, and HSP90 (Werner et al. 2007). Briefly, 25 µg of protein from each sample was electrophoresed on a 12% polyacrylamide gel and 5% stacking gel using sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE). The proteins were then transferred to a polyvinylidene fluoride membrane using an electroblotter (Millipore, Bedford, Massachusetts). Primary antibodies [sc-13132 (Santa Cruz Biotechnology, INC), H5147, and H1775 from Sigma‒Aldrich] were used for immunoblotting of HSP27, HSP70, and HSP90. For ß-actin, which is a housekeeping protein (control), monoclonal anti-ß-actin (A3854) and anti-mouse IgG-peroxidase conjugate (A2304, Sigma‒Aldrich) were utilized as primary and secondary antibodies, respectively. The primary antibody binds specifically to the protein of interest, while the secondary antibody recognizes and binds to the primary antibody, allowing for detection of the protein. Finally, 3,3′-diaminobenzidine (DAB; Sigma‒Aldrich, D-7304) and H2O2 were used as substrates to generate a visible signal indicating the presence of the protein. Note that gill and kidney cells were analyzed using western blots, and the enzyme assay was not performed on these cells.

Antioxidant assay in liver cells

GST activity was assessed by measuring the increase in absorption at 340 nm in 100 mM Na-phosphate buffer (pH 6.5) containing 1 mM 1-chloro-2,4-dinitrobenzene (CDNB, Sigma-Aldrich) and 1 mM GSH (Pacini et al. 2013).

GPx activity was measured at 340 nm using 100 mM Na-phosphate buffer (pH 7.5), 1 mM EDTA, 0.12 mM NADPH, 2 mM GSH, 1 mM NaN3, 1 U glutathione reductase (GR), and 0.6 mM H2O2 (ZellBio GmbH, Germany).

TAC levels were determined using an ELISA kit (ZellBio GmbH, Germany) at 490 nm, similar to GPx (Li et al. 2017).

Immune responses in liver cells

IgM was measured using an ELISA quantification kit (Hangzhou East Biopharm Co. Ltd.) (Valipour et al. 2018). IgM standards and supernatant samples were analyzed manually. The wavelength at 450 nm was determined within 15 min after all the necessary steps were completed. Three measurements were performed.

Quantitative C3 levels were determined using an ELISA sandwich (enzyme-linked immunosorbent assay) with a fish ELISA kit (Hangzhou East Biopharm Co. Ltd.) (Baharloei et al. 2020). The C3 fish monoclonal antibody was previously coated with an antibody-enzyme monoclonal well. The biotin-labeled C3 antibody was mixed with streptavidin–horseradish peroxidase and incubated at 37°C to form an immune complex. To remove the unmixed enzyme, the plate was washed. The liquid color changed to yellow when sulfuric acid was added. The OD at 450 nm was measured with a microplate reader, and the C3 concentration was determined.

LYZ activity was assessed using a turbidimetric method (Kim et al. 2019). Micrococcus lysodeikticus (Sigma‒Aldrich) was used as a substrate (0.2 mg/ml 0.05 M phosphate buffer, pH 6.6). In this study, lyophilized chicken egg white LYZ was used to construct a standard curve, and changes in turbidity at 530 nm were measured. Chicken egg white LYZ activity was determined from the results.

Cortisol assay

An enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Institute, Nanjing, China) was used to measure cortisol levels in liver cells. Color changes were detected using a spectrophotometric spectrophotometer (450 nm) (Long et al. 2019) (SI section 1-3).

Statistical analysis

For the statistical analysis, SPSS/PC + 23 (SPSS Inc.) and GraphPad Prism 8 were used. All experiments were repeated three times, and the numerical data are presented as the mean ± SEM. The first step was to check and confirm the normality of the data by performing a one-sample Kolmogorov‒Smirnov test. Significant differences between groups were analyzed using one-way analysis of variance (ANOVA) and Duncan's test for multiple comparisons. To evaluate the differences in the main components between the studied parameters and different temperature stress treatments, principal component analysis (PCA) was performed. Pearson correlation analysis was conducted to examine the possibility of a relationship between the biomarkers and fish cells. The significance level was set at P < 0.0001.

Results

Cell viability

Evaluation of the viability (%) of cells treated with HSPi

The liver, gill, and kidney cells were exposed individually to various concentrations of HSPi, including SZ, MZ, HN-P1, HN-P2, AMG, and NOP (Fig. 1; P < 0.0001). The concentrations of the compounds MZ, HN-P1, and HN-P2 (Fig. 1 b, c and d, respectively) strongly fluctuated in all three cell lines and did not follow a continuous increasing or decreasing trend. A sporadic change pattern was observed. These compounds were ignored in subsequent tests. SZ (at a concentration of 160 µM: 138.36% in the liver, 143.40% in the gill, and 141.88% in the kidney; Fig. 1 a) and AMG (at a concentration of 80 mM: 148% in the liver, 139.21% in the gill, and 143.66% in the kidney; Fig. 1 f) had the best survival rates in all three cell lines compared to the control group (100%). In contrast, individuals with higher NOP concentrations (at 1600 mM: 80.33% in the liver, 81.66% in the gill, and 78.29% in the kidney; Fig. 1 e) had lower survival rates (P < 0.0001). Furthermore, the optimal dose of HSPi was determined based on the cell survival results for the following concentrations of NOP: 800 mM NOP (N800), AMG: 80 mM (A80), and SZ: 80 µM (SZ80).

Overview of cell viability at different treatment doses (%) under temperature stress

We grouped the treatments as shown in Fig. 2 and assessed their survival rates (Fig. 3). The levels of cell survival differed among the treatments chosen for liver, gill, and kidney cells. The T26 treatment had a reduced effect on cell viability (%) in all three treated cell tissues (liver: 90%, gill: 89.66%, and kidney: 88.67%) compared to the control (100%; P < 0.0001). The T18 group showed a similar effect to the control (T22) group in all three cell lines. A temperature of 26°C had a decreasing effect on cell viability. Among all HSPi compounds, N800 had the lowest cell viability (liver: 86.10%, gill: 86.61%, and kidney: 82.88%). In all three cell lines, cell viability increased when the cells were first treated with AMG (A80) and then exposed to stress temperatures (T18 and T26).

Fig. 3
figure 3

The cell survival rate (%) of cells isolated from Acipenser ruthenus tissues (means ± SEM) treated with various doses and under various temperatures was investigated. A dose of 800 mM NOP (N800), 80 mM AMG (A80), and 80 µM SZ (SZ80) was selected as the optimal dose of HSPi. Additionally, the temperature stress groups (18, 22, and 26°C) were selected for the test. In the 12 treatment groups, each parameter was measured three times. Within each column, different letters indicate significantly different groups according to Duncan's test (a, b, c, and..; P < 0.0001)

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HSP response to temperature stress

HSP27

The expression of the HSP27 protein in liver, gill, and kidney cells varied significantly (Fig. 4; P < 0.0001). Compared to the control group (T22), the T26 group had increased HSP27 expression in liver cells (Fig. 4 a and d; P < 0.0001). Among all the HSPi compounds, SZ80 expressed the least amount of protein (SZ80 + T22: 1.153-fold, SZ80 + T26: 1.453-fold). A80 + T26 (2.546-fold) and A80 + T22 (2.448-fold) showed the highest HSP27 expression (P < 0.0001). In the gill, the T26 group (3.036-fold) showed maximal HSP27 expression compared with the control group (Fig. 4 b and d). Treatment with N800 + T26 also had a greater effect on HSP27 expression (2.377-fold; P < 0.0001). Similar to those in liver cells, the protein expression levels in gill cells in the SZ80 treatment groups were the lowest among all the HSPi compounds (P < 0.0001). In kidney cells (Fig. 4c and d), SZ80 + T22 (0.088-fold) and N800 + T26 (2.926-fold) cells expressed less and more HSP27, respectively, than did the controls. The HSP27 protein expression in the T26 group (1.631-fold) was slightly greater than that in the control group. In cells treated with HSPi compounds, especially N800 and A80, followed by temperature stress (T26), protein expression increased (HSPi + T26; P < 0.0001).

Fig. 4
figure 4

The relative protein expression of HSP27 was analyzed by western blotting. a) Liver, b) gill, and c) kidney cells. d) Eight groups were considered: 1) control cells without treatment at 22°C (T22) and HSPi groups containing 22°C, 2) 80 mM amygdalin (A80 + T22), 3) 80 µM SZ (SZ80 + T22), 4) 800 mM Nopal Endurance (N800 + T22), and 5) cells without treatment at 26°C (T26). The HSPi groups were incubated at 22°C for 24 h, after which they were transferred to a temperature of 26°C: 6) 80 mM amygdalin (A80 + T26), 7) 80 µM SZ (SZ80 + T26), and 8) 800 mM Nopal Endurance (N800 + T26). There were 8 treatment groups in which each parameter was measured three times. The HSP27 protein levels are shown as the fold change relative to the control values (means ± SEM), and within each column, different letters indicate significantly different groups according to Duncan's test (a, b, c, and…; P < 0.0001)

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HSP70

Under temperature stress conditions (T26), HSP70 expression was evaluated in sterlet sturgeon liver cells (Fig. 5a and d), gills (Fig. 5b and d), and kidneys (Fig. 5c and d). The changes in the expression of the HSP70 protein in all three cell lines were almost the same. The N800 + T26 group showed the greatest changes in HSP70 protein expression in liver, gill, and kidney cells. Compared with that of the other inducing compounds, the protein expression of the synthetic compounds (SZ80 + T22 and SZ80 + T26) was the lowest. HSP70 protein expression was slightly greater under stress conditions (group T26) than under the control conditions (P < 0.0001). In general, all three cell lines showed an increase in HSP70 expression when first treated with inducing compounds (especially N800) and then exposed to temperature stress (P < 0.0001).

Fig. 5
figure 5

The relative protein expression of HSP70 was analyzed by western blotting. a) Liver, b) gill, and c) kidney cells. d) Eight groups were considered: 1) control cells without treatment at 22°C (T22) and HSPi groups containing 22°C, 2) 80 mM amygdalin (A80 + T22), 3) 80 µM SZ (SZ80 + T22), 4) 800 mM Nopal Endurance (N800 + T22), and 5) cells without treatment at 26°C (T26). The HSPi groups were incubated at 22°C for 24 h, after which they were transferred to a temperature of 26°C: 6) 80 mM amygdalin (A80 + T26), 7) 80 µM SZ (SZ80 + T26), and 8) 800 mM Nopal Endurance (N800 + T26). There were 8 treatment groups in which each parameter was measured three times. The HSP70 protein levels are shown as the fold change relative to the control values (means ± SEM), and within each column, different letters indicate significantly different groups using Duncan's test (a, b, c, and…; P < 0.0001)

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HSP90

HSP90 protein expression was altered by HSPi and temperature treatments in liver, gill, and kidney cells (Fig. 6; P < 0.0001). In the liver, the highest HSP90 expression was observed in the A80 + T26 treatment group (2.553-fold), while the control group had the lowest HSP90 expression (Fig. 6a and d). Compared to those in the T26 treatment group, the HSP90 expression in the A80 + T26 and N800 + T26 treatment groups significantly increased (P < 0.0001). The liver cells expressed the highest concentration of the HSP90 protein as a result of AMG (A80). In the gills, the N800 + T26 treatment group (5.536-fold) had higher levels of HSP90 expression than did the control group, while SZ80 (SZ80 + T22 and SZ80 + T26) had the lowest protein expression (Fig. 6b and d). Compared with those in the T26 group, the groups that received the inducing compounds first and then underwent temperature stress showed significantly greater HSP90 expression (P < 0.0001). In the kidney, the N800 + T26 treatment group (2.870-fold) had higher levels of HSP90 expression, while the SZ80 + T22 treatment group (0.861-fold) had lower levels (Fig. 6c and d). The T26 group had increased HSP90 expression in kidney cells compared to that in the control group (T22). HSP90 expression was significantly greater in N800 + T26 (2.870-fold) than in A80 + T26 (2.156-fold) and SZ80 + T26 (1.742; P < 0.0001). Note that original photos of the western blot gel are available in SI Figs. 5, 6, 7 and 8.

Fig. 6
figure 6

The relative expression of HSP90 was analyzed via western blotting. a) Liver, b) gill, and c) kidney of sterlet cells. d) Eight groups were considered: 1) control cells without treatment at 22°C (T22) and HSPi groups containing 22°C, 2) 80 mM amygdalin (A80 + T22), 3) 80 µM SZ (SZ80 + T22), 4) 800 mM Nopal Endurance (N800 + T22), and 5) cells without treatment at 26°C (T26). The HSPi groups were incubated at 22°C for 24 h, after which they were transferred to a temperature of 26°C: 6) 80 mM amygdalin (A80 + T26), 7) 80 µM SZ (SZ80 + T26), and 8) 800 mM Nopal Endurance (N800 + T26). In eight treatment groups, each parameter was measured three times. The HSP90 protein levels are shown as the fold change relative to the control values (means ± SEM), and within each column, different letters indicate significantly different groups according to Duncan's test (a, b, c, and…; P < 0.0001)

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Antioxidant activity

Liver cell antioxidant activity was assessed using GST, GPx, and TAC measurements (Table 1; P < 0.0001). The changes in all the aforementioned enzymes were similar. Enzyme activity significantly increased at stress temperatures (T18 and T26) compared to that at the control temperature (T22). The N800 + T26 group exhibited the highest activity of all three enzymes. Among all the HSPi compounds, SZ80 had the lowest activity (SZ80 + T22 < SZ80 + T18 < SZ80 + T26). Overall, the enzyme activity was greater in the treatment groups at 26°C than at 18°C (HSPi + T26 ˃ HSPi + T18 ˃ HSPi + T22).

Table 1 Antioxidant activity in liver cells of sterlet sturgeons (means ± SEM; P < 0.0001)
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Immune responses

Table 2 shows the measured activities of C3, IgM, and LYZ in liver cells exposed to different treatments (P < 0.0001). The control group had an apparent increase in C3 activity (1.58 mg/g), while the other treatment groups showed a clear decrease in activity. C3 levels were greater at 18°C than at 26°C, and treatment groups at 22°C had higher C3 levels than those at 18°C (the order of changes in the groups was treatments + T22 > treatments + T18 > treatments + T26; P < 0.0001). Similarly, the highest level of IgM was observed in the control group (2.7 mg/g), and the other treatment groups had a decreasing effect. Changes in groups T18 and T26 were similar. Among the HSPi compounds, SZ80 had the highest level (P < 0.0001). LYZ activity in groups T18 and T22 was the same (0.196 mg/g), while it decreased in the other groups. The LYZ levels in the treatment groups were greater at 18°C than at 26°C, and the LYZ levels were greater at 22°C than at 18°C (the order of differences between the groups was groups + T22 > groups + T18 > groups + T26; P < 0.0001).

Table 2 Immune responses in liver cells from sterlet sturgeons (means ± SEM; P < 0.0001)
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Assessing PCA

PCA was conducted on 20 variables and 36 treatments, revealing that the first, second, and third principal components accounted for 59.20%, 20.26%, and 7.09% of the changes, respectively (Table 3). Variables with a score higher than 0.6 were used to interpret the components (SI Fig. 10, Tables 1 and 2). The variables measured during temperature stress played a significant role in determining the relationships between the treatments and the tested variables in the first and second components (Fig. 7). Finally, Pearson’s correlation analysis was performed on all measured parameters (SI Table 3).

Table 3 Eigenvalue, percentage of variance and cumulative percentage of variance related to all three components
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Fig. 7
figure 7

Principal component analysis (PCA) for temperature stress. The three components accounted for 86.56% of the total variation. The variables measured during temperature stress played a significant role in determining the relationships between the treatments and the tested variables in the first and second components. The abbreviations are L: liver, G: gill, K: kidney, LV: liver viability, GV: gill viability, and KV: kidney viability

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Discussion

Sturgeon in the Caspian Sea has been cultivated artificially in recent years due to extinction conditions (Baharloei et al. 2021). The Sefidroud River, which flows into the Caspian Sea, is one of the fish release stations in Guilan Province (Hajirezaee et al. 2017). The water temperature of this river has fluctuated in different seasons in recent years due to the increase in the Earth's temperature and decrease in rainfall (Abdullah et al. 2022; Khoshakhlagh et al. 2016). On the other hand, due to its ecological conditions, the temperature of the Caspian Sea has increased by approximately 8°C in the last 40 years (Leroy et al. 2020; Zakerinejad et al. 2022). Sturgeons are anadromous fish that migrate to rivers to spawn. During this migration between the river and the sea, the fish encounter water temperature changes, which are considered stressors (Afraei Bandpei et al. 2010; Khodabandeh et al. 2009; Prokopchuk et al. 2016). Additionally, when sturgeons migrate from fresh to saltwater, their blood plasma ion levels increase, causing severe stress (Khodabandeh et al. 2009; Kim et al. 2021). Different fish species react differently to climate change and environmental changes. Some species may adapt to changing conditions, while others struggle to survive. It is imperative to understand these differences to develop effective conservation strategies (Carosi et al. 2019).

HSP27, HSP70, and HSP90 are proteins that act as cellular chaperones and may be involved in environmental factors (Deane and Woo 2011). Temperature stimulates the upregulation of HSP proteins, particularly HSP27, HSP70, and HSP90. As a result, this upregulation is caused by cellular stress induced by temperature fluctuations (Ji et al. 2016; Mohanty et al. 2018; Wang et al. 2007). In fish and shellfish, HSP inducers play a significant role in stress responses (Baharloei et al. 2020; Baruah et al. 2014). Environmental factors can negatively affect fish health by generating reactive oxygen species (ROS), which disrupt the immune system (Eissa et al. 2017). Fish health can be determined by studying safety parameters (Banaee et al. 2013). Moreover, antioxidant parameters can indicate how fish respond to environmental changes (Hattori et al. 2009). By investigating the effects of stress factors on fish environments, these changes provide an effective tool for managing fish health (Werner et al. 2007).

An ecological model of Acipenser ruthenus was used to examine liver, kidney, and gill cell survival. It was hypothesized that inducing HSPs in sterlet cells could increase protection and readiness to cope with temperature stress in vitro. First, MTT assays were used to investigate cytotoxicity (Fig. 1). Temperature negatively affected sterlet cell viability (Fig. 3). Additionally, temperature treatment altered HSP27, HSP70, and HSP90 expression in all three cell lines (Figs. 4, 5 and 6). As a consequence, temperature stress increased the oxidative activity of GST, TAC, and GPx enzymes (Table 1). The immune parameters IgM, LYZ, and C3 were also reduced (Table 2).

Despite our initial findings about temperature stress and its effects on cells, we encountered significant challenges in the continuation of our study. We discovered that HSPs can cause similar alterations without reducing cell viability or increasing cell death. Furthermore, normal conditions can be restored by combining the HSP and temperature. To investigate whether HSPi could counteract the temperature effects on A. ruthenus cells, we tested NOP-, AMG-, and pirano piranazole-derived compounds (SZ, MZ, HN-P1, and HN-P2; SI Figs. 1 to 4). We examined the viability of cells after applying the compounds mentioned above to better understand this challenge. MTT assays revealed that AMG and SZ increased cell viability, while NOP decreased cell viability (Fig. 1). Furthermore, cells treated with the inducing compounds and then exposed to temperature showed increased viability (Fig. 3).

Fish farming is particularly important for the preservation of endangered fish populations. This can be achieved by bringing them to special farms and releasing them into freshwater once they reach maturity. However, after being raised on farms under optimal conditions, the fish are released into water that differs from the water they were raised in, which can be a disadvantage. By using HSPis for the first aspect, this problem may be alleviated. For the second aspect of the challenge, reducing or eliminating temperature stress, combining temperature with HSPis is essential (Afraei Bandpei et al. 2010; Salmani et al. 2024; Vahdatiraad et al. 2023b; Vahdatiraad et al. 2023a). In all three cell lines (Figs. 4, 5 and 6), AMG and NOP regulate all HSPs (HSP27, HSP70, and HSP90) and increase their expression (HSPi + stress).

In aquatic organisms, temperature increases affect parameters such as metabolic rate and oxygen consumption, causing oxidative stress (Madeira et al. 2013). Thus, the induction of antioxidant defenses is an important component of the stress response to oxidative stress in biological systems (Han et al. 2018; Lushchak and Bagnyukova 2006). Our results are generally in accordance with previous findings, where oxidative stress biomarkers (both degradation products and antioxidants) are highly sensitive to temperature due to temperature-induced ROS production. Salmani et al. showed that the use of an HSP inducer in combination with temperature exposure resulted in increased activity of superoxide dismutase, catalase, and TAC enzymes in sturgeon fingerlings. The increase in the activity of these enzymes, particularly on the first day, indicated the activation of antioxidant defense mechanisms in response to changes in temperature-induced oxidative stress (Salmani et al. 2024). HSPs, including HSP70, have been shown to have antioxidant properties and can help protect cells from oxidative damage (Eissa et al. 2017; Lu et al. 2022). Therefore, it is reasonable to speculate that HSPs may serve as a protective mechanism against the oxidative stress induced by changes in temperature, particularly during the initial days. Overall, our findings suggest that the use of HSP-inducing compounds in combination with a temperature of 26°C (HSPi + 26°C ˃ HSPi + 18°C) improves antioxidant enzyme activity (Table 1). In the cells of sterlet sturgeon subjected to environmental stress, ROS can be controlled, and antioxidant defense mechanisms can be enhanced, potentially reducing the effects of temperature stress and environmental stressors on aquatic organisms. Salmonids and tilapia exhibit species-specific immune responses to temperature changes (Bailey et al. 2017; Ndong et al. 2007). In another study, the presence of HSPi was found to moderate immune activity in sturgeon fish (Vahdatiraad et al. 2023a). Overall, there was a decreasing trend between the treatment and control groups with regard to the immune response (C3, IgM, and LYZ) according to the present study. Cells treated with NOP and AMG first and then subjected to temperature stress produced the best results (Table 2). Based on PCA and correlation analysis, HSP expression in different tissues was related to the most studied parameters (Table 3, SI Tables 1 to 3). As a result, this family plays a significant role in a variety of biological states. Using PCA, we identified the appropriate treatment for temperature stress. This study indicated that HSP expression is directly related to antioxidant parameters (GST and GPx) and the immune response (IgM; Fig. 7). Thus, NOP and AMG could improve temperature stress and restore normal cell function.

Conclusions

Using cells isolated from Acipenser ruthenus sturgeons, we investigated the modulatory effects of HSP inducers on temperature stress. In vitro, HSP inducers are capable of modifying HSP expression, cortisol levels, and antioxidant activity. As a result, the use of HSP inducers may be a powerful and reliable method for increasing A. ruthenus resistance to temperature stress. However, our results also suggest that these compounds can reverse the harmful effects of temperature stress. Further research is necessary to investigate the effects of these ecological factors, especially in vivo and in combination with other stresses, on the health status of species in real-life situations to effectively implement this approach.