Azoramide ameliorates cadmium-induced cytotoxicity by inhibiting endoplasmic reticulum stress and suppressing oxidative stress

Reagents, antibodies, and cell lines

Cadmium chloride (CdCl₂) (purity > 99%) was purchased from Aladdin (C116344; Shanghai, China), prepared as a stock solution in ultrapure water and stored at −20 °C. Azoramide was obtained from Selleck (S8304; Shanghai, China), dissolved in dimethyl sulfoxide (D8418; Sigma−Aldrich, St Louis, MO, USA) as a stock solution and stored at −20 °C in the dark. 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Sigma−Aldrich (28718-90-3). An inhibitor of the p38 mitogen-activated protein kinase (MAPK) SB203580 (S1076) and a c-Jun N-terminal kinase (JNK) inhibitor SP600125 (S1460) were obtained from Selleck.

Primary antibodies against immunoglobulin heavy chain binding protein (BiP) (11587-1-AP; 1:1,000), phosphorylated JNK (p-JNK) (80024-1-RR; 1:1,000), phosphorylated p38 MAPK (p-p38 MAPK) (28796-1-AP; 1:1,000), phosphorylated α subunit of eukaryotic initiation Factor 2 (p-eIF2α) (28740-1-AP; 1:1,000), p-PERK (29546-1-AP; 1:1,000), nuclear factor-like 2 (Nrf2) (80593-1-RR; 1:1,000) and GAPDH (60004-1-Ig; 1:5,000) were obtained from Proteintech (Wuhan, Hubei, China). An antibody against light chain 3B (LC3B) (L7543; 1:1,000) was purchased from Sigma−Aldrich, and an antibody against poly ADP-ribose polymerase-1 (PARP1) (9532S; 1:1,000) was provided by Cell Signaling Technology (Danvers, MA, USA).

The human retinal pigment epithelial cell line ARPE-19 and the human proximal tubule epithelial cell line HK-2 were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). Cells were maintained in DMEM/F12 medium (D0697; Sigma−Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, S-FBS-SA-015; Serana, Pessin, Brandenburg, Germany) and 1% (v/v) penicillin/streptomycin (15070063; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in a humid atmosphere of 5% CO₂ at 37 °C.

In vivo experiments

Specific pathogen-free male (SPF) ICR mice weighing 22 to 25 g (aged 5 weeks) (n = 10) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in individually ventilated cages under SPF conditions in a controlled environment (24 °C, 55% humidity, and a 12-h day/night cycle) and given free access to drinking water and diet. All mice were kept in SPF facilities of Hangzhou Hibio Technology Company, and the experimental protocol was approved by the Animal Care and Use Committee of Hangzhou Hibio Technology Company (approval number HB2108002). The study was performed according to international, national and institutional rules for animal experiments.

The mice were allowed to acclimate for 1 week and were randomly divided into two groups. The concentrations of Cd were selected based on previous studies (Luo et al., 2016; Zhao et al., 2021). For cadmium exposure, ICR mice (n = 5) were intraperitoneally administered CdCl₂ (5 mg/kg) once a day for 7 days. The normal control (n = 5) received the same volume of normal saline. Twenty-four hours after the last injection, all mice were anesthetized with pentobarbital sodium (70 mg/kg), and blood samples were collected for subsequent analyses. The mice were then sacrificed by cervical dislocation, and organs (heart, liver, kidney, lung, spleen, testis, and eyes) were dissected, weighed, and stored at −80 °C until analysis. No animal was excluded from analysis, but a mouse’s spleen weight, a mouse’s liver Cd concentration, and an animal’s serum Cd concentration in the control group were not included in the analysis because of the failure of sample collection or preparation. The organ coefficient (relative organ weight) was calculated with the organ-to-body weight ratio (%).

Measurement of the concentration of Cd in mouse blood and tissues

The concentrations of Cd in ICR mouse blood and tissues, including the liver, spleen, lung, kidney, testis, and eye were analyzed by ICP−MS. The method for Cd detection using ICP−MS was established according to previously reported methods with minor modifications (Egger et al., 2019; Tai et al., 2022). Tissues (100 mg) were homogenized in RIPA lysis buffer (1,000 μL) (P0013C; Beyotime Biotechnology, Shanghai, China) with a tissue grinder. Tissue homogenate was centrifuged at 4 °C, 5,000 rpm for 5 min. Then, 100 μL of supernatant or serum was digested with 68% HNO₃ in 3.9 mL and reacted in a 15 mL centrifuge tube for 5 min. Subsequently, the reaction solutions were diluted with deionized water and analyzed using a 7800 ICP−MS instrument (Agilent Instruments, Tokyo, Japan). The optimum instrument conditions were set as listed in Table 1. The protein concentration of the supernatant was determined using a BCA kit (P0010S; Beyotime, Shanghai, China). The Cd concentration in each organ or tissue was expressed in unit of μg/mg protein, while the serum Cd concentration was expressed in units of μg/L and μg/mg protein.

DAPI staining

Azoramide and Cd concentrations were selected according to previously reported works (Fu et al., 2015; Komoike, Inamura & Matsuoka, 2012; Zhang et al., 2019). HK-2 cells and ARPE-19 cells were incubated with azoramide (20 μM) for 5 h and then treated in the presence or absence of Cd (20 μM) for 24 h. After washing with phosphate-buffered saline (PBS) three times, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with DAPI for 5 min in the dark. Cell nuclei were observed and photographed by fluorescence microscopy (IX53, Olympus, Tokyo, Japan). The healthy cell nucleus is uniformly stained and clear-edged, while the apoptotic nuclei show irregular edges around the nucleus, heavier staining, and nuclear pyknosis. According to the previously reported method (Jeon et al., 2023), the ratio of condensed nuclei to total nuclei was calculated and expressed as the apoptosis rate (% apoptosis) in each group, and at least three photos were included in the analysis.

Cell apoptosis analysis

Cell apoptosis was examined with flow cytometry (DxFLEX; Beckman Coulter, Suzhou, Jiangsu, China) following annexin V/propidium iodide (PI) double staining (CA1020; Solarbio, Beijing, China). After the indicated treatment, the cells were trypsinized and rinsed with PBS and then resuspended in 100 μL of binding buffer. Five microliters of annexin V and 5 μL of PI were sequentially added and incubated for 5 min in the dark at room temperature. Subsequently, cells were diluted with 400 μL binding buffer, and at least 10⁴ cells were analyzed in each treatment. Annexin V-positive cells were regarded as apoptotic cells. Therefore, cells in quadrants 1 and 4 of the flow cytometry dot plots with quadrant markers were considered apoptotic. Apoptosis rate = (annexin V positive cell number/total cell number) × 100%.

Western blotting (WB)

Total proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China), which contains 1 mM phenylmethanesulfonyl fluoride (ST506; Beyotime, Shanghai, China) and 1% protein phosphatase inhibitor cocktail (P1260; Solarbio, Beijing, China). The protein concentrations were determined with a BCA protein quantitation kit (P0010S; Beyotime, Shanghai, China). Next, 10 μg of protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (ISEQ00010; Millipore, Burlington, MA, USA). After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were incubated overnight at 4 °C with the indicated primary antibodies. Following this, the membranes were incubated with their corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (115-035-044 and 111-035-003, respectively; dilution: 1:2,500 and 1:5,000, respectively; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. Finally, the protein bands were visualized using the ChemiDox™ XRS+ system (Bio-Rad, Hercules, CA, USA) after exposure to Western ECL Substrate (1705061; Bio-Rad, Hercules, CA, USA) for 2 min. The optical density of each WB band was measured using Image Lab™ software (Bio-Rad, Hercules, CA, USA) and normalized to the densities of GAPDH for the same samples. The results are expressed as fold changes relative to the corresponding control, which was always considered as 1.

Mitochondria-associated reactive oxygen species (ROS) measurements

After the designated treatment, mitochondrial ROS were assessed by MitoSOX™ Red (M36008; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) staining according to the manufacturer’s instructions. Cells were collected and resuspended in 100 μL DMEM/F12 medium and incubated with 2.5 μM MitoSOX™ Red in the dark at 37 °C for 15 min. After washed with PBS three times, the cells were analyzed by flow cytometry (DxFLEX), and at least 10⁴ cells were included in the analysis. CytExpert software (Beckman Coulter, Suzhou, Jiangsu, China) was used for measurement of the mean fluorescence intensity of each sample, and the results were expressed as fold changes relative to the control group.

Quantitative real-time PCR (qPCR)

Total RNA was extracted and purified with a RNAiso Plus kit (9109; TaKaRa bio-INC., Kusatsu, Shiga, Japan). cDNA was synthesized with a PrimeScript™ RT Master Mix Kit (RR036A; TaKaRa, Kusatsu, Shiga, Japan). Then, qPCR detection was conducted on an ABI Prism® 7500 real-time PCR detection system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with a TB Green® Premix Ex Taq™ (Tli RNaseH Plus) kit (RR420A; TaKaRa, Kusatsu, Shiga, Japan). The transcriptional expressions of C/EBP homologous protein (CHOP) and heme oxygenase 1 (HO-1) were measured by qPCR. The primers used in the present study were as follows: HO-1, 5′-CCAGCGGGCCAGCAACAAAGTGC-3′, 5′-AAGCCTTCAGTGCCCACGGTAAGG-3′; CHOP, 5′-GACCTGCAAGAGGTCCTGTC-3′, 5′-TGTGACCTCTGCTGGTTCTG-3′; and GAPDH, 5′-TGACGCTGGGGCTGGCATTG-3′ and 5′-GGCTGGTGGTCCAGGGGTCT-3′. Relative gene expression was calculated with the 2^−ΔΔCt method with GAPDH as a loading control. At least three independent samples were performed in each group, and each sample was measured in triplicate.

JC-1 staining

Disruption of mitochondrial inner transmembrane potential ( $\Delta \Psi m$) triggers the activation of intrinsic apoptotic pathway. In this study, $\Delta \Psi m$ was evaluated using JC-1, a dye that accumulates in mitochondria in a potential-dependent manner. When mitochondrial membrane integrity is compromised, the fluorescence emission of JC-1 shifts from red (aggregate) to green (monomer) (Li et al., 2016). The JC-1 staining kit (C2006) from Beyotime Company was used for this purpose. HK-2 cells and ARPE-19 cells were incubated with azoramide (20 μM) for 5 h and then treated with or without Cd (20 μM) for 24 h. JC-1 staining was performed according to the manufacturer’s instructions. Briefly, cells were washed with PBS, incubated with JC-1 for 30 min at 37 °C, and observed under an inverted fluorescence microscope (IX53, Olympus, Tokyo, Japan).

Inhibition of p38 MAPK and JNK

HK-2 cells and ARPE-19 cells were seeded in 6-well plates and incubated with Cd (20 μM) in the presence or absence of SB203580 (10 μM) or SP600125 (10 μM) for 24 h. Concentrations of the inhibitors were selected based on previous studies (Jin et al., 2023; Tang et al., 2023). Then, the cells were collected for WB analysis or stained with annexin V/PI and measured by flow cytometry.

Statistical analysis

Data are expressed as the means ± standard deviations (SD). For statistical analysis, the unpaired Student’s t test was used for data with only two groups; one-way analysis of variance followed by Tukey’s multiple comparisons test was used for data containing more than two groups. All data were analyzed with GraphPad Prism software (version 9.0; San Diego, CA, USA), and p < 0.05 was considered statistically significant.

Accumulation of Cd in different tissues of ICR mice

We observed significant body weight loss in ICR mice after repeated exposure to Cd for 7 days (Fig. 1A and Table 2). However, Cd exposure did not result in any statistically significant differences in liver (1.24 ± 0.14 g vs 1.14 ± 0.09 g), heart (0.12 ± 0.01 g vs 0.12 ± 0.04 g), or lung (0.15 ± 0.02 g vs 0.16 ± 0.02 g) weights (Table 2). Nevertheless, Cd exposure led to increased spleen weight (0.06 ± 0.01 g vs 0.15 ± 0.01 g) while inducing weight loss in the kidney (0.39 ± 0.02 g vs 0.34 ± 0.02 g) and testis (0.16 ± 0.01 vs 0.07 ± 0.04 g) (Table 2). The heart coefficient, liver coefficient, and kidney coefficient were not significantly altered (Figs. 1B–1D). However, the lung coefficient and spleen coefficient showed significant increases (Figs. 1E and 1F). Notably, the testis coefficient showed a dramatic decrease following Cd treatment (Fig. 1G).

To assess Cd concentration in various organs of ICR mice, we employed ICP-MS. Our findings demonstrated that Cd concentration in the serum of control mice was 2.95 ± 5.46 μg/L (or 0.04 ± 0.08 μg/g protein) (Figs. 1H and 1I). In the Cd exposure group, Cd concentration significantly increased to 44.22 ± 11.27 μg/L (or 0.72 ± 0.17 μg/g protein) in comparison to the control (Figs. 1H and 1I). Notably, we found that Cd accumulated in the eye at a higher concentration (10.83 ± 9.14 μg/g protein vs 2.11 ± 0.62 μg/g protein in the control) (Fig. 1J). Among the organs tested, Cd concentration ranked highest in the liver (741.68 ± 81.65 μg/g protein), followed by the kidney (358.35 ± 28.24 μg/g protein), testis (153.52 ± 45.67 μg/g protein), lung (45.69 ± 19.65 μg/g protein), spleen (37.61 ± 13.11 μg/g protein), eye (10.83 ± 9.14 μg/g protein), and serum (0.72 ± 0.17 μg/g protein) (Fig. 1K). These data indicate that Cd tends to accumulate in multiple vital organs, with a preference for the liver and kidney. Intriguingly, the testis showed higher Cd concentration compared to the lung or spleen, confirming that Cd is reproductively toxic (Fig. 1K).

Azoramide protects cells against Cd-induced toxicity

To investigate the protective role of azoramide in Cd-induced cytotoxicity, two widely used cell types, the human proximal tubule epithelial cell line HK-2 and the human retinal pigment epithelial cell line ARPE-19, were employed in the study. We found that Cd exposure could significantly induce apoptotic nuclear morphological changes, and the proportion of nuclear shrinkage and the intensity of DAPI staining increased significantly (Figs. 2A and 2B). Cleavage of PARP1 (C-PARP1), a widely used apoptotic marker protein, was elevated after Cd treatment in HK-2 cells (Fig. 2C). In contrast, azoramide coincubation significantly suppressed PARP1 cleavage (Fig. 2C), indicating that azoramide exerts a protective effect on Cd-exposed HK-2 cells. However, the expression of C-PARP1 was too low to be detectable in ARPE-19 cells in the present study. We further detected apoptosis by annexin V/PI double-staining. Cd treatment dramatically increased the cell apoptosis rate compared to the control group (Figs. 2D and 2E). However, azoramide significantly alleviated Cd-induced cell apoptosis in HK-2 and ARPE-19 cells, and the apoptosis rate was reduced by nearly 20% (Figs. 2D and 2E). Overall, these results suggest that azoramide can significantly protect against Cd-induced cytotoxicity.

Azoramide inhibits Cd-induced ER stress

Azoramide has been shown in previous studies to be a small molecule inhibitor of ER stress (Fu et al., 2015; Ke et al., 2020). In our investigation, we found that azoramide treatment significantly enhanced Cd-induced BiP expression (Figs. 3A and 3B). Additionally, our results showed that Cd increased the phosphorylation level of PERK, whereas azoramide incubation effectively attenuated this increase (Figs. 3C and 3D). Our data showed that Cd increased p-eIF2α expression in HK-2 and ARPE-19 cells (Figs. 3E and 3F). However, azoramide exacerbated the phosphorylation of eIF2α (Figs. 3E and 3F), which may inhibit the translation process and alleviate the pressure of protein folding and processing within the ER. Moreover, our study revealed that Cd treatment elevated the mRNA expression of CHOP, a key mediator of programmed cell death (Figs. 3G and 3H). Azoramide significantly inhibited the upregulation of CHOP (Figs. 3G and 3H). These results strongly suggest that the azoramide’s protective effect against Cd toxicity is closely associated with its ability to inhibit ER stress.

Azoramide inhibits Cd-induced intracellular oxidative stress

It is well-known that ROS can be generated from oxidative phosphorylation in mitochondria (Li et al., 2015). As byproducts in mitochondria, excess ROS may disrupt the electron transport chain and result in mitochondrial dysfunction. We therefore measured mitochondrial ROS with MitoSOX red staining and found that Cd exposure significantly upregulated ROS production in mitochondria, while azoramide suppressed mitochondrial ROS elevation (Figs. 4A and 4B). The Nrf2 pathway is one of the major cellular defense mechanisms against oxidative stress (Tonelli, Chio & Tuveson, 2018). To elucidate its role in azoramide-induced protection effects, we detected Nrf2 by WB. We found that Cd exposure promoted Nrf2 expression, whereas azoramide treatment diminished Cd-induced Nrf2 upregulation (Figs. 4C and 4D). As a consequence, the expression of downstream targeted genes, such as HO-1, was inhibited in the presence of azoramide (Figs. 4E and 4F). Furthermore, JC-1 staining revealed that normal cells exhibited an intact $\Delta \Psi m$, whereas Cd treatment induced markedly $\Delta \Psi m$ loss, as indicated by a shift in JC-1 fluorescence from red to green (Figs. 4G and 4H). Azoramide, however, dramatically attenuated Cd-induced $\Delta \Psi m$ loss and promoted cell survival (Figs. 4G and 4H). Collectively, these data suggest that azoramide may suppress ROS production in mitochondria and inhibit intracellular oxidative stress, thus inhibiting Cd-induced mitochondrial injury.

Azoramide inhibits the activation of p38 MAPK and JNK signaling

Evidence suggests that the MAPK signaling pathway may play a role in Cd-induced cell damage (Kalariya et al., 2009). In our study, we observed that Cd treatment significantly increased the phosphorylation levels of p38 MAPK and JNK (Figs. 5A–5D). However, azoramide dramatically inhibited p-p38 MAPK and p-JNK expression, suggesting that azoramide may inhibit the activation of MAPK signaling (Figs. 5A–5D). These findings also indicate that ER stress, as inhibited by azoramide, may be an upstream event in Cd-induced MAPK activation. To clarify the role of p38 MAPK and JNK in Cd-induced cell death in HK-2 and ARPE-19 cells, cells were treated with pharmacological inhibitors. We found that Cd-induced PARP1 cleavage in HK-2 cells was diminished by SB203580, an inhibitor of p38 MAPK (Fig. 5E). Conversely, the JNK inhibitor SP600125 significantly promoted Cd-induced cleaved PARP1 expression (Fig. 5F). Consistently, apoptosis analysis showed that SB203580 decreased Cd-induced apoptosis rate in HK-2 and ARPE-19 cells (Figs. 5G and 5I), whereas SP600125 aggravated Cd-induced cell apoptosis (Figs. 5H and 5J). These results suggest that p38 MAPK activation is toxic to HK-2 and ARPE-19 cells. In contrast, JNK activation is beneficial for cell survival.

Azoramide inhibits Cd-induced autophagy

Autophagy may also play a certain role in Cd-induced cell apoptosis (Zhang et al., 2019). Our study found that Cd exposure significantly upregulated the expression of autophagy-related marker proteins, such as BECN1 and LC3BII (Fig. 6). However, azoramide treatment inhibited Cd-induced BECN1 expression, and markedly suppressed LC3BII elevation in HK-2 and ARPE-19 cells (Fig. 6). These findings suggest that azoramide alleviates Cd-induced autophagy.