Pyrroloquinoline quinone protected autophagy-dependent apoptosis induced by mono(2-ethylhexyl) phthalate in INS-1 cells

Abstract

Mono(2-ethylhexyl) phthalate (MEHP) is the main metabolite of di(2-ethylhexyl) phthalate (DEHP) in organisms and is commonly used as a plasticizer. Exposure to DEHP impairs the function of islet beta cells (INS-1 cells), which is related to insulin resistance and type 2 diabetes. At present, some research data have also confirmed that MEHP has a certain damage effect on INS-1 cells. In our experiment, we found that MEHP would lead to the increase of reactive oxygen species (ROS) and the upregulation of autophagy. And downregulated ROS production by N-acetyl-L-cysteine could also reduce autophagy. In addition, MEHP-induced lysosomal membrane permeability (LMP) subsequently released cathepsin D. Additionally, MEHP induced the collapse of mitochondrial transmembrane potential and release of cytochrome c. Addition of autophagy inhibitor 3-methyladenine relieved MEHP-induced apoptosis as assessed by the expression of cleaved caspase 3, cleaved caspase 9, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, indicating that MEHP-induced apoptosis was autophagy dependent. Cathepsin D inhibitor, pepstatin A, suppressed MEHP-induced mitochondria release of cytochrome c and apoptosis as well. Meanwhile, pyrroloquinoline quinone (PQQ), a new B vitamin, improved the above phenomenon. Taken together, our results indicate that MEHP induces autophagy-dependent apoptosis in INS-1 cells by lysosomal–mitochondrial axis. PQQ improved this process by downregulating ROS and provided a degree of protection. Our study provides a new perspective for MEHP on the cytotoxic mechanism and PQQ protection in INS-1 cells.

Introduction

Di(2-ethylhexyl) phthalate (DEHP), a commonly used synthetic chemical, is used as a plasticizer. It is found in consumer products, medical devices, cosmetics, food packaging materials and can also be used for dispersion, varnish, paint, and emulsions. ^1,2 Due to its extensive use and high output, it has now caused considerable enthusiasm. Once DEHP enters the body through ingestion, inhalation or skin contact, it is rapidly metabolized into its main metabolite, mono(2-ethylhexyl) phthalate (MEHP), which is preferentially absorbed. ³ MEHP is the bioactive monoester metabolite of DEHP and excreted in urine. ⁴ Previous research has shown that DEHP unleashes its toxic effects through MEHP. ⁵ Phthalates are considered to be endocrine disruptors, and studies have shown that exposure to phthalates is associated with glucose homeostasis and lower testosterone levels. ⁶ In young adult males, urinary MEHP metabolites are associated with impaired glucose homeostasis and decreased testosterone levels. ⁷ Epidemiological studies have shown that phthalate levels are associated with type 2 diabetes and obesity. The development and progression of type 2 diabetes are mostly dependent on the progressive failure of beta cells to provide sufficient insulin. ⁸ In view of the important role of pancreatic β-cells in maintaining glucose homeostasis, this study aimed to investigate the role of MEHP-induced pancreatic cell (INS-1 cells) apoptosis and its molecular mechanisms.

Pyrroloquinoline quinone (PQQ), a universal, anionic water-soluble compound, was found in methylotrophic bacteria for the first time in 1979 as a coenzyme for methanol dehydrogenase and named as methoxatin. ^9,10 Previous researches have shown that PQQ can prevent oxidative stress, reduce free radical levels, and lipid peroxidation. ¹¹ And recently, it was reported that oral administration of PQQ ameliorated glucose tolerance abnormalities in type 2 diabetic mice. ¹² Currently, the presence of PQQ has been detected in various foods and others. ¹³ Thence, PQQ was defined as a new type of vitamin B. ¹⁴ As an essential nutrient, there is increasing evidence that PQQ has many beneficial biological functions such as anti-inflammatory, ¹⁵ hepatoprotective, ¹⁶ cardioprotective, ¹⁷ and antioxidant properties. ¹⁸ Though PQQ is controversial as a vitamin in animal or human nutrition, there is increasing evidence that PQQ plays an important role in regulating cell signaling and redox balance. According to reports, PQQ plays an effective anti-oxidation role through the removal of reactive oxygen species (ROS) activity and other mechanisms. ¹⁹

Autophagy is a catabolic process that relies on lysosomes in eukaryotic cells to degrade intracellular substrates and participates in a variety of physiological and pathological processes, which is responsible for preserving cell homeostasis. ²⁰ Autophagy is usually a protective process in cells. However, under certain conditions, it is harmful to cells and induces cell death through apoptosis or necrosis. The disorder of autophagy has been confirmed in the pathogenesis of several diseases, such as neurodegenerative diseases, heart disease, cancer, and aging. ^21,22 Apoptosis is a well-known natural programmed cell death process, administered by the family of cysteine aspartate proteases called caspases, which plays a key role in physiological development and homeostasis. ²³ More and more studies have confirmed that there is a close relationship between autophagy and apoptosis. Autophagy and apoptosis control the renewal of intracellular organelles and proteins. In addition, many studies have shown that autophagy occurs before apoptosis, ²⁴ autophagy can directly induce apoptosis. ²⁵ The integrity of islet β-cell function and quality is critical for the pathogenesis of diabetes. ²⁶ Research data show that autophagy is associated with diabetes through its effects on islet β-cells. ²⁷ It has been previously reported that dysregulation of autophagy leads to apoptosis, indicating that autophagy protects pancreatic β-cells. ²⁸ In this experiment, we aimed to investigate the relevant mechanism of autophagy inducing apoptosis in rat pancreatic β-cells.

The function of lysosomes determines the progression of the autophagy process, because lysosomes play a key role in the degradation of this process. Lysosomes contain a variety of hydrolytic enzymes that can digest most of the large molecules in cells. ²⁹ The research results suggest that autophagy-dependent apoptosis is mediated by cathepsin D released from lysosomes. ³⁰ In addition, dysfunction of lysosomal function usually destroys autophagy and promotes changes in mitochondrial function and the occurrence of apoptosis. Mitochondria are also involved in cascade activation of apoptosis. ³¹

In summary, in this experiment, we focused on whether MEHP induces autophagy-dependent apoptosis via lysosomal–mitochondrial axis in INS-1 cells. We also aim to explore whether PQQ plays a protective role in this process and how it can be protected.

Materials and methods

Cell culture and treatment

The INS-1 cells line was obtained from the China Center for Type Culture Collection (CCTCC, GDC192). The INS-1 cells are derived from X-ray irradiated rats with canine tumors. They are insulin-positive and can synthesize insulin I and II. It can be used for the study of islet beta cell function. INS-1 cells were grown in DMEM medium (Gibco, BRL-Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Biological Industries), at 37°C in a humidified atmosphere of 5% CO₂. After 2–3 days of culture, the cells were propagated and the logarithmic phase cells were taken for experiments. MEHP was derived from Sigma Aldrich (CAS no. 4376-20-9, assay: 97%) and dissolved in dimethyl sulfoxide (DMSO, Sigma, assay: 99.5%) at 100 mM for stock. PQQ (Lot. H-0221A01) supplied by Eisai Food &Chemical Co. (Tokyo, Japan) was dissolved in three distilled water to a stock solution of 5 mM for use. In intervention experiments, INS-1 cells were treated by different agents: 3-methyladenine (3MA, 5 mM for 2 h, Santa Cruz Biotechnology, California, USA), pepstatin A (100 μM for 4 h, Selleck Chemicals, Houston, Texas, USA), chloroquine (CQ, 10 μM for 2 h, Sigma Aldrich, St. Louis, Missouri, USA), and N-acetyl-L-cysteine (NAC, 10 mM for 1 h, Wako, Osaka, Japan) before exposed to MEHP for 24 h. The DMSO concentration was 0.14% for all exposure groups and solvent controls.

Western blot analysis

At the end of the designated treatments, the INS-1 cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysates were prepared using lysis buffer provided with a protein extraction kit (Keygen Biotech, Nanjing, Jiangsu, China). The pyrolysis of the cell was centrifuged for 5 min at 4°C and 15,000 r/min, and the upper clearing containing the total protein was separated. The concentration of protein was determined by the bicinchoninic acid method. Decomposition of samples with an appropriate ratio of sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinyl fluoride membrane. After closing with 10% nonfat milk, the membranes were incubated with primary antibodies against LC3 (Sigma, CAT no. L7543), p62 (Abways Technology, Shanghai, China), cathepsin D (Cell Signaling Technology, Danvers, Massachusetts, USA), cytochrome c (Abways, CY5734), caspase 9 (Cell Signaling Technology), caspase 3 (Cell Signaling Technology, CAT no. #9662), and the β-actin (Cell Signaling Technology, CAT no. #3700) was used as an internal control. The membranes were then incubated with horseradish peroxidase-conjugated as secondary antibodies and the bound antibody was visualized using the Super-Signal West Pico Kit (Thermo Scientific, Rochford, IL, USA). Using the Bio-Rad ChemiDoc™ MP imaging system, the expected protein bands were detected. Relative abundance of target protein (normalized to β-actin) was measured with the Gel-Pro Analyzer 4.0 software. The experiment was repeated at least twice.

Measurement of intracellular ROS

Detection of intracellular ROS production by dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. ³² After different treatments, INS-1 cells (5 × 10⁵ cells/mL) were washed twice with cold PBS then treated with DCFH-DA at a final concentration of 5 mM for an additional 40 min at 37°C in darkness. After the smear, the cells were washed twice and photographed with an inverted fluorescence microscope. Finally, fluorescence density of images was analyzed with Image-Pro Plus 4.1 software.

Detection the level of superoxidase dismutase and malondialdehyde

Superoxidase dismutase (SOD) is an active substance derived from life, which can eliminate the harmful substances produced in the process of metabolism. Malondialdehyde (MDA) is a metabolite of free radical-induced polyunsaturated fatty acids in the body. To investigate the level of oxidative stress, the SOD activity and MDA level in the culture medium were measured by using SOD and MDA assay kit (Kaiji Bioengineering, China). Following the introduction of kit, SOD activity was measured at 550 nm and expressed as U/mL; MDA level was monitored at 532 nm using 1,1,3,3-tetraethoxypropane as standard and expressed as nmol/mg port.

Determination of the intracellular glutathione content

Glutathione (GSH) is an important antioxidant in cells. The content of intracellular GSH is usually used to indicate the production of ROS in cells. Intracellular GSH levels were determined using a GSH assay kit (Kaiji Bioengineering, China). According to the instructions of the kit, the level of GSH is measured at 405 nm and expressed by μmol/g prot.

Lysosomal membrane permeability assessment

Stability of lysosomal membrane was evaluated by acridine orange (AO) shift test. AO is a metachromatic fluorophore and a lysosomotropic base. In intact lysosomes, accumulation of oligomeric protonated AO causes high concentrations of red fluorescence, while diffusion of lysosomal contents into the cytosol associated with lysosomal membrane permeability (LMP) leads to the formation of monomeric deprotonated form of AO showing green fluorescence. ³³ In short, after different treatments, cells were harvested. After being washed twice with PBS, INS-1 cells were exposed to 1 mg/mL AO (Amresco) for 15 min at 37°C in darkness. The sample was immediately observed and photographed with a fluorescence microscope (Olympus BX63). The stability of the lysosomal membrane was estimated by red fluorescence, using Image-pro Plus 6.0 software (Media Cybernetics).

Mitochondrial membrane potential assessment

The mitochondrial membrane potential (ΔΨm) of MEHP-treated INS-1 cells was evaluated by the potentiometric fluorescent dye JC-1. JC-1 shows a shift from green fluorescence, which corresponded to depolarized/low ΔΨm (JC-1 monomers), to red fluorescence, which corresponded to polarized/normal ΔΨm (JC-1 aggravates). Briefly, after different treatments, cells were harvested. Then the cells were treated with 5 g/mL JC-1 (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) for 20 min in darkness at 37°C. The sample was immediately observed and photographed under a fluorescence microscope (Olympus BX63). The change of ΔΨm was determined by the ratio of red/green fluorescence intensity, ³⁴ using Image-pro Plus 6.0 software (Media Cybernetics).

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay

In situ apoptosis detection kit (Keygen Biotech), to detect apoptosis, following the manufacturer’s protocol. After terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling, nucleus was counter stained with 4′,6-diamidino-2-phenylindole (DAPI) (Keygen Biotech). After different treatments, cells were harvested, fixed with 4% paraformaldehyde for 25 min, and permeatilized in 0.1% Triton X-100 for 5 min. Then, cells were exposed to 50 μL TUNEL reaction mixture for 1 h at 37°C in a humidified atmosphere in the dark. After cleaning with PBS, cells were added DAPI incubation for 10 min. Use of fluorescence microscopy to capture fluorescence images (Olympus BX63), and images for TUNEL-stained cells and DAPI-labeled nuclei were observed on five randomly chosen fields for each section. Image-Pro Plus 6.0 software (Media Cybernetics) was used to quantify the number of TUNEL (+) cells.

Statistical analysis

All values were presented as mean ± standard deviation (SD) from at least two independent experiments. The results were analyzed statistically using a one-way analysis of variance followed by Student–Newman–Keuls test and SPSS 19.0 software. The level of significance was set at p < 0.05 for all statistical analysis.

Results

PQQ reduced apoptosis induced by MEHP in INS-1 cells

TUNEL staining was performed to evaluate cellular apoptosis. We found that apoptosis enhanced with the increasing concentration of MEHP (0–25 μM) in INS-1 cells. Co-treatment of PQQ with MEHP (25 μM) for 24 h, it was found that apoptosis was reduced compared with only MEHP-treated group (Figure 1(a)). In addition, we examined the expression of the activated cleaved form of caspases 9 and 3 by Western blot as a marker of apoptosis. The content of the cleaved caspases 3 and 9 increased with the increasing concentration of MEHP (Figure 1(b)). Similarly, after 24 h of PQQ and MEHP co-treatment, the expression of the cleaved caspases 3 and 9 decreased in contrast to the only MEHP-treated group (Figure 1(c)). These findings suggested that PQQ reduced apoptosis induced by MEHP.

Figure 1.

PQQ reduced apoptosis induced by MEHP in INS-1 cells. Cells were treated with 0–25 μM MEHP and 5 μM PQQ co-processed with 25 μM MEHP for 24 h. (a) Apoptosis was evaluated using TUNEL assay by fluorescence microscopy (scale bar = 20 μm) in INS-1 cells after different treatments. Green indicates TUNEL positive staining and blue indicates DAPI nuclear counterstaining. Quantitative analysis of TUNEL positive cells is shown in (a). Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against caspase 9, caspase 3, and β-actin. Relative expression of caspases 9 and 3 was shown as a percentage of β-actin. (b) INS-1 cells were treated with 0–25 μM MEHP for 24 h. Protein quantitative analysis of caspases 9 and 3 is shown. (c) INS-1 cells were treated with 25 μM MEHP and 5 μM PQQ co-processed with 25 μM MEHP for 24 h, and their protein quantitative analysis is shown. Each bar represents mean ± SD (n = 3) (*p < 0.05 vs. control, ^# p < 0.05 vs. the group treated with MEHP). PQQ: pyrroloquinoline quinine; MEHP: mono(2-ethylhexyl) phthalate; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI: 4′,6-diamidino-2-phenylindole; SD: standard deviation.

PQQ ameliorated collapse of ΔΨm in MEHP-treated INS-1 cells

Downstream events of lysosomal cathepsin D release and trigger events of apoptosis are the collapse of ΔΨm. ³⁵ We performed JC-1 staining to detect the ΔΨm. The ΔΨm was decreased with increasing concentration of MEHP. Co-treatment of PQQ with MEHP for 24 h, the ΔΨm was reversed (Figure 2(a)). Cytochrome c, a pro-apoptotic protein, once released from the mitochondria into the cytoplasm, it will lead to caspase activation and apoptosis. ³⁶ The protein level of cytochrome c was augmented with increasing concentration of MEHP (Figure 2(b)). Cytochrome c expression was reduced after 24 h of PQQ and MEHP co-treatment (Figure 2(c)).

Figure 2.

PQQ restored ΔΨm in MEHP-treated INS-1 cells. Cells were treated with 0–25 μM MEHP and 5 μM PQQ co-processed with 25 μM MEHP for 24 h. (a) The ΔΨm changes was measured by JC-1 staining which observed by a fluorescence microscopy. The red fluorescence represents polarization (high potential) and green fluorescence represents depolarization (low potential). Quantitative analysis of JC-1 staining in INS-1 cells is shown in (a). Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against cytochrome c and β-actin. Relative expression of cytochrome c was shown as a percentage of β-actin. (b) INS-1 cells were treated with 0—25 μM MEHP for 24 h. Protein quantitative analysis of cytochrome c is shown. (c) INS-1 cells were treated with 25 μM MEHP and 5 μM PQQ co-processed with 25 μM MEHP for 24 h, and the protein quantitative analysis is shown. Each bar represents mean ± SD (n = 3) (*p < 0.05 vs. control and ^# p < 0.05 vs. the group treated with MEHP). PQQ: pyrroloquinoline quinine; MEHP: mono(2-ethylhexyl) phthalate; SD: standard deviation.

PQQ restored LMP and cathepsin D release in MEHP-treated INS-1 cells

The stability of the lysosomal membrane in MEHP-treated cells was estimated by AO staining. The results showed that the percentage of cells with red fluorescence (complete lysosomes) decreased as the concentration of MEHP increased. However, this condition was alleviated after PQQ treatment (Figure 3(a)). Cathepsin D released by lysosome participates in apoptosis signal conduction. ³⁷ Western blot analysis pointed that the cathepsin D expression was augmented with increasing concentration of MEHP (Figure 3(b)). Similarly, cathepsin D expression was reduced after 24 h of PQQ and MEHP co-treatment (Figure 3(c)).

Figure 3.

PQQ restored LMP and cathepsin D release in MEHP-treated INS-1 cells. Cells were treated with 0–25 μM MEHP and 5 μM PQQ co-processed with 25 μM MEHP for 24 h. (a) LMP was detected by AO staining and visualization by fluorescence microscopy (scale bar = 20 mm). Quantitative analysis of AO staining is shown in (a). Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against cathepsin D and β-actin. Relative expression of cathepsin D was shown as a percentage of β-actin. (b) INS-1 cells were treated with 0–25 μM MEHP for 24 h. Protein quantitative analysis of cathepsin D is shown. (c) INS-1 cells were treated with 25 μM MEHP and 5 μM PQQ co-processed with 25 μM MEHP for 24 h, and the protein quantitative analysis is shown. Each bar represents mean ± SD (n = 3) (*p < 0.05 vs. control and ^# p < 0.05 vs. the group treated with MEHP). PQQ: pyrroloquinoline quinine; LMP: lysosomal membrane permeability; MEHP: mono(2-ethylhexyl) phthalate; AO: acridine orange; SD: standard deviation.

The reduction of cathepsin D release caused by pepstatin A can alleviate the apoptosis and mitochondrial damage induced by MEHP in INS-1 cells

To examine the relationship between cathepsin D, cytochrome c, cleaved caspases 3 and 9, cathepsin D inhibitor pepstatin A was used. The experimental data showed that the protein expression levels of cathepsin D, cytochrome c, cleaved caspases 3 and 9 were significantly decreased after pepstatin A treatment (Figure 4(a)). Meanwhile, TUNEL assay also showed that apoptosis decreased in MEHP-treated INS-1 cells after pepstatin A treatment (Figure 4(b)). The ΔΨm of the INS-1 cells was reversed after pepstatin A treatment (Figure 4(c)). Based on the above data, cathepsin D released is positively correlated with MEHP-induced apoptosis and mitochondrial damage in INS-1 cells.

Figure 4.

The reduction of cathepsin D release caused by pepstatin A can alleviate the apoptosis and mitochondrial damage induced by MEHP in INS-1 cells. INS-1 cells were pretreated with 100 μM pepstatin A for 4 h and then incubated with 25 μM MEHP for 24 h. (a)Western blot was performed on the cytoplasmic protein fraction of cells to determine the expression of cathepsin D, cytochrome c, caspase 9, and caspase 3. Relative expression of these proteins was expressed as a percentage of the level of β-actin. (b) Apoptosis was assessed using TUNEL assay by fluorescence microscopy (scale bar = 20 µm). Green indicates TUNEL positive staining and blue indicates DAPI counterstaining of nuclei. Quantitative analysis of TUNEL positive cells is shown in the above figure. (c) The ΔΨm was evaluated by JC-1 staining (scale bar = 20 µm). Quantitative analysis of the ratio of red/green fluorescence is shown. Each bar represents mean ± SD (n = 3) (*p < 0.05 vs. control and ^# p < 0.05 vs. the group treated with MEHP). MEHP: mono(2-ethylhexyl) phthalate; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI: 4′,6-diamidino-2-phenylindole; SD: standard deviation.

PQQ reduced autophagy level induced by MEHP in INS-1 cells

The most reliable biochemical marker of autophagy is the conversion of protein LC3 and p62 levels. ³⁸ The expression of LC3 and p62 was detected by Western blot. After treatment with 0–25 μM MEHP for 24 h, the expression of LC3-II and p62 was significantly increased in INS-1 cells (Figure 5(a)). To assess the effect of MEHP on autophagy flux, we examined the expression of LC3 and p62 proteins in the presence of the lysosomal inhibitor CQ. After pretreatment with CQ, it was found that the expression of LC3-II and p62 was further increased after MEHP treatment (Figure 5(b)). Co-treatment of PQQ with MEHP for 24 h, the contents of LC3 and p62 were significantly reduced (Figure 5(c)). These data revealed that PQQ reduced autophagy activation.

Figure 5.

PQQ downregulated autophagy level induced by MEHP in INS-1 cells. Western blot was performed on the cytoplasmic protein fraction of cells using antibodies against LC3, p62, and β-actin. Relative expression of LC3 and p62 was shown as a percentage of β-actin. (a) INS-1 cells were treated with 0–25 μM MEHP for 24 h. Quantitative analysis of LC3 and p62 protein content is shown. (b) Cells were pretreated with 10 μM CQ for 2 h, then incubated with 25 µM MEHP. Relative expression of LC3 and p62 was expressed as a percentage of the level of β-actin. (c) INS-1 cells were co-treated with 5 μM PQQ exposed to 25 μM MEHP for 24 h. Quantitative analysis of LC3 and p62 protein content is shown. Each bar represents mean ± SD (n = 3) (*p < 0.05 vs. control, ^# p < 0.05 vs. the group treated with MEHP). PQQ: pyrroloquinoline quinine; MEHP: mono(2-ethylhexyl) phthalate; CQ: chloroquine; SD: standard deviation.

MEHP-induced autophagy-dependent apoptosis through lysosomal–mitochondrial axis in INS-1 cells

To verify the role of autophagy in apoptosis, we used autophagy inhibitor 3MA. The results showed that the protein expression levels of LC3, p62, cathepsin D, cytochrome c, and cleaved caspase 3 were significantly lower after pretreatment with 3MA (Figure 6(a)). Simultaneously, TUNEL assay also showed that apoptosis decreased by pretreating with 3MA (Figure 6(b)). The ΔΨm was reversed after 3MA treatment (Figure 6(c)). The AO staining showed that the percentage of cells with red fluorescence (complete lysosomes) increased after 3MA treatment (Figure 6(d)). These data indicate that MEHP induces autophagy-dependent apoptosis via the lysosomal–mitochondrial axis.

Figure 6.

MEHP induces autophagy-dependent apoptosis through lysosomal–mitochondrial axis in INS-1 cells. INS-1 cells were pretreated with 1 mM 3MA for 2 h and then incubated with 25 μM MEHP for 24 h. (a) Western blot was performed on the cytoplasmic protein fraction of cells to determine the expression of LC3, p62, cathepsin D, cytochrome c, and caspase 3. Relative expression of these proteins was expressed as a percentage of the level of β-actin. (b) Apoptosis was assessed using TUNEL assay by fluorescence microscopy (scale bar = 20 µm). Green indicates TUNEL positive staining and blue indicates DAPI counterstaining of nuclei. Quantitative analysis of TUNEL positive cells is shown in the above figure. (c) The ΔΨm was evaluated by JC-1 staining (scale bar = 20 µm). Quantitative analysis of the ratio of red/green fluorescence is shown. (d) LMP was detected by AO staining and visualization by fluorescence microscopy (scale bar = 20 mm). Quantitative analysis of AO staining is shown in the above figure. Each bar represents mean ± SD (n = 3) (*p < 0.05 vs. control and ^# p < 0.05 vs. the group treated with MEHP). MEHP: mono(2-ethylhexyl) phthalate; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; LMP: lysosomal membrane permeability; DAPI: 4′,6-diamidino-2-phenylindole; AO: acridine orange; SD: standard deviation.

PQQ attenuated the ROS and oxidative stress caused by MEHP in INS-1 cells

We used ROS fluorescent probe to detect ROS content in INS-1 cells. The experimental results showed that the content of ROS increased with the increase of MEHP concentration. However, the content of ROS decreased after 24 h of co-treatment with PQQ and MEHP (Figure 7(a)). To assess the oxidative stress, we examined GSH, MDA levels, and SOD activity. It turned out that intracellular GSH levels and SOD activity decreased with increasing MEHP concentration, but MDA levels were opposite (Figure 7(b)). The intracellular GSH content and SOD activity were increased by co-treatment with PQQ and MEHP. In contrast, a significant decrease of MDA level was observed in cells by co-treatment with PQQ and MEHP (Figure 7(c)).

Figure 7.

PQQ attenuated the ROS and oxidative stress caused by MEHP in INS-1 cells. Cells were treated with 0–25 μM MEHP and 5 μM PQQ co-processed with 25 μM MEHP for 24 h. (a) DCFH-DA assay was used for detecting ROS levels in INS-1 cells after different treatments by a fluorescence microscopy. Quantitative analyses of DCFH-DA assay in INS-1 cells are shown in the above figure. (b) The histograms represent quantitative analysis of GSH, MDA levels, and SOD activity in INS-1 cells after 0–25 μM MEHP treatment. (c) The histograms represent quantitative analysis of GSH, MDA levels, and SOD activity in INS-1 cells after 5 μM PQQ co-processed with 25 μM MEHP for 24 h. Each bar represents mean ± SD (n = 3) (*p < 0.05 vs. control and ^# p < 0.05 vs. the group treated with MEHP). PQQ: pyrroloquinoline quinine; ROS: reactive oxygen species; MEHP: mono(2-ethylhexyl) phthalate; DCFH-DA: dichloro-dihydro-fluorescein diacetate; GSH: glutathione; SOD: superoxidase dismutase; MDA: malondialdehyde; SD: standard deviation

Downregulated ROS production induced by NAC reduced autophagy levels, restored LMP, and reduced mitochondrial damage and apoptosis induced by MEHP in INS-1 cells

To demonstrate the effects of ROS production on autophagy and apoptosis, we used the ROS scavenger, NAC. The results showed that after NAC treatment, GSH levels and SOD activity increased, but MDA levels decreased (Figure 8(a)). At the same time, the results indicated that the content of ROS decreased after NAC treatment (Figure 8(b)). Western blot results showed that the expression of LC3, p62, cathepsin D, cytochrome c, and cleaved caspase 3 were significantly decreased after NAC treatment (Figure 8(c)). Simultaneously, TUNEL assay also showed that apoptosis decreased after treated with NAC and MEHP than only MEHP group (Figure 8(d)). These data suggest that ROS production leads to increased autophagy, lysosomal permeability, mitochondrial damage, and apoptosis.

Figure 8.

Downregulated ROS production induced by NAC reduced autophagy levels, restored LMP, and reduced mitochondrial damage and apoptosis induced by MEHP in INS-1 cells. INS-1 cells were pretreated with 10 mM NAC for 1 h and then incubated with 25 μM MEHP for 24 h. (a) Histograms represent quantitative analysis of GSH, MDA levels, and SOD activity in INS-1 cells after pre-treatment with NAC for 2 h followed by incubation with 25 μM MEHP for 24 h. (b) DCFH-DA assay was used for detecting ROS levels in INS-1 cells after different treatments by a fluorescence microscopy. Quantitative analyses of DCFH-DA assay in INS-1 cells are shown in the above figure. (c) Western blot was performed on the cytoplasmic protein fraction of cells to determine the expression of LC3, p62, cathepsin D, cytochrome c, and caspase 3. Relative expression of these proteins was expressed as a percentage of the level of β-actin. (d) Apoptosis was assessed using TUNEL assay by fluorescence microscopy (Scale bar = 20 µm). Green indicates TUNEL positive staining and blue indicates DAPI counterstaining of nuclei. Quantitative analysis of TUNEL positive cells is shown in the above figure. (e) The ΔΨm was evaluated by JC-1 staining (scale bar = 20 µm). Quantitative analysis of the ratio of red/green fluorescence is shown. (f) LMP was detected by AO staining and visualization by fluorescence microscopy (scale bar = 20 mm). Quantitative analysis of AO staining is shown in the above figure. Each bar represents mean ± SD (n = 3). (*p < 0.05 vs. control and ^# p < 0.05 vs. the group treated with MEHP). ROS: reactive oxygen species; NAC: N-acetyl-L-cysteine; LMP: lysosomal membrane permeability; MEHP: mono(2-ethylhexyl) phthalate; GSH: glutathione; SOD: superoxidase dismutase; MDA: malondialdehyde; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI: 4′,6-diamidino-2-phenylindole; DCFH-DA: dichloro-dihydro-fluorescein diacetate; AO: acridine orange; SD: standard deviation.

Discussion

MEHP is a biologically active metabolite of DEHP, mainly found in the intestine and also in the liver, kidney, lung, and pancreas. ³⁹ It is widely used as a plasticizer for plastic products. More and more studies have reported that DEHP and its metabolites can destroy endocrine and can be found in many biological fluids of fetuses and adults. ⁴⁰ Many epidemiological studies have shown that various phthalate metabolites are associated with insulin resistance and even diabetes. ^41,42 The cytotoxicity of MEHP has previously been evaluated in a variety of cell types, and some data demonstrate that MEHP induces cytotoxicity and apoptosis in several cell types. At present, MEHP has received extensive attention because MEHP is 20 times more toxic in rats than DEHP. ⁴³ Therefore, in this experiment, we mainly investigate the toxic effects of MEHP on rat islet cells. PQQ is a new vitamin B, and more and more data to prove that PQQ has a variety of beneficial effects. However, the underlying mechanism of PQQ protection against MEHP-induced apoptosis in INS-1 cells has not been well explained. Here, we demonstrate that MEHP induces autophagy-dependent apoptosis via the lysosomal–mitochondrial axis. PQQ improves apoptosis by inhibiting this signaling pathway.

Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia caused by defects in insulin secretion or action. Pancreatic dysfunction and cell death are considered important factors in the pathogenesis of diabetes. The pathology of several human diseases involves free radical mechanisms, including DM. ⁴⁴ In fact, free radicals and other ROS are formed excessively in the DM.

ROS are products of normal and exogenous exposures and play important roles under normal physiological conditions because they are important second messengers that regulate cellular redox status. ⁴⁵ However, excessive ROS will lead to oxidative stress, leading to cell dysfunction and even cell death. ⁴⁶ GSH is the main reducing agent in cells, which can reduce various disulfides by hydrogen transfer. It is an important ROS scavenger. ⁴⁷ SOD is an active substance derived from life, which can eliminate the harmful substances produced in the process of metabolism, and is the main material for scavenging free radicals in the body. MDA is a metabolite of free radical-induced polyunsaturated fatty acids in the body. Recent studies have shown that MEHP has toxic effects on the deregulation of NADH-ubiquinone oxidoreductase chain 1 (Nd1) gene, which may lead to the increase of ROS and the upregulation of SOD1 gene in cells. ⁴⁸ In this research, the effects of MEHP on ROS were investigated by measuring ROS production and oxidative stress by fluorescent probes DCFH-DA and MDA, SOD, and GSH detection kits. The results showed that the ROS increased with the dose of MEHP, and the changes in GSH, SOD, and MDA levels also indicated that MEHP increased ROS content.

Many studies have discussed the link between autophagy and apoptosis, but there seems to be no clear conclusion. At present, the interaction between the two is mostly manifested as apoptosis changing autophagy. But how autophagy controls apoptosis at a mechanistic level is unclear. ⁴⁹ Previous studies have shown a balance between autophagy and apoptosis, maintaining homeostasis under normal conditions or under mild stress conditions. Inactivation of autophagy can lead to accumulation of proteins and organelles, which in turn drive the induction of apoptosis. Afterward, strong autophagic activity induced by strong stimulation can destroy most of the cytoplasm and organelles, dysfunctional cells, promote abnormal cell morphology, and ultimately lead to apoptosis. ⁵⁰ Autophagy is an evolutionarily conserved catabolic process in which organelles and cytosol macromolecules self-degrade. In this study, it was found that MEHP acted on INS-1 cells, autophagy was upregulated, which was manifested as changes in LC3-II and p62 proteins. This indicates that MEHP induces the occurrence of autophagy. There are many studies that prove that ROS can be used as an upstream factor of autophagy. ⁵¹ In our study, clearing ROS helped to reduce the autophagy in the INS-1 cells treated by MEHP. Therefore, it can be logically inferred that ROS are located upstream of autophagy.

Autophagy is an important mechanism that relies on lysosomes to maintain cell stability, isolates defective organelles and proteins from autophagosomes, and then fuses with lysosomes to break down and recover the contents. ⁵² In this experiment, it was found that MEHP-induced LMP led to the release of cathepsin D. After inhibition of autophagy with 3MA, it was found that LMP was improved and the release of cathepsin D was reduced. These results indicate that MEHP-induced autophagy can lead to LMP in INS-1 cells. In other words, MEHP-induced LMP is autophagy dependent.

Cathepsin D released by lysosomes is an important mediator of apoptosis. In LMP, cathepsins are released into the cytosol where they interact with Bid and Bax, followed by mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release. ⁵³ MOMP is an important parameter of mitochondrial function and has been used as an indicator of cell health. In this experiment, it was found that after MEHP acted on INS-1 cells, the ΔΨm of collapsed and cytochrome c was released. Pepstatin A, an inhibitor of cathepsin D, inhibits MOMP and the release of cytochrome c. This indicates that LMP is located upstream of the mitochondria-related death signal. After the intervention of NAC and 3MA, the results consistent with the above appeared.

Apoptosis refers to a highly regulated and controlled death process following the physiological or pathologically stimulating organism’s cells. The loss of MOMP is a sign of apoptosis. ⁵⁴ Cytochrome c from mitochondria binds to the Apaf-1 protein, which in turn activates caspase 9, leading to the death of caspase 3-dependent cells. ⁵⁵ We confirmed that MEHP induced apoptosis in INS-1 by TUNEL and protein changes in caspases 9 and 3. After the intervention of NAC, 3MA, and Pepstatin A, respectively, the occurrence of apoptosis was alleviated. Based on the above data, it can be inferred that MEHP induces ROS and autophagy, autophagy-dependent LMP drives MOMP and the release of cytochrome c, and eventually triggers apoptosis.

At present, there are more and more researches on the mechanism of PQQ. Studies have shown that PQQ (5 μM) combined with DEHP reduces DEHP-induced DNA damage in pancreatic beta cells. ⁵⁶ PQQ has always been shown to act as an antioxidant by inhibiting oxidative stress. ⁵⁷ In this experiment, we first hypothesized that PQQ inhibited the production of ROS, which led to the alleviation of apoptosis. The final experimental data, as we hypothesized, reduced ROS production after PQQ action, and subsequent autophagy was inhibited, which in turn expressed LMP, cathepsin D, MOMP, cytochrome c downregulation, and finally reduced MEHP-induced apoptosis.

In conclusion, this study analyzed the toxic effects of MEHP on INS-1 cells. Our experimental data indicate that MEHP-induced ROS overdose is an upstream event that subsequently induces upregulation of autophagy, which ultimately leads to autophagy-dependent apoptosis via the lysosomal–mitochondrial axis. PQQ protects INS-1 cells from MEHP to some extent by this signaling pathway. However, there is still a need to further study the biological effects of MEHP in different cells and organisms and their underlying mechanisms, ultimately eliminating the harm that plasticizers bring us.