Pterostilbene attenuates oxidative stress induced by hydrogen peroxide in MAC-T cells through activating PINK1/Parkin-mediated mitophagy

Abstract

High‐producing dairy cows are susceptible to altered redox balance owing to their high secretion and strong metabolism during transition to lactation. Previously reports demonstrated that pterostilbene (PTE) alleviate hydrogen peroxide (H₂O₂)-induced oxidative damage. However, molecular mechanism underlying protective role of PTE in H₂O₂-elicited oxidative stress in bovine mammary epithelial cells (MAC-T cells) remains unclear. The aims of our research is to clarify the potential molecular mechanisms of PTE in H₂O₂-triggered oxidative damage. MAC-T cells were pre-treated with PTE (0, 10, 25 and 50 μM) for 12 h and then cultured with H₂O₂ (0, 100, 200, 400, 600, 800 and 1000 μM) for another 24 h. The results were interpreted by CCK8, western blot, and immunofluorescence assay. Results showed that PTE significantly attenuated H₂O₂-mediated reduction in T-AOC, SOD and GSH-Px activity. Mechanistic studies found that PTE restricted the H₂O₂-induced protein expression of HO-1, GPX4 and SOD1, and PTE reversed intracellular MDA and ROS generation and decreased the MMP in MAC-T cells. Moreover, H₂O₂ exposure markedly inhibited mitophagy, whereas PTE pre-treatment activated PINK1/Parkin-mediated mitophagy pathway, which further limited ROS production. Overall, PTE can be used to improve oxidative stress and as a novel antioxidant agent in dairy cows.

Highlights

• PTE restored the levels of H₂O₂-induced oxidative stress markers (T-AOC, SOD, GSH-Px and MDA) in MAC-T cells.

• PTE effectively blocked the increase of protein expression (HO-1, SOD1 and GPX4) in H₂O₂-induced MAC-T cells.

• PTE remarkably decreased the accumulation of ROS and improved MMP in H₂O₂-induced MAC-T cells.

• PTE exerted a significant protective effect against oxidative stress injury by reducing the inhibition of H₂O₂ on mitophagy.

Keywords:

• Pterostilbene
• oxidative stress
• PINK1/Parkin
• mitophagy
• MAC-T

Introduction

Owing to increasing demand for milk and other dairy products, dairy cows health has attracted considerable interest. As an essential structure for the secretion and synthesis of milk protein, mammary tissue has strong metabolism in cows and rapidly produce a large amount of ROS and reactive nitrogen free radicals, which can lead to redox imbalance and oxidative stress and thereby trigger various adverse effects, such as mitochondrial dysfunction, inflammation, apoptosis and cell death and ultimately leading to metabolic disorders (Kan et al. 2021; Yu et al. 2022; Qi et al. 2023; Zhang X et al. 2023). Therefore, finding a target to reduce the event of oxidative stress in dairy cow is necessary.

Mitophagy (mitochondria autophagy) is a selective form of macroautophagy that eliminates the excessive accumulation of ROS by selectively inducing lysosomal degradation in mitochondria, considered as a defensive response to keep mitochondrial homeostasis in response to infection (Onishi et al. 2021; Verbeke et al. 2023). The activation or inhibition of mitophagy plays a vital role in oxidative damage alleviation and inflammatory response (Ko et al. 2021; Cao et al. 2023). The PINK1/Parkin signalling pathway is considered one of the ubiquitin-dependent mitochondrial degradation mechanism (Li J et al. 2023) that plays a key role in the regulation of mitophagy triggered by several pathogens. For example, Lactobacillus rhamnosus GR-1 protects MAC-T cells against Escherichia coli-triggered apoptosis, oxidative damage and ROS production through PINK1/Parkin-mediated mitophagy (Li Y et al. 2021), which are also activated in Staphylococcus aureus-challenged model to promote its survival and contribute to persistent S. aureus infection within MAC-T cells (Liu K et al. 2022; Xu et al. 2023). These findings indicate that PINK1/Parkin-mediated mitophagy is crucially involved in mediating oxidative stress injury.

Pterostilbene (PTE), a resveratrol dimethyl ether derivative, has strong bioactivity and is present in Dalbergia and Vaccinium spp (Liu Y et al. 2020). Similar to resveratrol, PTE has a wide range of various pharmacological properties, including anti-inflammation (Abd-Elmawla et al. 2023), anti-apoptotic (Surien et al. 2023), and anti-oxidative (Chen X et al. 2023). Furthermore, PTE can protect porcine alveolar macrophages (Jin J et al. 2022) or IPEC-J2 cells (Chen et al. 2022) from oxidative stress and mitochondrial damage. Additionally, PTE can be used to prevent oxidative damage triggered by deoxynivalenol in MAC-T cells (Zhang et al. 2021) and avoid oxidative insult to bovine spermatozoa and preserve the sperm quality parameters (Sapanidou et al. 2023). Therefore, the effects of PTE on oxidative damage and its mechanism in MAC-T cells are still unknown.

Hence, the purpose of this study was to examine the effect and specific mechanism of action of PTE on cell dysfunction and provide a new reference for the treatment of oxidative damage. The findings provided evidence that PTE promotes mitophagy and inhibits ROS production and ameliorates H₂O₂-triggered oxidative stress injury by regulating mitochondrial function.

Material and methods

Cell culture and treatment

MAC-T cells were provided by Professor Yi Yang of Yangzhou University and maintained in a DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin in a humid atmosphere at 37 °C with 5% CO_2. The MAC-T cells were passaged, treated at 80% confluence, and seeded into 6-, 12- or 96-well plates for subsequent experiments and pre-treatment with PTE for 12 h. Then, the cells were subjected to H₂O₂ for 24 h for the construction of an oxidative stress model.

Cell viability assay

CCK-8 kit (K1018, ApexBio Technology, Houston) was used to measure cell viability. The MAC-T cells were seeded into a 96-well plate at a density of 5000 cells per well and then exposed to various PTE or H₂O₂. Subsequently, 10 μL of CCK-8 solution was added to each well for 2 h, and absorbance at 450 nm was measured by a microplate reader.

Measurement of antioxidative activity

After the stimulation of MAC-T cells, samples were collected, and the total antioxidant capacity (T-AOC, A015) and glutathione peroxidase (GSH-Px, A005), superoxide dismutase (SOD, A001) and malondialdehyde (MDA, A003) levels were detected with commercial kits purchased from Nanjing Jiancheng Biotechnology according to the manufacturer instructions.

Measurement of ROS

After stimulation, 2,7-dichloro-dihydrofluorescein diacetate (DCFH‐DA, D6883, Sigma-Aldrich, USA), an ROS indicator, was diluted in a serum-free medium to a final concentration of 10 μM for 30 min. Then, the cells were washed three times with PBS to remove DCFH‐DA. Finally, the cells were observed and imaged using an inverted fluorescence microscope (Leica-DMi8-M, Germany) through multi band fluorescence filter system. ImageJ was used to measure the fluorescence intensity. Briefly, the target image should be transferred to 8 bits and ajust the threshold. Next, we should set the measurements, including ‘area, mean, integrated density, mean gray value, and limited to threshold’ and then measure the integrated density values to analyse.

Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was detected by an MMP assay kit (E-CK-A301, Elabscience Biotechnology Co., Ltd., China) containing JC-1 fluorescent probes. After treatment, MAC-T cells were incubated with JC-1 reagent for 30 min in the dark and then washed twice with a solution buffer (PBS). Subsequently, the images were captured under an inverted fluorescence microscope (Leica, Germany). The green monomer JC-1 and red aggregated JC-1 were detected at emission wavelengths of 530 nm and 590 nm, respectively.

Western blot analysis

After treatment, the cells were collected and lysed with RIPA buffer (P1003B, Beyotime Biotechnology, China). Total protein was determined according to a previous study (Jiang et al. 2020). The denatured protein samples were separated by 10% SDS-PAGE gel (150 V, 50 min) and transferred to a PVDF membrane for 30 min using 400 mA (activated with methanol for 1 min, FFP19, Beyotime, China). Subsequently, the membranes were blocked with 3% defatted milk powder (P0216, Beyotime, China) for 2 h at room temperature and incubated with primary antibodies at 4 °C overnight against HO-1 (27282-1-AP, Proteintech, China), SOD1 (10269-1-AP, Proteintech), GPX4 (14432-1-AP, Proteintech), Parkin (14060-1-AP, Proteintech), PINK1 (23274-1-AP, Proteintech), beclin-1 (11306-1-AP, Proteintech) and GAPDH (60004-1-Ig, Proteintech). The membrane was washed three times with TBST buffer for 5 min each and then incubated with the recommended dilution of conjugated secondary antibody at room temperature for 2 h. Finally, the protein bands were imaged through ECL chemiluminescence (YWB003, YIFEIXUE Biotechnology, China) and analysed by ImageJ software. Briefly, open an image and transfer to 8 bits. Secondly, process the subtract background and set measurements (pick area, mean gray value, and integrated density) and scale (fill ‘unit of length’ with ‘pixels’). Thirdly, we should switch the image to bright bands and choose the target area on the image for measurement. After measuring all of the bands, copy the integrated density values to analyse.

Immunofluorescence assay

The MAC-T cells were seeded into 12-well plates, and a coverslip was placed at the bottom of each well. In subsequent experiments, cells were initially treated with PTE for 12 h and then exposed to H₂O₂ for 24 h. To label the mitochondria, Mito-Tracker Red CMXRos (C1035, Beyotime Biotechnology, China) was added to living MAC-T cells at 37 °C for 30 min. The MAC-T cells were washed three times with cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Next, 0.1% Triton X-100 were used to permeabilize the cells for 10 min, and the cells were blocked with 3% BSA for 30 min. Afterwards, the cells were incubated with LC3 (14600-1-AP, Proteintech) or Parkin (14060-1-AP, Proteintech) in a humidified chamber at 4 °C overnight and then incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (A0423, Beyotime Biotechnology, China) at room temperature for 1 h in the dark. After washing, DAPI (C1005, Beyotime Biotechnology, China) was added to label cell nuclei. Eventually, the cells were observed and imaged using an inverted fluorescence microscope (Leica-DMi8-M, Germany) through multi band fluorescence filter system. MitoTracker Red CMXRos, DAPI and secondary antibodies were detected at their emission wavelengths of 599 nm, 488 nm and 519 nm, respectively.

Statistical analysis

All data were presented as means ± SEM. Statistical comparisons between groups were performed using one-way ANOVA followed by the post-hoc StudentNewman-Keuls test or Least Significance Difference test. GraphPad Prism 8 software was utilised for data analyses and operated to generate bar plots. In this experiment, p value < 0.05 was considered statistically significant.

Results

Effects of PTE or H₂O₂ on the cell viability of MAC-T cells

The toxicity range of PTE or H₂O₂ stimulation concentration on MAC-T cells was determined, and cell viability was then detected. The CCK-8 results showed that PTE did not affect the viability of MAC-T cells at 25 μM, whereas the effect of PTE showed a significant increase or decrease at 10 and 50 μM, respectively (Figure 1(a)).

Figure 1. Effects of pterostilbene (PTE) or H₂O₂ on the cell viability of MAC-T cells. (a) The chemical structure of PTE. (b) MAC-T cells were treated with different concentrations of PTE (0, 10, 25 and 50 μM) for 12 h and then cell viability was detected using CCK8. (c) Different concentrations of H₂O₂ (0, 100, 200, 400, 600, 800 and 1000 μM) stimulated MAC-T cells and then viability was measured. (d) MAC-T cells were pre-treated with PTE for 12 h and subsequently incubated with H₂O₂ (600 μM) for 24 h. The cell viability of PTE on H₂O₂-induced MAC-T were determined by CCK8 assay. Data were presented as mean ± SEM (n = 3), **p < 0.01 versus control group, ^##p < 0.01 versus H₂O₂ group.

The results showed that H₂O₂ above 400 μM significantly inhibited the cell viability of MAC-T cells. Thus, 600 μM H₂O₂ was used in subsequent experiments (Figure 1(b)). Then, the MAC-T cells were pre-treated with different dose of PTE (10, 25 and 50 μM) for 12 h and followed by H₂O₂ (600 μM) for another 24 h to investigate the impact of PTE on H₂O₂-triggered oxidative damage. The results showed that reduction in H₂O₂ viability was rescued at 10 or 50 μM PTE (Figure 1(c)). Therefore, according to previous studies (Zhang et al. 2021), we selected PTE of 10 μM as the experimental concentration.

PTE restored the levels of H₂O₂-induced oxidative stress markers in MAC-T cells

Whether the antioxidant effects of PTE after H₂O₂ treatment remains unknown. We observed that H₂O₂ stimulation significantly decreased the activities of T-AOC, SOD and GSH-Px as opposed to control group. However, these changes were not observed when the MAC-T cells were pre-treated with PTE (p < 0.01, Figures 2(a–c)). In addition, the PTE supply markedly downregulated increase in MDA content in the H₂O₂-induced group (Figure 2(d)). These data suggested that PTE exhibited antioxidative ability.

Figure 2. Pterostilbene (PTE) restored the levels of H₂O₂-induced oxidative stress markers in MAC-T cells. Pre-treatment with PTE (10 μM) was performed for 12 h, followed by H₂O₂ (600 μM) treatment of MAC-T cells for 24 h. (a) T-AOC level, (b) SOD activity, (c) GSH-Px activity and (d) MDA content. Data are present as mean ± SEM (n = 3), **p < 0.01 versus control group, ^##p < 0.01 versus H₂O₂ group.

PTE attenuated H₂O₂-triggered oxidative damage in MAC-T cells

To further confirm the effect of PTE on H₂O₂-triggered oxidative damage, we first performed Western blot to evaluate the protein expression related to oxidative stress mediators. The outcomes demonstrated that HO-1 expression significantly changed in the presence of PTE compared with H₂O₂ group (p < 0.01, Figure 3(b)). Moreover, H₂O₂ exposure significantly decreased SOD1 and GPX4 protein expressions in comparison to control group, whereas pre-treatment with PTE effectively blocked the above phenomenon (p < 0.01, Figure 3(c)) or resulted in an upward trend (p = 0.06, Figure 3(d)). Additionally, PTE up-regulated the expression of NRF2 protein in H₂O₂-induced MAC-T cells (Figure 3(e)). The data further mentioned that PTE mitigated H₂O₂-triggered oxidative stress and damage in MAC-T cells.

Figure 3. Pterostilbene (PTE) attenuated H₂O₂-triggered oxidative damage to MAC-T cells. Pre-treatment with PTE (10 μM) was performed for 12 h, followed by H₂O₂ (600 μM) treatment of MAC-T cells for 24 h. (a) Representative Western blot bands for HO-1, SOD1 and GPX4 proteins. (b) Quantitation of HO-1 protein level in MAC-T cells. (c) Quantitation of SOD1 protein level in MAC-T cells. (d) Quantitation of GPX4 protein level in MAC-T. (e) Quantitation of NRF2 protein level in MAC-T data are present as mean ± SEM (n = 3), **p < 0.01 versus control group, ^##p < 0.01 versus H₂O₂ group.

Next, we measured the intracellular levels of ROS and MMP in MAC-T cells through immunofluorescence, which are important indicators for evaluating the health of mitochondria. The results revelled that H₂O₂ exposure significantly led to the accumulation of ROS in comparison to control group, whereas pre-treatment with PTE remarkably reduced the adverse effect (p < 0.01, Figures 4(a–b)). The decrease of MMP is a landmark event of early cellular apoptosis. In this experiment, we found that pre-treatment with PTE resumed the ratio of red to green fluorescence intensities and tended mitochondrial membrane permeability towards normal levels (p < 0.01, Figures 4(c–d)). Notably, PTE partly alleviated H₂O₂-induced abnormal changes in ROS level and MMP in MAC-T cells.

Figure 4. Pterostilbene (PTE) resumed H₂O₂-induced ROS and MMP levels in MAC-T cells. Pre-treatment with PTE (10 μM) was performed for 12 h, followed by H₂O₂ (600 μM) treatment of MAC-T cells for 24 h. (a) Immunolabelling and quantification of ROS level by DCFH-DA staining in MAC-T cells. Green: DCFH-DA, scale bar: 100 µm. (b) MMP was detected using JC-1 staining. Red: aggregate, green: monomer, scale bar: 100 µm. Data are present as mean ± SEM (n = 3), **p < 0.01 versus control group, ^##p < 0.01 versus H₂O₂ group.

PTE improved mitophagy in H₂O₂-stimulated MAC-T cells

Mitophagy is a host defense system that plays a critical role in mitochondria homeostasis. To verify whether PTE eliminates H₂O₂-stimulated intracellular ROS production through mitophagy mediated by PINK1/Parkin, we first investigated mitophagy-related protein expression. After 24 h, H₂O₂ treatment markedly downregulated beclin-1 (p < 0.01), PINK1 (p < 0.01) and Parkin (p = 0.07) expression levels, whereas PTE pre-treatment blocked this decrease (p < 0.01, Figure 5(a–b)). We also observed that PTE attenuated H₂O₂-induced reduction in the number of LC3 puncta (Figure 5(c)). To confirm these findings and assess the formation of mitophagosomes, we performed dual immunostaining for Parkin and Mito-Tracker in MAC-T cells. These results suggested that exposure to H₂O₂ downregulated the colocalisation of Parkin and Mito-Tracker in MAC-T, whereas PTE alleviated this inhibition (Figure 5(d)). These data indicated that PINK1/Parkin-mediated mitophagy were involved in alleviating PTE in H₂O₂-induced oxidative damage.

Figure 5. Pterostilbene (PTE) improved mitophagy in H₂O₂-stimulated MAC-T cells. Pre-treatment with PTE (10 μM) was performed for 12 h, followed by H₂O₂ (600 μM) treatment of MAC-T cells for 24 h. (a–b) Western blot images and quantification showing beclin-1, PINK1 and Parkin protein levels in MAC-T cells. (c) Representative immunofluorescence images of LC3 (green). DAPI (blue)-stained nucleus. Scale bar: 100 µm. (d) Representative images of immunofluorescence double-labelling Parkin (Green) and mitochondrial marker (Mito-Tracker, red). DAPI (blue)-stained nucleus. Scale bar: 100 µm. Data were presented as mean ± SEM (n = 3), **p < 0.01 versus control group, ^##p < 0.01 versus H₂O₂ group.

To further investigate the correlations among PTE, H₂O₂ and mitophagy, we used 3-methyladenine (3-MA, autophagy inhibitor) to assess. MAC-T cells were incubated with 3-MA and PTE for 12 h before exposure to H₂O_2. Interestingly, increase in oxidative stress-related or mitophagy-related protein expression in PTE + H₂O₂ were markedly decreased by 3-MA (p < 0.01, Figures 6(a–b)). Intracellular ROS production and the localisation of LC3 were reversed before pre-treatment with 3-MA (Figures 6(c–d)). Overall, these data confirmed that PTE had a significant protective effect against oxidative damage through reducing the inhibition of H₂O₂ on mitophagy.

Figure 6. Effect of 3-MA incubation on the efficacy of pterostilbene (PTE) treatment. MAC-T cells were incubated with 3-MA (autophagy inhibitor, 5 mM) and PTE (10 μM) for 12 h before exposure to H₂O₂ (600 μM)_. (a–c) Western blot images and quantification showing HO-1, SOD1, beclin-1, PINK1 and Parkin protein levels in MAC-T cells. (d) ROS levels. Scale bar: 100 µm. (e) LC3 staining of each group of MAC-T cells. Scale bar: 100 µm. Data were presented as mean ± SEM (n = 3), *p < 0.05, **p < 0.01 versus control group. ^#p < 0.05, ^##p < 0.01 versus H₂O₂ group. ^&p < 0.05, ^&&P < 0.01versus PTE group.

Discussion

Oxidative stress has been implicated to play a role in pathogenesis of many disease conditions in animals (Patra et al. 2011). To date, oxidative stress of mammary tissue has gradually become an urgent problem in high‐producing dairy cows during transition into lactation, negatively affects milk performance and mammary gland health and causes huge economic loss (Khan et al. 2023; Zhou M et al. 2023). Therefore, it is essential to find plant additives to reduce the occurrence of oxidative stress in dairy cow mammary glands.

Natural plant polyphenol, especially resveratrol, protects multiple cells against oxidative stress (Machado et al. 2023; Zamanian et al. 2023). Resveratrol has beneficial effects that inhibit inflammatory response, oxidative stress and apoptosis in MAC-T cells (Jin X et al. 2016; Zhou et al. 2019; Zhou et al. 2020; Ouyang et al. 2023). PTE is a natural analog of resveratrol and has a longer half-life and wide range of stronger pharmacological effects than resveratrol (Vankova et al. 2020). Additionally, growing evidences have reported that PTE, as a protective antioxidant, can be used to piglets (Zhang, Chen, Li, et al. 2020) and chickens (Zhang, Chen, Chen, et al. 2020). However, the application of PTE in dairy cows remains unclear. Thus, it is worth investigating whether PTE could relieve oxidative stress in MAC-T cells.

Mitochondrial are important mediators of cellular metabolism, and producers or targets of ROS, which thereby disrupting mitochondrial permeability transition pores and alters mitochondrial membrane potential (Zhang Z et al. 2023). ROS accumulation can result in an increase in MDA and decrease in T-AOC, SOD and GSH levels (Cui et al. 2006). Abnormal ROS production and MDA and imbalance in the antioxidant defense system in MAC-T cells subjected to deoxynivalenol were rescued by PTE (Zhang J et al. 2021), and in other types of pathological models (Amin et al. 2022; Chen Y et al. 2023). In the present study, PTE pre-treatment inhibited H₂O₂-induced the production of ROS and MDA and facilitated T-AOC, SOD and GSH-Px activity and improved MMP. Furthermore, we found that PTE significantly upregulated the protein expressions of HO-1, SOD1 and GPX4, which are the main antioxidant protein and acts directly downstream of Nrf2 (Lee et al. 2021). PTE facilitated accumulation of Nrf2 into the nucleus and enhanced the levels of PRDX4, SOD2 and GPX1 in tunicamycin-exposed jejunum in piglets (Chen et al. 2022). It is possible that the Keap1-Nrf2 mechanism becomes disrupted and results in cell and tissue damage under unusually severe oxidative stress, which can be restored by PTE. These outcomes indicate that PTE can attenuate H₂O₂-induced oxidative damage in MAC-T cells possibly through the Nrf2/HO-1 signalling pathway.

Growing studies reveale that mitophagy plays crucial roles in preventing diseases such as non-alcoholic fatty liver disease (Shen B et al. 2023), skin carcinogenesis (Tsai et al. 2012), and acute tubular necrosis (Chen J et al. 2023), and is considered a key intracellular process that improves mitochondrial biogenesis and eliminates ROS formation. More importantly, PINK1/Parkin-dependent mitophagy is a classic pathway to maintain mitochondrial homeostasis. Mechanistically, the accumulation of PINK1 onto mitochondria accelerates Parkin recruitment from cytosol to mitochondria in response to a decline in membrane potential (Pereira et al. 2023). In this study, we found that H₂O₂ exposure markedly decreased the beclin-1, PINK1 and Parkin expressions and the puncta number of LC3, as well as the colocalization of Parkin and mitochondrial markers. Likewise, Escherichia coli infection inhibited PINK1/Parkin-mediated mitophagy in MAC-T cells and brought about mitochondrial damage and subsequent enhanced ROS production (Li Y et al. 2021). However, Staphylococcus aureus infection of bovine macrophages (Zhou X et al. 2023) or MAC-T cells (Liu K et al. 2022; Xu et al. 2023) generates oxidative damage and enhances mitophagy through the PINK1/Parkin pathway. Additionally, the newcastle disease virus reprograms host cell energy metabolism by activating PINK1/Parkin-dependent mitophagy (Gong et al. 2022). These inconsistent findings may be attributed to the types and levels of stimulated factors, time of supplementation and cell types. Our results showed that PTE pre-treatment significantly alleviated H₂O₂-triggerated oxidative damage, accompanied by the increase in PINK1/Parkin-mediated mitophagy. This result is consistent with a previous report (Chen et al. 2022). Therefore, our outcomes demonstrate that PINK1/Parkin-dependent mitophagy is a possible mechanism by PTE alleviates H₂O₂-induced oxidative stress injury.

Limitation

There are several limitations of this research that should be noted. Firstly, the limitation of the current study is that the mechanisms of mitophagy receptors (such as Nix, BNIP3 and FUNDC1) (Liu L et al. 2014; Shen Y et al. 2023), which are important was not identified. Secondly, a major limitation of the present study is the lack of measurement of NRF2 activity, which is an important transcriptional regulatory factor. Finally, we found that the level of GPX4, a main regulatory component involved in ferroptosis, significantly changed in the presence of PTE or H₂O₂, indicating that ferroptosis has a significant impact on oxidative stress (Song and Long 2020). PTE can ameliorate oxidative stress in human ovarian granulosa cells through Nrf2/HO-1-mediated ferroptosis (Chen X et al. 2023). Thus, HO-1/ROS-mediated ferroptosis will receive our great interest in next experiment.

Conclusion

Overall, this study demonstrated that PTE can ameliorate oxidative stress in response to H₂O₂ in MAC-T cells. The underlying mechanism is that PTE can effectively reduce the inhibition of H₂O₂ on PINK1/Parkin-mediated mitophagy. These outcomes suggest that PTE acts a potent therapeutic agent for oxidative stress.

Ethical approval

All procedures were approved by the Animal Care and Use Committee of Xuzhou University of Technology, Xuzhou, China.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The original data of the paper are available upon request from the corresponding author.

Additional information

Funding

The present study were supported by the Natural Science Foundation of Jiangsu Province [BK20210077, BK20211051, and BK20210078], the Natural Science Foundation of the Jiangsu Higher Education Institutions of China [22KJB230008 and 22KJA240003] and Key Research and Development Plan of Xuzhou City [KC23034, KC21296 and KC22133].