Protection effect of cis 9, trans 11-conjugated linoleic acid on oxidative stress and inflammatory damage in bovine mammary epithelial cells | Scientific Reports

Scientific Reports volume 14, Article number: 26295 (2024) Cite this article

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The present study was conducted to observe the protective effects of c9, t11- conjugated linoleic acid (CLA) on oxidative stress and inflammation in bovine mammary epithelial cells (BMECs) exposed to H2O2. The BMECs were treated with different concentrations of H2O2 for 8 h, 600 µmol/L was determined to be the damage concentration. Using different concentrations of c9, t11-CLA to process BMECs for 24 h, 50 and 100 µmol/L were determined to be the effective concentrations for subsequent analyses. Thus, four BMEC groups were established: Control group; H2O2 group; 50 µmol/L c9, t11-CLA + H2O2 group; 100 µmol/L c9, t11-CLA + H2O2 group. We observed that the H2O2 group exhibited significantly lower total antioxidant activity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and significantly higher secretions of malondialdehyde (MDA), interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α and expressions of IL-1β, IL-6, and IL-8 than the control group (p < 0.05). Pretreatment with c9, t11-CLA enhanced SOD, CAT, and GPx activities and SOD mRNA expression and repressed IL-6 and IL-8 secretion and expression in H2O2-treated BMECs (p < 0.05). In conclusion, c9, t11-CLA treatment efficiently enhanced antioxidant capacity and decreased inflammation induced by H2O2 in BMECs.

In recent years, the continuous advancement in modern feeding has significantly enhanced the production performance of dairy cows in China. However, during the perinatal period and the peak of lactation, the metabolic rate of mammary cells increases obviously, and the enhancement of metabolic activity leads to the accumulation of free radicals in the mammary tissues of dairy cowsShifts in thioredoxin reductase activity and oxidant status in mononuclear cells obtained from transition dairy cattle. J. Dairy. Sci. 90, 1186–1192. https://doi.org/10.3168/jds.S0022-0302(07)71605-3 (2007)." href="/articles/s41598-024-77711-0#ref-CR1" id="ref-link-section-d86797637e415">1,2. Free radical accumulation could reduce the feed utilization rate and milk yield in dairy cows3. The reduced antioxidant defense in cows makes them prone to oxidative stress, leading to the incidence of several diseases, such as bovine mastitis. Metabolic stress in dairy cows leads to lipid mobilization, inflammation, oxidative stress, and subsequent metabolic stress in the prenatal period, which affects milk production and health of dairy cows, resulting in enormous economic losses to the dairy industry4,5. Oxidative stress reflects an imbalance between free radical production and the body’s ability to detoxify via antioxidants. Under physiological conditions, the antioxidant systems maintain the oxidant-antioxidant balance by rectifying the altering levels of oxidants. In over-conditioned cows, an inflammatory state is caused by the oxidative stress frequently induced by excessive lipolysis that enhances reactive oxygen species (ROS) production via the β-oxidation process. Excessive ROS accumulation enhances the expression of pro-inflammatory mediators, including cytokines6. Therefore, several researchers have focused on reducing the metabolic stress in the mammary glands of dairy cows in order to improve the antioxidants or the anti-inflammatory capacity in the mammary glands and improve milk quality.

Fatty acids play an important role in regulating oxidative stress and inflammation. Oxidative stress levels can be measured by quantifying several oxidative stress markers, such as malondialdehyde (MDA), total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)7. Several polyunsaturated fatty acids (PUFAs) are known to exhibit an anti-inflammatory potential in various cell types. Some studies have observed that the T cells treated with fatty acids modulated the secretion of various cytokines, including tumor necrosis factor- α (TNF- α), interleukin (IL)-1β, IL-6, IL-8, IL-108,9. Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid with several important physiological functions, such as anti-inflammation, anti-oxidation, anticancer, body fat reduction, and immunity improvement. Most of the beneficial properties of CLA are elicited by its two isomers: c9, t11-CLA and t10, c12-CLA. CLA primarily exists in the form of the c9, t11-CLA isomer, accounting for more than 90% of the total CLA, and present at relatively higher levels in the meat and milk fat of ruminant animals10. CLA plays an important role in anti-inflammatory responses11,12, that CLA regulates the production of cytokines involved in inflammatory responses, including IL-6, TNF-α, and IL-1β13. The c9, t11-CLA is a potential foodborne drug that can regulate the immune system and inflammatory response in porcine14. Low concentrations of c9, t11-CLA significantly decreased concentrations of IL-6 and IL-8 in endothelial cell, have modest anti-inflammatory effects15. The dietary-supplemented rumen-protected CLA has been found to alleviate mild inflammation and oxidative stress in cows after calving16. Furthermore, CLA treatment only mildly impacted the antioxidative and anti-inflammatory status of mid-lactating dairy cows17. In addition, CLA and essential fatty acids might enhance the anti-inflammatory effects of calves18. CLA supplementation reduced IL-1β, IL-6, and TNF-α activities in bovine mammary epithelial cells (BMECs) which exhibits lipopolysaccharide (LPS)-induced inflammation19. It has been found that CLA could protect BMECs against oxidants by enhancing activities of SOD, GPx1, and glutathione S-transferase (GST)20.

Oxidative stress and inflammation are responsible for the pathogenesis of several diseases. The underlying pathogenetic mechanisms are exceedingly complex and play a crucial role in mammalian physiology. Currently, some drugs are used to suppress inflammation in animals; however, these drugs are usually characterized by poor therapeutic efficacy, significant side effects, and adverse compensatory mechanisms21. Therefore, CLA might be used as a novel alternative and complementary intervention to disrupt oxidative stress and inflammatory processes without undesired adverse effects. The present study was aimed at investigating the protective efficacy of c9, t11-CLA supplementation on oxidative stress and inflammation status of BMECs after H2O2 stimulation.

This study’s protocol was reviewed and approved by the Animal Care and Ethics Committee of the Inner Mongolia Minzu University, Tongliao, China (NO. 20221026). All experiments were performed in accordance with relevant named guidelines and regulations and complied with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. BMECs were isolated from the mammary glands of Chinese Holstein dairy cows at a local abattoir. Mammary tissues for cell culture were obtained from the deep layer of the mammary gland and soaked in ice-cold phosphate-buffered saline (PBS) before cell culture. BMECs were cultured according to the methods previously described by Zhang et al.22 Briefly, mammary tissues were thoroughly rinsed with PBS, finely minced with eye scissors, and incubated in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium containing equal volume of type II collagenase for 1 h at 37 ℃ in a 5% CO2 incubator. Then, the culture was filtered and centrifuged to obtain BMECs. The cells were then cultured in basal culture medium, comprising DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 U/mL penicillin, 1 µg/mL hydrocortisone, 4 µg/mL prolactin, 0.5% v/v of insulin-transferrin-selenium-x supplement, and 10 ng/mL epidermal growth factor (EGF). The BMECs were subcultured when the cultures reached 80% confluence. Cells from the third generation were used for subsequent analyses.

First, we intended to establish the damage model by determining the most effective H2O2 concentration and treatment duration. Similarly, we also exposed the cells to varying c9, t11-CLA concentrations to determine the most effective concentration for subsequent experiments. We used the Cell Counting Kit-8 (CCK-8) assays to assess BMEC viability after subjection to varying H2O2 and c9, t11-CLA treatments. Briefly, BMECs were seeded in 96-well microplates at a density of 1 × 104 cells/cm2 for 24 h. When the cultures reached 80% confluence, the cells were subjected to different treatments. BMECs were treated with 100 µL of complete media supplemented with either varying c9, t11-CLA concentrations (0, 50, 100, 200, 400, and 800 µmol/L) for 24 h or varying H2O2 concentrations (0, 200, 400, 600, and 800 µmol/L) for 8 h. In addition, some cells were treated with 600 µmol/L H2O2 for 0, 4, 8, 12, 16, and 24 h. Next, 10 µL of CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 4 h. Then, the absorbance of the culture in each well was measured spectrophotometrically by using a BioTek Synergy H4 Hybrid Reader at 550 nm. The optical density of each well was used to determine the cell viability after each treatment. Based on the cell viability assays, we finally selected four cell groups for subsequent analyses:

Control group: Cells were not treated with either c9, t11-CLA or H2O2.

H2O2 group: Cells were treated with 600 µmol/L H2O2 for 8 h.

c9, t11-CLA (50) + H2O2 group: Cells were pretreated with 50 µmol/L c9, t11-CLA for 24 h, washed twice with PBS, and treated with 600 µmol/L H2O2 for 8 h.

c9, t11-CLA (100) + H2O2 group: Cells were pretreated with 100 µmol/L c9, t11-CLA for 24 h, washed twice with PBS, and treated with 600 µmol/L H2O2 for 8 h.

BMECs were seeded in each well of 6-well microplates at a density of 1 × 104 cells/well. The MDA content and the activities of antioxidant enzymes (T-AOC, SOD, GPx, and CAT) and pro- and anti-inflammatory interleukins (IL-6, IL-8, IL-1β, and TNF-α) secreted by BMECs were detected respectively using bovine enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.) according to the manufacturer’s instructions.

The mRNA abundances of the genes encoding bovine pro- and anti-inflammatory interleukins (IL-1 β, IL-6, and IL-8) and antioxidant enzymes (CAT, SOD, and GPx1) in BMECs were detected using qRT-PCR.

Briefly, the BMECs were seeded in 6-well microplates at the concentration of 1 × 104 cells/well in DMEM/F12 medium for 48 h and subjected to the four treatments stated above. The total RNA of the cells was extracted using the RNAprep Pure Cell/Bacteria kit (DP430, Tiangen Biotech, China) according to the manufacturer’s instructions and stored at − 80 °C. The RNA purity and concentrations were determined by measuring the 260 nm/280 nm ratio using a BioTek Synergy H4 Hybrid Reader. The RNA purity results of 260 nm/280 nm ratio were all between 1.8 and 2.0. The RNA integrity was assessed using 1% agarose gel electrophoresis. Next, 100 ng total RNA was added in the reaction system to reverse transcribed into cDNA by a PrimeScript RT Master Mix Kit (TaKaRa, RR820A, Dalian, China). RT-qPCR was conducted on a Light Cycler 480 (Roche Holding AG, Basel, Switzerland) with a SYBR Premix Ex TaqTM II kit (TaKaRa, RR047A, Dalian, China) following the manufacturer’s recommendations. The PCR conditions were set as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 30 s, respective annealing temperature (Table 1) for 20 s, and 72 °C for 30 s. Relative gene abundances were measured using the 2−ΔΔCT method, with Glyceraldehyde phosphate dehydrogenase (GAPDH), β-actin, ribosomal protein S9 (RPS9) as the internal control of the mRNA expression in our experiments. These were synthesized by Shanghai Sangon Biological Engineering and Technology Service Co. Ltd. The primer sequences used in this study were previously reported by Basiricò et al.20 and Ma et al.23 and the primer sequences were provided in Table 1.

At least three biological replicates from each treatment group were analyzed. All data were collected as mean ± SEM for each variable. The differences between all groups were determined by one-way ANOVA using the SAS software (SAS Version 9.0; SAS Institute Inc., Cary, NC). For all transcript abundance of genes determined, data was obtained as cycle threshold (CT) values. 2−∆∆Ct values were calculated. The differences were considered significant at p < 0.05.

First, we assessed BMEC viability after treatment with varying H2O2 concentrations for 8 h by CCK-8 assays. As shown in Figs. 1 and 2, the viability of BMECs decreased significantly with increasing H2O2 concentrations, When H2O2 concentration reached 200 µmol/L, cell viability significantly decreased (p < 0.05), and when H2O2 concentration reached 800 µmol/L, cell death was excessive (p < 0.01). A cell viability of 67.58% was observed after treatment with 600 µmol/L H2O2. This cell viability could induce oxidative stress and inflammation, while the irreversible cellular damage owing to excessive cell death could be avoided, so 600 µmol/L H2O2 was used in subsequent experiments.

Effects of varying H2O2 concentrations on bovine mammary epithelial cells (BMECs) viability. The cells were treated with varying H2O2 concentrations (0, 200, 400, 600, and 800 µmol/L) for 8 h. Absorbance was measured at 550 nm. The results are expressed as the mean ± SEM. Different superscript letters indicate that the difference between groups is significant (p < 0.05). These data are representative of three independent experiments.

Effects of H2O2 stimulation on bovine mammary epithelial cells (BMECs). Cells before H2O2 stimulation (A), cells after 600 µmol/L H2O2 stimulation (B).

Then, we assessed BMEC viability after treatment with 600 µmol/L H2O2 concentrations for varying durations (0, 4, 8, 12, 16, 24 h). As shown in Fig. 3, the cell viability decreased significantly with increasing treatment duration (p < 0.01), which decreased to 84%, 67%, 56%, 50%, 39%, respectively; and with excessive cell death at 12 h (p < 0.01). Therefore, the damage model of BMECs was established after treatment with 600 µmol/L H2O2 for 8 h.

Effect of varying H2O2 durations on BMECs viability. The cells were treated with 600 µmol/L H2O2 for 0, 4, 8, 12, 16, and 24 h. Absorbance was measured at 550 nm. The results are expressed as the mean ± SEM. Different superscript letters indicate that the difference between groups is significant (p < 0.05). These data are representative of three independent experiments.

Next, we measured BMEC viability after exposure to different doses of c9, t11-CLA for 24 h. As shown in Table 2, exposure of BMECs to less than 200 µmol/L of c9, t11-CLA increased the cell viability (p < 0.05). However, exposure to higher c9, t11-CLA concentrations decreased cell viability, with a significant decrease after exposure to 400 µmol/L c9, t11-CLA (p < 0.01). Therefore, c9, t11-CLA concentrations of 50 µmol/L and 100 µmol/L were selected for subsequent analyses.

As shown in Table 3, the H2O2 group significantly increased MDA content compared with the control group (p < 0.01). But the c9, t11-CLA-pretreated groups exhibited significant redunction in MDA levels than the H2O2 group (p < 0.05). And the Fig. 4 both A and B showed that the H2O2 group exhibited significantly lower CAT, SOD, T-AOC and GPx activities than the control group (p < 0.05). However, pretreatment with 100 µmol/L c9, t11-CLA significantly reversed the effects of H2O2 on CAT, SOD, T-AOC, and GPx activities (p < 0.05). In contrast, pretreatment with 50 µmol/L c9, t11-CLA significantly alleviated the effects of H2O2 on CAT, T-AOC, and GPx activities (p < 0.05).

Effects of c9, t11-CLA pretreatment on the levels of antioxidant enzymes in H2O2-treated BMECs; CAT and SOD (A), GPx and T-AOC (B). The absorbance of antioxidant enzymes CAT and SOD, GPx and T-AOC was measured at 450 nm. The data are expressed as the mean ± SEM. Different superscript letters indicate that the difference between groups is significant (p < 0.05). These data are representative of three independent experiments.

Furthermore, the Fig. 5 showed that the H2O2 group exhibited significantly higher levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6; p < 0.05) and chemokine (IL-8; p < 0.05) than the control group. Pretreatment with 50 µmol/L c9, t11-CLA significantly reversed the effects of H2O2 group on IL-8 and IL-6 levels (p < 0.05). However, the levels of pro-inflammatory cytokines TNF-α and IL-1β were comparable between the H2O2 group and the c9, t11-CLA pretreated groups (p > 0.05).

Effects of c9, t11-CLA pretreatment on the levels of inflammation and cytokine in H2O2-treated BMECs. The absorbance of the cytokines IL-6, IL-8, IL-1β and TNF-α was measured at 450 nm. The data are expressed as the mean ± SEM. Different superscript letters indicate that the difference between groups is significant (p < 0.05). These data are representative of three independent experiments.

As shown in Fig. 6, the H2O2 group exhibited significant downregulation of antioxidant enzymes SOD and GPx1 mRNAs expression compared to the control group (p < 0.05). The mRNA expression of SOD was restored in BMECs pretreated with 50 µmol/L and 100 µmol/L c9, t11-CLA with remarkably higher expressions than the H2O2 group (p < 0.05).

Effects of c9, t11-CLA on the expressions of GPx1, SOD, and CAT mRNAs in BMEC groups. Gene expression related to antioxidant enzymes in BMECs exposed to H2O2 or pretreated with CLA (50 µmol/L or 100 µmol/L). The gene expression results were normalized to the gene expression of GAPDH. All results were expressed as 2−∆∆Ct values. Different superscript letters indicate that the difference between groups is significant (p < 0.05).

Furthermore, as shown in Fig. 7, the H2O2 group exhibited significant upregulation of IL-1β, IL-6, and IL-8 mRNAs expression than the control group (p < 0.05). However, most of the expressions of these genes were restored in BMECs pretreated with 50 µmol/L and 100 µmol/L c9, t11-CLA, with remarkably lower expressions than the H2O2 group (p < 0.05), except for IL-6 expression in 50 µmol/L c9, t11-CLA pretreatment group.

Effects of c9, t11-CLA on the expressions of IL-6, IL-8, and IL-1β mRNAs in BMEC groups. Gene expression related to inflammation and cytokine secretion in BMECs exposed to H2O2 or pretreated with CLA (50 µmol/L or 100 µmol/L). The gene expression results were normalized to the gene expression of GAPDH. All results were expressed as 2−∆∆Ct values. Different superscript letters indicate that the difference between groups is significant (p < 0.05).

Oxidative stress is caused by the imbalance between levels of ROS and antioxidants in cells and tissues24. Among ROS, H2O2 is generated relatively more easily and is relatively stable as a strong oxidizer, forming highly active free radicals25. Therefore, H2O2 was used in this study to induce oxidative stress in BMECs and establish damage model. Generally, a damage model is established using an H2O2 concentration that reduces cell viability to 55–70%26. On account of this concentration range, H2O2 induces significant oxidative stress and inflammation in the cells. Meanwhile, it can be avoided the irreversible cellular damage owing to excessive cell death might lead to impaired protective effect of c9, t11-CLA test results26. The treatment of BMECs with 600 µmol/L H2O2 for 6 h induced marked oxidative stress damage23. In the present study, treatment of BMECs with 600 µmol/L H2O2 for 8 h reduced cell viability to 67.58% which was in the suitable concentration range. Thus, we established the cellular damage model using 600 µmol/L H2O2.

Though c9, t11-CLA reportedly exhibits anticarcinogenic, antioxidant, anti-inflammatory and other biological functions, an optimum beneficial concentration of c9, t11-CLA has not yet been reported. In addition, previous studies have shown that at concentrations of 50 µM or 100 µM, CLA does not affect cell viability, while at 1000 µM, it exhibits marked toxic effects on the cells23,27. The results of the present study were in line with the previous findings. We observed that at 50 or 100 µmol/L, c9, t11-CLA exerted its beneficial effects; however, treatment with over 200 µmol/L c9, t11-CLA significantly reduced cell viability. Therefore, we used concentrations of c9, t11-CLA at 50 and 100 µmol/L as the appropriate concentration for subsequent analyses.

CLA has been shown to significantly increase SOD and GPx activities in the serum and liver, enhancing the clearance of hydroxyl radicals and superoxide anions in laying hens28. And treatment with 100 µmol/L c9, t11-CLA significantly upregulated the mRNA expression of genes encoding SOD and CAT in the liver cells of laying hens29. SOD and GPx are interconnected enzymatic antioxidants that protect periparturient cows against excessive oxidative stress30. Compared with essential fatty acids, the CLA isomer exhibits stronger protective effects against oxidative stress in BMECs20. When BMECs exposed to oxidative stress, CLA has been found to reduce ROS production by enhancing the expressions of SOD1, GPx and GST, and inhibiting the transcription of genes encoding pro-inflammatory cytokines31. CLA treatment has been found to enhance the levels of antioxidant enzymes, such as GPx and glutathione reductase, indicating a protective effect against oxidative stress32. CLA increases the total antioxidant capacity by increasing the intracellular levels of antioxidants, such as glutathione and uric acid, and the expression of intracellular antioxidant enzymes, such as SOD, CAT, and GPx33. In the present study, the H2O2 group exhibited markedly lower SOD, CAT, T-AOC and GPx activities and the mRNA expression of SOD and GPx1 genes, but 50 or 100 µmol/L c9, t11-CLA pretreatment significantly reverted the downregulation of H2O2 group on antioxidant enzymes activities and the mRNA of SOD. In conclusion, CLA improved the antioxidant capacity of BMECs by protecting relevant antioxidant enzymes activities and the mRNA expression to resist H2O2 stimulation.

MDA, a byproduct of lipid peroxidation, was used as a marker of oxidative stress. Increasing proportion of CLA reduces the absolute MDA content produced during lipid peroxidation34. A previous study showed that long-term CLA administration at a dose of 3 g/day in atherosclerosis patients decreased the MDA levels and significantly increased the GPx levels35. In the present study, the H2O2 group exhibited significantly higher MDA levels, and c9, t11-CLA pretreatment significantly reversed the H2O2-induced effects.

Inflammation, which can exacerbate oxidative stress, is a complex physiological response to harmful stimuli or toxins36. A growing number of studies have demonstrated that CLA, especially c9, t11-CLA, exhibits anti-inflammatory potential in several cell and disease models. 50 µM c9, t11-CLA and t10, c12-CLA isomers inhibited the transcription of pro-inflammatory cytokines in BMECs27. In vitro studies showed that c9, t11-CLA exerted an anti-inflammatory effect in cultured peripheral blood mononuclear cells by suppressing the TNF-α, IL-1β, and IL-6 levels37. The protective effects of c9, t11-CLA on BMECs exposed to Escherichia coli -induced inflammation were affected by suppressing the upregulation of IL-1β, IL-6 and IL-8 while enhancing the expression of the anti-inflammatory cytokine23. Cell culture studies have demonstrated that c9, t11-CLA suppresses the synthesis of pro-inflammatory cytokines, including IL-6, TNF-α, and IL-1β, playing an important role in the pathogenesis of many chronic inflammation-mediated diseases38. In the present study, BMECs pretreated with 50 µmol/L c9, t11-CLA exhibited marked reduction of IL-6, IL-8 levels and IL-8, IL-1β than the H2O2 group. Similarly, BMECs pretreated with 100 µmol/L c9, t11-CLA showed significant downregulation in the mRNA expression of IL-6, IL-8 and IL-1β than the H2O2 group. Therefore, c9, t11-CLA protected BMECs by limiting the outbreak of the inflammatory induced by H2O2, which is in accordance with the previous studies.

The present study showed that pretreatment with c9, t11-CLA enhanced the activities of antioxidant enzymes, and decreased the MDA content and pro-inflammatory cytokines IL-1β and IL-6 and chemokine IL-8 levels. These findings corroborate an antioxidant and anti-inflammatory role of c9, t11-CLA in bovine mammary gland cells. The results provide data support for study on the role of CLA in dairy cows.

All data generated or analysed during this study are included in this published article and its supplementary information file.

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This study was funded by the Inner Mongolia Natural Science Foundation (2019LH03013, 2023LHMS03060, 2023LHMS03009), and Doctoral Research Start-up Fund of Inner Mongolia Minzu University (BS674).

College of Animal Science and Technology, Inner Mongolia Minzu University, Tong Liao, People’s Republic of China

Hang Zhang, Yu-qiong Wang & Chang-long Gou

College of Life Sciences and Food Engineering, Inner Mongolia Minzu University, Tong Liao, People’s Republic of China

Ni Dan

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H.Z wrote the original main manuscript text and conducted the investigation and computational experiments. Y.Q.W did the investigation, validation, and visualization; C.L.G. analyzed and interpreted the data; and N.D. acquired funding and drafted and critically reviewed the manuscript. All authors have read and approved the final manuscript.

Correspondence to Ni Dan.

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Zhang, H., Dan, N., Wang, Yq. et al. Protection effect of cis 9, trans 11-conjugated linoleic acid on oxidative stress and inflammatory damage in bovine mammary epithelial cells. Sci Rep 14, 26295 (2024). https://doi.org/10.1038/s41598-024-77711-0

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Received: 25 July 2024

Accepted: 24 October 2024

Published: 01 November 2024

DOI: https://doi.org/10.1038/s41598-024-77711-0

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