Zearalenone (ZEN) is an estrogenic mycotoxin from Fusarium
fungi, and it is a common contaminant of cereal grains such as oat, barley, and sorghum worldwide. Due to the estrogenic activity of ZEN and its metabolites, some countries including the USA, Japan, and France have a guideline to limit the intake of ZEN, varying from 50 to 1000 μg/kg per day depending on the country [1
]. Despite the regulation limit of ZEN for human food intake, humans are at risk of ZEN and its metabolites by intaking animal products. Mauro et al. reported that ZEN and its metabolites are present in the serum samples of nearly all surveyed participants, which were healthy women at ages 25–69 years recruited in Rutgers-New Brunswick, New Jersey, USA [2
]. Their concentrations are positively correlated with meat intake and body mass index, suggesting that humans can easily be exposed to ZEN through the consumption of livestock fed with contaminated corn/grains, which could commonly occur in animal industries in any attempt to reduce production cost.
Many studies have pointed out the danger of ingesting ZEN and its metabolites, causing various toxic effects on reproductive cells/tissues [3
] as well as hepatocytes and kidney cells [7
]. Most toxicity studies of ZEN have been focused on animals, yet some reported toxic effects on human embryonic stem cells and SNO human esophageal carcinoma cells [9
] due to the potential risk of ZEN exposure from intaking animal products [2
]. Once ZEN is ingested by animals or humans, it must initially pass through hepatic and systemic blood vessels before reaching other target sites. Nevertheless, no such study has been published concerning vascular cells/tissues. To understand better the potential risk posed by ZEN exposure, it would be important to assess its potential toxicological effects on blood vessels as well as endothelial cells (ECs).
Endothelial cells (ECs) are an essential component of the cardiovascular system that constructs selective blood–tissue barriers of vascular tissues. ECs regulate vessel integrity by releasing its specific bioactive molecules, particularly nitric oxide (NO). NO is catalyzed by NO synthase (NOS), majorly endothelial NOS (eNOS) in ECs, and thus eNOS regulation is the key to maintain blood vessel homeostasis. Therefore, studying the regulation of eNOS by ECs is an important aspect to understand better about blood vessel function. The eNOS activity is commonly regulated by phosphorylation and dephosphorylation at its specific sites; for example, the phosphorylation of eNOS at serine 1179 (eNOS-Ser1179
; in bovine sequence) increases eNOS activity and NO production, leading to vessel dilation [11
]. On the contrary, the phosphorylation of either eNOS-Thr497
decreases its activity leading to vessel constriction [15
]. Apart from eNOS phosphorylation and dephosphorylation, eNOS activity can be regulated through changes in gene and protein expressions of eNOS itself, where 17β-estradiol increases eNOS protein and activity in ovine fetal pulmonary artery ECs [18
Epidemiological studies showed that the prevalence of cardiovascular diseases dramatically increases in women after menopause, indicating a potential association of estrogen on vascular function. Considering that ZEN has estrogenic effects and ECs are likely to be exposed to ZEN once it is ingested by humans and animals before reaching other target tissues of ZEN, it is important to find potential toxic effects and mechanisms of ZEN on EC function. In this study, we looked at the effects of ZEN on eNOS activity and NO production using BAECs. We also characterized the mechanism underlying ZEN-induced alteration in eNOS activity and expression and then observed changes of ZEN-stimulated vessel relaxation using an ex vivo aortic mouse model.
eNOS is a key regulator of NO production, which is involved in maintenance of blood vessel homeostasis [30
]. Many studies have demonstrated that phosphorylation/dephosphorylation of eNOS at specific sites is a major regulatory mechanism to alter eNOS activity. Some studies have also reported that eNOS activity can be affected by changes in protein and mRNA expression of eNOS upon exposure to external stimuli. For example, H2
increases the stability of eNOS mRNA, thereby increasing eNOS mRNA transcription and activity [32
]. On the contrary, tumor necrosis factor-α promotes binding of eukaryotic elongation factor 1-α 1 (eEF1A1) [33
] or microRNA-155 [34
] to the 3′-UTR of eNOS mRNA, thereby reducing mRNA stability and decreasing eNOS expression. Our current findings indicate that ZEN can decrease eNOS activity by different mechanisms from those reported previously. In particular, our results demonstrate that ZEN promotes binding of PXR to Sp1, which then increases the binding affinity of Sp1 to specific G/C-rich regions of the eNOS promoter (−113 to −12), consequently decreasing eNOS expression in BAECs.
The human eNOS promoter has positive regulatory domains I (PRD I; −104 to −95) and II (−144 to −115) where transcription factors such as Ets-1, YY1, Elf-1, MAZ, and Sp1 bind to regulate eNOS mRNA transcription [32
]. The results from our study demonstrate that Sp1 could function as a repressor of eNOS mRNA transcription to decrease eNOS expression. In this regard, oxidative stress has also been reported to decrease eNOS expression by increasing Sp1 binding to specific human eNOS promoter regions (−1386, −632, −104) [35
]. Our results were largely consistent with these previous study results with a few exceptions; oxidative stress has been reported to increase the level of Sp1 protein and its phosphorylation, yet we failed to find any alteration in Sp1 protein expression in BAECs treated with ZEN. These differences are probably because of the use of different cell types and external stressors. The authors in the previous study used human hepatocarcinoma cells transfected with the human eNOS promoter gene, while we used BAECs naturally expressing eNOS. Furthermore, it is difficult to compare directly the results of the two studies since different stressors were used. It would be interesting to investigate the reason for these differences further, but this is beyond the scope of the current study.
Understanding the detailed molecular mechanism by which Sp1 inhibits eNOS promoter activity warrants further investigation. Various cofactors expressed in response to external stimuli interact with Sp1 to regulate eNOS promoter activity; for example, HDACs, NCoR1, and/or SMRT regulate gene activity by binding to Sp1. Binding of HDAC1 to Sp1 decreases murine thymidine kinase (TK) promoter activity in Swiss 3T3 fibroblasts [25
], while HDAC1 has been shown to compete with E2F1 for binding to the carboxyl-terminal of Sp1 in the regulation of murine TK promoter activity [24
]. Other studies showed that NCoR1 and SMRT bind to Sp1 to regulate the promoter activities of the vascular endothelial growth factor receptor 2 (VEGFR2) gene in MCF-7 cells [27
] and the UAS gene in African green monkey kidney cells (CV-1) [26
], respectively. In contrast to these previous studies, none of these transcription factors including HDAC1, NCoR1, and SMRT affected the ZEN-mediated decrease in eNOS protein and mRNA in BAECs.
Instead, we found that ZEN induced an interaction between PXR, a ligand-activated nuclear receptor, and Sp1, thereby altering eNOS expression. PXR is primarily known as a sensor of exogenous chemicals that can regulate gene expression, enzyme biotransformation, and drug transportation [36
]. Similar to these studies, ZEN mediates the expression of xenobiotic-inducible genes such as CYP3A through increasing PXR activation in human hephatocellular carcinoma HepG2 cells [28
]. At the same time, our study clearly showed that PXR could function as a corepressor of eNOS transcription. In support of this, PXR was shown to suppress the transcription of the rifampicin-mediated G6Pase gene by directly interfering with the binding of cAMP response element-binding protein (CREB) to the CRE response element of this gene in Huh7 cells [39
]. Another study also reported that PXR had a negative effect on eNOS expression; indole 3-propionic acid, a ligand of PXR, decreased eNOS expression in mice with an intact PXR but not mice with PXR knocked out, indicating the importance of PXR in regulating eNOS expression. Consistent with this recent study, we found firstly that PXR indirectly inhibits eNOS expression in ECs through interaction with Sp1.
So far, we have discussed how both Sp1 and PXR can decrease eNOS expression. The next question was whether Sp1 and PXR interact physically. When ECs were exposed to ZEN, we found that PXR interacted with Sp1 as a cofactor to decrease eNOS expression, indicating that it affects the gene transcription of eNOS indirectly by altering the binding affinity of Sp1 to the promoter of eNOS. Several studies have reported that cofactors of Sp1 bind to the Sp1 inhibitory domain (ID) or C2H2-type zinc finger DNA-binding domain (ZFDBD) to regulate Sp1 binding affinity, thereby changing target gene expression [26
]. Additionally, some ligand-activated receptors such as the thyroid hormone receptor, retinoic acid receptor, and vitamin D3
receptor (VDR) retain a DNA binding domain (DBD) and ligand-binding domain (LBD). These domains are important for interaction with Sp1 to allow a complex of nuclear receptors to form; this complex subsequently binds to G/C-rich regions in the promoter of IL-1β, altering gene transcriptions [40
]. In addition, VDR was shown to bind to C-terminal sites of Sp1 (622 to 788). Similar to these receptors, PXR also retains a DBD and LBD [41
], suggesting that PXR is likely to interact with Sp1 to increase the affinity of Sp1 for G/C-rich regions of the eNOS promoter. Therefore, we speculate that ZEN promotes binding of PXR to Sp1, which increases the binding affinity of Sp1 for the bovine eNOS promoter region (−113 to −12), resulting in decreased transcription of eNOS in BAECs. The ZEN-medicated decrease in expression of eNOS protein and NO production would eventually decrease vessel relaxation, as we observed in our ex vivo mouse aortic model. Nonetheless, the concentration of ZEN used in this ex vivo study was higher compared to those circulating in animals exposed to ZEN-contaminated food [42
]. Different cells and animals respond differently to a wide range of concentrations of ZEN, causing diverse effects. Like concentration ranges of ZEN between 10 pM and 300 μM used in many in vitro studies [43
], the adequate concentrations responsible for in vivo toxicological effects are difficult to define due to a variety of times, doses, and routes of injections. A recent in vivo study showed that intratesticular injection of 300 ng ZEN/day (estimated to be 0.5 μM/day) for 21 days decreased regeneration of Leydig cells [44
]. Considering that continuous exposure of ZEN could lead to accumulation in host tissues [45
], there is a potential chance that chronic exposure of ZEN for a long time may lead to a similar effect as what we observed in our study, disrupting vascular function.
It is also interesting to note that the observed effects of ZEN on eNOS expression were independent of ERs in our study. ZEN has been reported to bind competitively to genomic or nongenomic ERs and then activate transcription of several estrogen-responsive genes [46
]. However, we found that the effect of ZEN on eNOS expression was mediated by neither genomic nor nongenomic ERs in BAECs. Another study reported that ZEN induces neutrophil extracellular trap production via NADPH oxidase, extracellular signal-regulated kinase, and p38-dependent signaling pathways, but not through genomic ER signaling pathway in primary bovine neutrophils [49
]. Although the previous study did not examine the involvement of nongenomic ER, our data suggest that some toxicological effects of ZEN, including the reduction in eNOS expression, are likely to be mediated via other receptors or molecules that are independent of ER-mediated signaling pathways. We did not identify specific upstream target(s) of binding of PXR to Sp1 in ZEN-inhibited eNOS expression in the current study; these targets should be identified in future studies.
5. Materials and Methods
Zearalenone (ZEN), trichostatin A (TSA, HDAC inhibitor), and mithramycin A (Sp1 inhibitor) were purchased from Merck Millipore (Darmstadt, Germany). ICI 182,780 (genomic estrogen receptor (ER) antagonist) and G-15 (nongenomic ER antagonist) were acquired from Tocris bioscience (Bristol, UK). G-36 (nongenomic ER antagonist) was acquired from Cayman Chemical (Ann Arbor, MI, USA). eNOS-specific antibody was acquired from BD Transduction Laboratories (Lexington, KY, USA). α-tubulin antibody was acquired from AbFrontier (Seoul, Korea). Antibodies for Sp1 and pregnane X receptor (PXR) as well as all HRP-conjugated secondary antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA). Minimal essential medium (MEM), newborn calf serum (NCS), Dulbecco’s phosphate-buffered saline (DPBS), L-glutamine, penicillin–streptomycin antibiotics, and trypsin-EDTA were acquired from Thermo Fisher Scientific (Waltham, MA, USA).
5.2. Full-Length and 5′-Deleted Human eNOS Promoter
Full-length human eNOS promoter (−1600 to +22; eNOS
(−1600)) and serially 5′-deleted promoters of eNOS
(−873), and eNOS
(−428) were prepared as described previously [50
(−135) was amplified by polymerase chain reaction (PCR) with the following forward (F) and reverse (R) primers: eNOS
(−135)-F, 5′-GGC TTG TTC CTG TCC CAT TG-3′ and eNOS
(−135)-R, 5′-TGC TGC CTG CTC GAG CAG AGC -3′. Synthesized PCR products containing each progressive 5′-deleted human eNOS promoter sequence were inserted into a luciferase reporter gene plasmid (pGL2). Corresponding eNOS
–luciferase promoter constructs were designated pGL2-eNOS
(−428), and pGL2-eNOS
5.3. Site-Directed Mutagenesis
Site-directed mutagenesis of pGL2-eNOS(−135) plasmid was prepared using a QuikChange II site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. Sequences of mutagenic primer pairs were as follows (mutagenized bases are identified by lowercase letters): Sp1(mut1)-F, 5′-GTG TAT GGG ATA GGt GCG atG CGA GGG CCA GCA C-3′; Sp1(mut1)-R, 5′-GTG CTG GCC CTC GCa TCG caC CTA TCC CAT ACA C-3′; Sp1(mut2)-F, 5′-CGG GGC GAG GGC CAG CAC Taa gaA GCC CCC Ttt tAC TGC CCC CTC- 3′; Sp1(mut2)-R, 5′-ACC GAG AGG AGG Gct CAG TGt cAG GGG cCT CTC CAG TGC TG-3′.
5.4. Cell Culture and Drug Treatments
BAECs were obtained as previously described [51
] and cultured in MEM supplemented with 5% NCS in a humidified atmosphere chamber containing 5% CO2
at 37 °C. BAECs established a typical cobblestone structure of ECs and EC-specific marker von Willebrand factor VIII. BAECs between passages 5 and 9 were used in all experimental trials. When the cells were at ~80% confluence, 1% NCS MEM containing the indicated concentrations of ZEN was added. In some experimental trials, the cells were pretreated various factors for 1 h prior to ZEN exposure.
BAECs were transfected with 3 μg of pGL2-eNOS(−1600), pGL2-eNOS(−1400), pGL2-eNOS(−962), pGL2-eNOS(−873), pGL2-eNOS(−428), or pGL2-eNOS(−135) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. For all pGL2 transfection experiments, BAECs were cotransfected with 70 ng of Renilla luciferase reporter vector for normalizing transfection efficiency. For small interfering RNA (siRNA) transfection, two or three potential siRNA oligonucleotides for nuclear receptor corepressor 1 (NCoR1), silencing mediator of retinoid and thyroid hormone receptor (SMRT; also referred to as NCoR2), and pregnane X receptor (PXR) were purchased from GenePharma (Shanghai, China) and mixed for better efficiency. siRNA oligonucleotide sequences were as follows: NCoR1, 5′-CAU UUG GAG UCA AAC AUG AAG-3′, 5′-GAA GAA AAA GUA GAA GAA AAG-3′, 5′-GUC AAU CUG CCA UCA AAC ACA-3′; SMRT, 5′-CCA UGA AGG UGU ACA AAG ACC-3′, 5′-GGG AAA AGA CUC AAA GUA AAC-3′; PXR, 5′-CUG GUU AUC ACU UCA AUG UCA UG-3′, 5′-GGG AAG AUC UGU GUU CAA UGA AG-3′, 5′-AUC AUU ACA CAC UGA CAA UAA GC-3′. Negative control siRNA oligonucleotide with the sequences 5′-UUC UCC GAA CGU GUC ACG UTT-3′ was also obtained from GenePharma. Commercially available siRNAs targeting Sp1, PXR, and negative control siRNA were obtained from Santa Cruz. siRNA (100 nM) was transfected to BAECs using DharmaFECT 4 transfection reagent (Horizon Discovery Ltd. Cambridge, UK) according to the manufacturer’s protocols.
5.6. Luciferase Assay
After the transfection of pGL2-eNOS plasmids, the transfected cells were maintained for 24 h, washed twice with DPBS, and lysed by adding 200 μL of 1 × passive lysis buffer (Promega, Madison, WI, USA). Lysates were collected in 1.5 mL tubes and centrifuged at 16,000× g for 20 min. Protein (20 μg) in a total of 20 μL supernatant was used for the luciferase assay. The Dual-Luciferase Reporter Assay System (Promega) was used to assess the luciferase activity of the transfectants. All data from firefly luciferase were normalized with Renilla luciferase.
5.7. Reverse Transcription (RT)-PCR
BAECs were homogenized with 1 mL of TRIzol reagent (Thermo Fisher Scientific) to extract total RNA from BAECs as previously described [12
]. The total RNA was then converted to cDNA using SuperScriptTM
III reverse transcriptase (Thermo Fisher Scientific). PCR for each target gene was carried out using the following primers: eNOS
-F, 5′-GCC TCC TGT GAG ACC TTC TG- 3′; eNOS
-R, 5′- TCT CTG GGA AGT CAC CTT GG- 3′; NCoR1
-F, 5′- GAA ACA CCT GGC GAT GCT AT- 3′; NCoR1
-R, 5′- GCT TCT CAT GCA CAA CAG GA- 3′; SMRT
-F, 5′- TCT GGG GAA GAC AAT GAT GA-3′; SMRT
-R, 5′- GTG TCG GAG CTG TTG TTG AC-3′; PXR
-F, 5′- TAG GTC TGT GGA GAG CCA AG-3′; PXR
-R, 5′- CCA GGA TCC ATG TCT GAG TC-3′; GAPDH
-F, 5′- TCA CCA GGG CTG CTT TTA AT- 3′; GAPDH
-R, 5′- GGT CAT AAG TCC CTC CAC GA- 3′. The amplified gene products were separated using a 1.0% agarose gel in TAE buffer (40 mM Tris-acetate, pH 8.0, 1 mM EDTA), tagged with RedSafeTM
Nucleic acid Staining Solution (iNtRON Biotechnology, Gyeonggi-do, Korea), and visually observed under UV.
5.8. Western Blot Analysis
BAECs exposed to ZEN with or without various pretreatments were washed with ice-cold DPBS. The cells were then lysed with lysis buffer (20 mM Tris-HCl at pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA) supplemented with Protease Inhibitor CocktailTM (Merck Millipore), 1 mM NaF and 1 mM Na3VO4, 1 mM β-glycerophosphate, and 1 mM phenylmethanesulfonyl fluoride. In a separate experiment, mouse aortic tissues were isolated and used as an ex vivo model to examine the effect of ZEN on vessel relaxation. Dissected aortas were exposed to either 20 μM of ZEN or vehicle and incubated at 37 °C under 5% CO2 for 16 h. The aortas were then chopped in pieces with iris scissors and incubated in lysis buffer to extract aortic protein. The concentrations of aortic protein were assessed using a BCA assay. Equal amounts of isolated protein (20 µg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. Blots were tagged with the appropriate primary antibodies and the corresponding secondary antibodies. The blots were developed using enhanced chemiluminescence reagent (ECL, GE Healthcare, Pittsburgh, PA, USA).
5.9. Assessment of Nitric Oxide (NO) Production
NO production by BAECs was assessed by measuring the concentration of nitrite, a stable metabolite of NO, in cell culture supernatant as previously described [12
]. The culture medium was removed, and the cells were incubated in Kreb’s buffer (pH 7.4; 118 mM NaCl, 4.6 mM KCl, 27.2 mM NaHCO3
, 1.2 mM KH2
, 2.5 mM CaCl2
, 1.2 mM MgSO4
, 11.1 mM glucose) at 37 °C for 1 h. Each 200 µL of supernatant was transferred into a 96-well plate, and additional 100 µL of Griess reagent (50 µL of 0.1% N
-(1-naphthyl) ethylenediamine and 50 µl of 1% sulfanilamide containing 5% phosphoric acid) was added into each well. The plate was incubated at room temperature for 15 min, and optical density was measured using a microplate reader at a wavelength of 530 nm.
5.11. Chromatin Immunoprecipitation (ChIP) Assay
A ChIP assay was done using a ChIP assay kit (Merck Millipore), according to the manufacturer’s protocols as previously described [52
]. Formaldehyde (1%) was used to cross-link the cells, and the reaction was quenched by adding 0.1 M glycine. The cells were then suspended in SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCl at pH 8.1). The samples in SDS-lysis buffer were sonicated and precleared by protein A-agarose/Salmon Sperm DNA beads and then immunoprecipitated with 400–500 μg of sample and 2 μg of Sp1 or non-immune mouse IgG antibody as a negative control (Santa Cruz). DNA was isolated from total chromatin extract (Input) and ChIP samples and then used for PCR analysis. PCRs were performed using bovine eNOS promoter-specific primers: eNOS
-F, 5′- CTC AGA GCG GAA CCC AGG -3′ and eNOS
-R, 5′- AGC AGA GCT TCG GGC CTT -3′.
5.12. Preparation of Nuclear and Non-Nuclear Fractions
Nuclear and non-nuclear fractionations were carried out as previously described [53
]. Cells were lysed in nuclear extract buffer I (NEBI) (10 mM HEPES at pH 7.9, 10 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA) containing protease inhibitor. The suspension was incubated on ice for 15 min and lysed with additional 0.5% NP-40 followed by vortexing for 10 s. The cell lysate was subsequently centrifuged at 16,000× g
for 30 s, and the supernatant was collected and designated as the “non-nuclear fraction,” which was kept at 4 °C until use. The remaining pellet was re-suspended in NEBII (20 mM HEPES at pH 7.9, 400 mM NaCl, 1 mM DTT, 1 mM EDTA, 1 mM EGTA) containing protease inhibitor. The re-suspected pellets were incubated on ice for 30 min and centrifuged at 16,000× g
at 4 °C for 20 min. The supernatant was collected and designated as “nuclear fraction”.
5.13. Co-Immunoprecipitation (co-IP)
The nuclear fraction (200 μg) of cells treated with ZEN or vehicle was immunoprecipitated overnight using 1 μg of specific antibodies against Sp1, PXR, or non-immune mouse IgG as a negative control (Santa Cruz) at 4 °C. Protein-G sepharose beads were incorporated into the samples and incubated for 2 h at 4 °C. After the incubation, the beads were washed three times with NEBII. Lastly, these samples were mixed with SDS loading buffer, boiled for 5 min, and prepared for Western blot analyses.
All animal experiments were carried out under the approval of institutional guidelines for animal care and use in Yeungnam University (YUMC-AEC2019-003). Male C57BL/6 mice (eight weeks of age) were housed in a temperature- and humidity-controlled cage (22 ± 1 °C and 50 ±10%, respectively) under 12 h alternating light/dark cycle for 1 week at the beginning of the experiment. All mice were fed standard chow (Purina Mills, St. Louis, MO, USA) ad libitum and water during the experimental trials.
5.15. Measurement of Endothelium-Dependent Vessel Relaxation
Endothelium-dependent vascular relaxation was determined in thoracic aortic rings as previously described [15
], with slight modifications. Male C57BL/6 mice were sacrificed, and adventitial connective and fat tissues were carefully removed. The thoracic aorta was cut into 2 mm rings under a dissecting microscope and treated with either 20 μM of ZEN or DMSO as a control for 16 h. After the treatment, these rings were suspended by a small vessel wire myograph containing 37 °C Krebs-bicarbonate buffer (118 mM NaCl, 4.6 mM KCl, 27.2 mM NaHCO3
, 1.2 mM MgSO4
, 1.2 mM KH2
, 2.5 mM CaCl2
, 11.1 mM glucose) in a chamber maintaining 5% CO2
at 37 °C. Isometric tension was measured by using a force transducer. Aortic rings were equilibrated in Krebs-bicarbonate buffer for 30 min under resting tension of 0.5 g and then constricted by adding 0.1 μM phenylephrine (PE) until a steady-state was reached. Endothelium-dependent relaxations were determined by measuring the dilatory response of arteries to acetylcholine (ACh) (from 0.001 to 1 μM). The endothelium of the aortic ring was considered to be intact when complete relaxation (100%) was observed with 1 μM ACh treatment.
5.16. Statistical Analysis
All of the results, except the ex vivo aorta relaxation assay, were expressed as mean ± standard deviation (S.D.) with n indicating the number of repeated trials. Results from the ex vivo aorta relaxation trial were expressed as the mean ± standard error (S.E.), with n indicating the number of trials. Statistical significance was determined using Student’s t-test for paired data. A value of p < 0.05 was considered significant.
5.17. Ethical Approval
In brief, the animal study we applied in the manuscript was approved by the Institutional Animal Care and Use Committee (IACUC) as follows. Approval number: YUMC-AEC2019-003. Title of Project: Effect of zearalenone and telmisartan on eNOS and muscle tension of arteries in vascular endothelial cells and smooth muscle. Periods of Projects: 2019. 4.15–2019.10.31 Date of Approval: 15 April 2019.