The present study was approved by the Ethics Committee of Lianyungang Oriental Hospital Affiliated to Xuzhou Medical University (approval no. KY-2020-003-01). All procedures were performed in accordance with the 1964 Declaration of Helsinki and its later amendments. The patients or their guardians (for patients with reduced brain capacity) were informed of the project and provided written informed consent. Blood samples were collected from 15 patients with PD (nine male patients and six female; age, 63.4±7.76 years) and 15 healthy patients (controls; 11 male patients and four female patients; age, 63.07±9.56 years) who were admitted to the Lianyungang Oriental Hospital Affiliated to Xuzhou Medical University (Lianyungang, China) between April 2020 and August 2020. Inclusion criteria for patients with PD were as follows: i) Clinical features of the patient were in line with the clinical diagnostic criteria of the Parkinson's Society of England Brain Bank; and ii) PD was clearly diagnosed by two physicians at or above the deputy director level. The exclusion criteria for patients with PD were as follows: i) Cerebrovascular disease; ii) Parkinson's syndrome; iii) family-related PD; iv) Parkinson's superimposed syndrome; and v) hepatolenticular degeneration caused by external factors, such as drugs, encephalitis, and brain injury. A total of 5 ml fasting venous blood was drawn from the study subjects and placed into tubes containing anticoagulant. Subsequently, total RNA was extracted from these samples, as previously described (16).

MES23.5 cells were purchased from American Type Culture Collection and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% Penicillin-Streptomycin. Cells were grown at 37˚C with a 5% CO₂/95% air atmosphere. 6-OHDA (purity ≥97%; Merck & Co., Inc.) was dissolved in sterile deionized water to prepare a 60 mmol/l stock solution, which was stored at -20˚C in the dark. MES23.5 cells pretreated with 6-OHDA for 24 h served as the 6-OHDA group, whereas untreated cells served as the control group.

MES23.5 cells were plated into 10-cm dishes (2x10⁶ cells/dish) and then transfected with 25 nM pcDNA3.1-FOXA1 vector plasmid [overexpression (Ov)-FOXA1; Hunan Fenghui Biotechnology Co., Ltd.], empty vector plasmid (Ov-NC; Hunan Fenghui Biotechnology Co., Ltd.), 100 nM small interfering RNA (siRNA) targeted against trefoil factor 1 (TFF1; siRNA-TFF1-1, 5'-GGCCCAGGAAGAAACATGTAT-3'; siRNA-TFF1-2, 5'-GGCCATCGAGAACACTCAAGA-3'; Genepharm Biotech Corp.) or scrambled siRNA (siRNA-NC, 5'-GAAGACATCCTGCGGAAGTAA-3'; Genepharm Biotech Corp.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C according to the manufacturer's instructions. Following 48 h of transfection, the expression levels of FOXA1 and TFF1 were determined.

MES23.5 cells (2x10⁶) were suspended and homogenized in 0.75 ml TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min on ice. The quantity of total RNA was measured using a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a TaqMan^™ RT kit (cat. no. N8080234; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was performed using SYBR Green PCR Master Mix (Beijing Solarbio Science & Technology Co., Ltd.) on an ABI PRISM 7300 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95˚C for 5 min; followed by 38 cycles of 95˚C for 15 sec, 55˚C for 30 sec and 72˚C for 90 sec. The relative mRNA expression levels of each specific gene were quantified using the 2^-ΔΔCq method (17) and normalized to the internal reference gene GAPDH. The sequences of the primers used for RT-qPCR are listed in Table I.

MES23.5 cells (2x10⁶) were lysed using RIPA buffer (cat. no. P0013C; Beyotime Institute of Biotechnology) for 10 min on ice, and total protein was quantified using a BCA protein assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Protein samples (25 µg/lane) were subjected to 10% SDS-PAGE and then transferred onto PVDF membranes. After blocking in 5% fat-free milk for 2 h at room temperature, the membranes were incubated at 4˚C overnight with primary antibodies (all purchased from Abcam) targeted against FOXA1 (cat. no. ab170933; 1:1,000), TFF1 (cat. no. ab92377; 1:1,000), TH (cat. no. ab137869; 1:5,000), Bcl-2 (cat. no. ab32124; 1:1,000), Bax (cat. no. ab32503; 1:1,000), cleaved caspase 9 (cat. no. ab2324; 1:200), caspase 9 (cat. no. ab202068; 1:2,000) and GAPDH (cat. no. ab9485; 1:2,500). Subsequently, the membranes were incubated with an HRP-labelled goat anti-rabbit secondary antibody (cat. no. ab6721; 1:5,000; Abcam) for 2 h at room temperature. Protein bands were visualized using an ECL kit (cat. no. P0018S; Beyotime Institute of Biotechnology). Protein expression was semi-quantified using ImageJ software (version 1.8; National Institutes of Health) with GAPDH as the loading control.

MES23.5 cells (5x10³ cells/well) were seeded into a 96-well plate. Before the addition of 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) to each well, the cells were cultured for 24 h at 37˚C. Following a further incubation for 2 h at 37˚C after the addition of CCK-8, the optical density was measured at a wavelength of 450 nm using a microplate reader (Molecular Devices, LLC).

MES23.5 cells (2x10⁴ cells/well) were plated in 24-well plates, fixed with 4% paraformaldehyde (Macklin, Inc.) for 1 h at room temperature, and blocked in 1% BSA (Beijing Solarbio Science & Technology Co., Ltd.) for 0.5 h at room temperature. Next, the cells were incubated in the presence of a primary antibody against TH (cat. no. ab137869; 1:100; Abcam) at 4˚C overnight, followed by incubation with the Alexa Fluor^® 488-labelled goat anti-rabbit secondary antibody (cat. no. ab150077; 1:200; Abcam) for 1 h at room temperature. The nuclei were counterstained with DAPI for 5 min at room temperature. The results were observed under a fluorescence microscope (magnification, x200; Olympus Corporation).

The level of reactive oxygen species (ROS; cat. no. S0033S) and malondialdehyde (MDA; cat. no. S0131S), and the activity of superoxidase dismutase (SOD; cat. no. S0101S) in MES23.5 cells (2x10⁶) were quantified using commercial ELISA kits (all purchased from Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The optical density was measured at a wavelength of 525 nm for ROS, 550 nm for SOD and 532 nm for MDA using a microplate reader (Molecular Devices, LLC).

MES23.5 cells (2x10⁴ cells/well) were seeded into a 24-well plate. Subsequently, the TUNEL assay was performed using a TUNEL assay kit (cat. no. C1086; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The results were observed under a fluorescence microscope (magnification, x200; Olympus Corporation) at five fields of view.

ChIP assay was performed with a ChIP assay kit (cat. no. P2078; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Briefly, MES23.5 cells (1x10⁶) were cross-linked with 1% formaldehyde followed by centrifugation at 1,000 x g for 1 min at 4˚C. The precipitated cells were lysed with SDS lysis buffer containing 1 mM PMSF for 10 min, and sonicated in an ice bath. Cell lysates (2 ml) were incubated with 70 µl Protein A + G agarose for 30 min at 4˚C before incubation with 60 µl Protein A + G beads coated with 1 µg anti-FOXA1 antibody (cat. no. ab170933; Abcam) for 1 h at 4˚C. Anti-rabbit IgG (cat. no. 172730; Abcam) served as a NC. After the incubation, the sample was centrifuged at 1,000 x g for 1 min at 4˚C and the precipitate was collected. Cross-linked DNA released from the precipitate was purified using a DNA purification kit (cat. no. D0033; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The eluted DNA was subjected to RT-qPCR according to the aforementioned protocol.

The TFF1 promoter region containing a FOXA1 putative binding site [wild-type (WT), 5'-AGGTCACGGTGGCCAC-3') or mutant (MUT) site (5'-TCCAGTGCCACCGGTG-3')] was cloned into the pGL3-based vector (BioVector NTCC, Inc.). MES23.5 cells were seeded (1x10⁵ cells/well) into 24-well plates and co-transfected with 0.5 µg aforementioned reporter vector and 0.5 µg Ov-FOXA1 or Ov-NC vectors using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) at room temperature. Following 48 h of transfection, the relative luciferase activity was normalized to Renilla luciferase and measured using a dual-luciferase reporter assay system (Promega Corporation) according to the manufacturer's instructions.

Data are presented as the mean ± SD from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). Statistical differences between two groups were analyzed using an unpaired Student's t-test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of FOXA1 in the samples from patients with PD and healthy subjects were determined using RT-qPCR. The results revealed that the expression levels of FOXA1 in patients with PD were significantly lower compared with those in the controls (Fig. 1A).

Subsequently, the expression levels of FOXA1 in MES23.5 cells and pretreated cells (6-OHDA group) were determined using RT-qPCR and western blotting. FOXA1 expression was significantly downregulated in the 6-OHDA group compared with that in the control group (Fig. 1B and C).

A FOXA1 overexpression plasmid was constructed to explore the role of FOXA1 in MES23.5 cells, and the transfection efficiency was verified by RT-qPCR and western blotting (Fig. 1D and E). The expression level of FOXA1 in the Ov-FOXA1 group was significantly upregulated compared with that in the Ov-NC group. Subsequently, MES23.5 cells were divided into four groups: i) Control; ii) 6-OHDA; iii) 6-OHDA + Ov-NC; and iv) 6-OHDA + Ov-FOXA1. Cell viability was assessed using the CCK-8 assay. The results revealed that cell viability in the 6-OHDA group was significantly downregulated compared with that in the control group, whereas FOXA1 overexpression significantly attenuated this decrease (Fig. 1F).

As aforementioned, the absence of TH can affect the formation of DA (10); therefore, the expression of TH in these groups was evaluated using immunofluorescence, RT-qPCR and western blotting. The staining results revealed that the fluorescence intensity of the 6-OHDA group was markedly lower compared with that in the control group (Fig. 1G). FOXA1 overexpression notably increased the fluorescence intensity compared with that in the 6-OHDA + Ov-NC group, suggesting that FOXA1 overexpression could alleviate the downregulation of TH expression induced by 6-OHDA. In addition, the RT-qPCR and western blotting results also indicated that FOXA1 overexpression could significantly inhibit 6-OHDA-induced downregulation of TH expression (Fig. 1H).

Cells were grouped as previously described, and the effect of FOXA1 overexpression on cell inflammation and oxidative stress was subsequently determined. The mRNA expression levels of inflammatory factors, including TNF-α, IL-1β and IL-6, were determined using RT-qPCR (Fig. 2A). The expression of these molecules was significantly increased in the 6-OHDA group compared with that in the control group, whereas FOXA1 overexpression significantly reversed these effects. Furthermore, the levels of SOD, ROS and MDA were determined using ELISA kits. The results demonstrated that the level of SOD was significantly decreased in the 6-OHDA group compared with that in the control group, but significantly increased after FOXA1 overexpression (Fig. 2B). The levels of ROS and MDA were significantly increased in the 6-OHDA group compared with those in the control group, whereas FOXA1 overexpression significantly decreased these levels. Furthermore, cell apoptosis was determined using a TUNEL assay and western blotting. The fluorescence of apoptotic cells was more intense in the 6-OHDA group compared with that in the control group, and FOXA1 overexpression significantly downregulated the fluorescence intensity (Fig. 2C). The western blotting results showed that the protein expression levels of Bcl-2 were significantly decreased in the 6-OHDA group compared with those in the control group, which was accompanied by significantly increased expression levels of Bax and cleaved caspase 9 (Fig. 2D). FOXA1 overexpression significantly alleviated the effects of 6-OHDA on Bcl-2, Bax and cleaved caspase 9 expression.

It has been reported that TFF1 co-localizes with TH in midbrain neurons in rats, and TFF1-positive neurons in a 6-OHDA-induced model were dead (18). Therefore, in the present study, the expression levels of TFF1 in MES23.5 cells were determined using RT-qPCR and western blotting. The results revealed that TFF1 expression was significantly decreased in the 6-OHDA group compared with that in the control group (Fig. 3A and B). FOXA1 overexpression significantly increased the expression levels of TFF1 in 6-OHDA-induced MES23.5 cells (Fig. 3C). In addition, FOXA1 was considered to be able to bind to the site 405-390 bp of the TFF1 promoter (Fig. 3D). Therefore, a ChIP assay was performed to verify the interaction of FOXA1 with TFF1 promoter, and the results indicated that FOXA1 could bind to the promoter of TFF1 (Fig. 3E). Next, the binding site was mutated, and luciferase activities were measured using a luciferase reporter assay. The activity of the WT group was significantly higher compared with that of the MUT group, indicating that FOXA1 did bind to the aforementioned site (Fig. 3F).

To study the role of TFF1 in MES23.5 cells, TFF1 was silenced via siRNA, and its expression was determined using RT-qPCR and western blotting. The results revealed that the expression levels of the siRNA-TFF1-1 group were lower compared with those of the siRNA-TFF1-2 group; thus, the siRNA-TFF1-1 group was selected for subsequent experiments (Fig. 4A and B). MES23.5 cells were divided into the following five groups: i) Control; ii) 6-OHDA; iii) 6-OHDA + Ov-FOXA1; iv) 6-OHDA + Ov-FOXA1 + siRNA-NC; and v) 6-OHDA + Ov-FOXA1 + siRNA-TFF1. Cell viability of these groups was assessed using a CCK-8 assay, and the results revealed that the cell viability of the siRNA-TFF1 group was significantly reduced compared with that of the siRNA-NC group, indicating that TFF1 silencing reversed the increase in cell viability induced by FOXA1 overexpression (Fig. 4C). Next, the expression of TH in these groups was evaluated using immunofluorescence, RT-qPCR and western blotting. The expression of TH was significantly decreased after TFF1 silencing, which indicated that TFF1 silencing could reverse the effect of FOXA1 overexpression on TH expression (Fig. 4D-F). Furthermore, the effect of TFF1 silencing on cell inflammation was assessed by measuring the levels of TNF-α, IL-1β and IL-6. The RT-qPCR results suggested that TFF1 silencing could significantly elevate the levels of inflammatory cytokines in 6-OHDA-induced cells overexpressing FOXA1, which is equivalent to blocking the inhibitory effect of FOXA1 overexpression on cell inflammation (Fig. 5A). TFF1 silencing significantly reversed FOXA1 overexpression-induced alterations in the activity of SOD, ROS and MDA, which indicated that TFF1 could mediate oxidative stress (Fig. 5B). The effect of TFF1 silencing on cell apoptosis was determined using a TUNEL assay and western blotting. The fluorescence intensity in the siRNA-TFF1 group was enhanced compared with that of the siRNA-NC group (Fig. 5C). Moreover, the expression levels of Bax and cleaved caspase 9 were significantly increased and that of Bcl2 was decreased in the siRNA-TFF1 group compared with those in the siRNA-NC group (Fig. 5D). These findings suggested that TFF1 silencing reversed the effect of FOXA1 overexpression on cell apoptosis.