Bone marrow and peripheral blood were donated by 18 patients who were newly diagnosed with PV and 10 healthy adult volunteers at the Affiliated Zhuzhou Hospital Xiangya Medical College CSU (Zhuzhou, China) between August 2017 and April 2019. All patients met the World Health Organization diagnostic criteria for PV (1). Patient characteristics are shown in Table I. The healthy volunteers were eligible if they were 18–69 years of age and in healthy condition without active infections, and serious liver, kidney, heart and other diseases. Bone marrow and peripheral blood from 10 healthy volunteers were used as normal controls. The volunteers included 6 women and 4 men. The mean age was 41.5 years, and the age ranged between 23 and 69 years. All participants provided written informed consent according to the protocol approved by the Medical Ethics Committees of the Affiliated Zhuzhou Hospital Xiangya Medical College CSU (Zhuzhou, China) and in accordance with the principles outlined in the Declaration of Helsinki. Mononuclear cells were separated from bone marrow samples at 440 × g for 30 min at room temperature using Ficoll-Hypaque density gradient centrifugation (GE Healthcare). An EasySep™ CD34-positive selection kit (Stemcell Technologies, Inc.) was used to enrich the CD34⁺ cell population according to the manufacturer's protocol. CD34⁺ cells with a purity ≥85% were used in each experiment.

Plasma samples from patients with PV and healthy volunteers were collected to detect LTB4 levels using a leukotriene B4 Express ELISA kit (cat. no. 10009292; Cayman Chemical Company). Briefly, the standard or plasma sample, LTB4 AchE tracer and anti-LTB4 antibody were sequentially added to each well of a 96-well plate. The plates were then incubated for 60–90 min at room temperature before measuring the absorbance at 405 nm using an ELISA microplate reader (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from the purified CD34⁺ cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using a PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (cat. no. RR047A; Takara Bio, Inc.) according to the manufacturer's protocols. Reactions were incubated at 37°C for 15 min followed by heat inactivation for 5 sec at 85°C for reverse transcription. The PCR amplification was performed using TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (cat. no. RR820A; Takara Bio, Inc.). The primer sequences of the human 5-LO gene and the GAPDH gene are listed in Table II. The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 31 sec. Amplification was performed using an ABI 7300 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The 2^−ΔΔCq formula (15) was used to analyze the relative mRNA expression levels of 5-LO, which were normalized to the expression levels of GAPDH.

RIPA lysis solution (cat. no. P0013B; Beyotime Institute of Biotechnology) was used to extract total proteins from purified CD34⁺ cells. BCA assays (cat. no. 23227; Pierce; Thermo Fisher Scientific, Inc.) were used to determine the protein concentration. An equal amount of total proteins (30 µg/lane) was separated by 10% SDS-PAGE and then transferred onto PVDF membrane (cat. no. IPVH00010; EMD Millipore). The membranes were blocked using TBS with 0.05% Tween-20 (TBST) containing 5% non-fat milk at room temperature for 2 h, and then incubated with primary antibodies against 5-LO (dilution, 1:500; cat. no. sc-136195; Santa Cruz Biotechnology, Inc.) and β-actin (dilution, 1:1,000; cat. no. ab8226; Abcam) overnight at 4°C. After washing with TBST, the membranes were incubated with secondary antibodies (dilution, 1:2,000; cat. no. 7076; Cell Signaling Technology, Inc.) conjugated to horseradish peroxidase at room temperature for 1 h. Finally, the protein blots were visualized using a Hypersensitive ECL chemiluminescence kit (cat. no. P10018FS; Beyotime Institute of Biotechnology). The results were semi-quantified using ImageJ software (v1.52; National Institutes of Health).

The colony-forming ability of the cells was estimated by inoculating 500 CD34⁺ cells in MethoCult H4435 (Stemcell Technologies, Inc.). Various concentrations of zileuton (50, 100, 250 and 500 µM; Cayman Chemical Company) and/or 50 nM ruxolitinib (Cayman Chemical Company) were added for 14 days at 37°C. After 14 days, the presence of colonies (>40 cells) was scored under a light microscope (Zeiss LSM 800 with Airyscan; magnification, ×25; Zeiss AG).

CD34⁺ cells (1×10⁶ cells/well) from patients with PV and healthy volunteers were seeded into a 6-well plate and treated with 100 µM zileuton, 50 nM ruxolitinib or the combination of 100 µM zileuton and 50 nM ruxolitinib for 48 h at 37°C. After 2 days, 5×10⁵ cells were collected and applied to the flow cytometry detection. The apoptosis assay was performed using an Annexin V-FITC Apoptosis Detection Kit (cat. no. 556547; BD Biosciences). In brief, cells were stained with Annexin V-FITC and PI for 15 min at room temperature, and detected using a flow cytometer. Stained cells were analyzed using a FACSCalibur instrument (BD Biosciences) using a 488 nm excitation wavelength and emission was detected at 530 nm (for FITC) and 575 nm (for PI). The data were analyzed using the BD FACSuite™ version 1.01 (BD Biosciences). The phases of the cell cycle and DNA synthesis activity of CD34⁺ cells were determined using a FITC BrdU Flow kit (cat. no. 559619; BD Biosciences) according to the manufacturer's protocol. Briefly, cells were incubated with BrdU, a nucleoside analogue of thymidine, and then stained with anti-human CD34-allophycocyanin (dilution, 1:5; cat. no. 560940; BD Biosciences). After fixing in BD Cytofix/Cytoperm Buffer for 30 min on ice and permeabilizing in BD Cytoperm Permeabilization Buffer Plus for 10 min on ice, cells were treated with DNase to expose BrdU epitopes and then incubated with a FITC-conjugated anti-BrdU antibody (provided in kit; BD Biosciences) for 20 min at room temperature. DNA was counterstained with 7-aminoactinomycin D (7AAD; provided in kit; BD Biosciences) for 15 min at room temperature. Stained cells were then analyzed using a FACSCalibur instrument using a 488 nm excitation wavelength and emission was detected at 530 nm (for FITC-conjugated anti-BrdU) and 610 nm (for 7AAD). The data were analyzed using BD FACSuite™ version 1.01.

All experiments were repeated three times. Data are presented as medians and interquartile ranges. Differences in 5-LO expression and LTB4 levels between groups were compared using a two-tailed Mann-Whitney test, while data derived from the same samples after different treatments were analyzed using the Friedman test, and the Nemenyi post hoc test was subsequently used for pairwise comparisons between groups. GraphPad Prism 7 software (GraphPad Software, Inc.) and SPSS 26.0 software (IBM Corp.) were utilized for statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

To determine the potential effects of 5-LO inhibitor zileuton in the treatment of human PV, the present study first assessed the basal protein and mRNA expression levels of 5-LO in CD34⁺ cells from the bone marrow of patients with PV and healthy volunteers. Western blot analysis revealed that CD34⁺ cells from patients with PV exhibited higher protein expression levels of 5-LO than those from healthy volunteers (Fig. 1A and B; Table SI). Consistent with the results observed for protein expression, 5-LO mRNA expression was also increased in CD34⁺ cells from patients with PV compared with in those from healthy volunteers, as demonstrated by the results of RT-qPCR (Fig. 1C; Table SII).

Plasma levels of LTB4, a metabolite of the 5-LO signaling pathway, were measured in 14 patients with PV and 10 healthy volunteers using ELISA. Higher LTB4 levels were observed in patients with PV compared with those in healthy volunteers (Fig. 2; Table SIII).

A CFA was performed to assess the effect of zileuton treatment on the colony formation of primary CD34⁺ cells in vitro. As shown in Fig. 3, zileuton treatment inhibited granulocytes and monocytes (CFU-GM)- and burst-forming unit-erythroid (BFU-E)-derived colony formation by PV CD34⁺ cells in a dose-dependent manner, with an IC₅₀ of 460.4 µM for CFU-GM and 233.5 µM for BFU-E (Fig. 3B and C; Table SIV). By contrast, zileuton treatment did not markedly alter the colony formation of the CD34⁺ cells from healthy volunteers (Fig. 3A and C; Table SIV).

To explore whether the effect of zileuton on colony formation of hematopoietic cells was associated with the levels of 5-LO, 5-LO protein expression in CD34⁺ cells from patients with PV and healthy volunteers was measured with and without treatment with increasing concentrations of zileuton using western blotting. As shown in the Fig. 4, zileuton dose-dependently decreased 5-LO protein expression in CD34⁺ cells from a patient with PV, which exhibited high 5-LO expression, but had no significant effects on 5-LO expression in CD34⁺ cells from a healthy volunteer.

The present study investigated the effect of combination treatment with zileuton and ruxolitinib on the formation of colonies of primary CD34⁺ cells from 10 patients with PV and 6 healthy volunteers in vitro. The doses selected for the present study were 100 µM zileuton and 50 nM ruxolitinib. In the CD34⁺ cells from healthy volunteers, treatment with 100 µM zileuton, 50 nM ruxolitinib or 100 µM zileuton combined with 50 nM ruxolitinib did not alter colony formation (Fig. 5A and C; Table SV). By contrast, the yields of CFU-GM and BFU-E in CD34⁺ cells from patients with PV treated with 50 nM ruxolitinib were decreased by 19 and 35%, respectively, and the yields of CFU-GM and BFU-E in CD34⁺ cells from patients with PV treated with 100 µM zileuton were decreased by 12 and 20%, respectively (Fig. 5B and C; Table SV). However, the combination of ruxolitinib with zileuton had a greater effect on the colony formation of CD34⁺ cells from patients with PV, with a 36% a reduction in CFU-GM and a 55% reduction in BFU-E (Fig. 5B and C; Table SV).

The apoptosis rate of CD34⁺ cells from patients with PV and healthy volunteers after treatment with zileuton and/or ruxolitinib was detected using flow cytometry. The apoptosis rate of CD34⁺ cells from patients with PV was slightly increased following zileuton or ruxolitinib treatment (Fig. 6; Table SVI). However, combination treatment with zileuton and ruxolitinib induced apoptosis in a greater number of CD34⁺ cells from patients with PV than treatment with either individual drug. By contrast, neither ruxolitinib nor zileuton alone or in combination induced apoptosis of CD34⁺ cells from healthy volunteers (Fig. 6; Table SVI).

Cell cycle arrest is an important effect of numerous anticancer agents. The present study investigated the effects of zileuton and/or ruxolitinib on the cell cycle of CD34⁺ cells from patients with PV in vitro. As shown in Fig. 7A and C, treatment with zileuton and ruxolitinib alone slightly increased the number of CD34⁺ cells from patients with PV that were arrested in the G₀/G₁ phase of the cell cycle, with a concomitant decrease in the percentage of cells in S phase (Table SVII). However, combination treatment with zileuton and ruxolitinib caused more CD34⁺ cells from patients with PV to be arrested in the G₀/G₁ phase and a significant decrease in the proportion of cells in S phase compared with either monotherapy. By contrast, the percentage of CD34⁺ cells from healthy volunteers in each phase of the cell cycle did not markedly change, regardless of whether they were treated with zileuton, ruxolitinib or the combination treatment (Fig. 7A and B; Table SVII).