Cell culture

Mouse leukemic macrophage cell line RAW264.7 (Cat. No. CL-0190) was purchased from Procell Life Science & Technology. It was cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Cat. No. 11965092, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Cat. No. SA301.02, CellMax) and 1% Penicillin–Streptomycin (P/S; Cat. No. P1400, Solarbio). Human prostate hyperplasia cell line BPH-1, kindly provided by Prof. Long Wang from The Third Xiangya Hospital of Central South University in Changsha, China, was cultured in RPMI 1640 medium (Cat. No. C3010-0500, VivaCell) supplemented with 10% FBS and 1% PS. All cells were maintained at 37 °C in a fully humidified atmosphere of 95% air and 5% CO2.

Preparation of conditioned culture medium from inflammatory macrophages

We induced inflammation in RAW264.7 macrophages by using 10 ng mL−1 of pro-inflammatory cytokine TNF-α (Cat. No. 300-01A, PeproTech), as our previously reported (Deng et al. 2022). Subsequently, the residual culture medium was replaced and the cells were incubated in fresh CM supplemented with either 300 ng mL−1 of recombinant ITLN-1 protein (Cat. No. abx067296, Abbexa) or an equal volume of PBS for 24 h. After incubation, the conditional medium (CM) from inactivated (treated with Veh; RAWVeh − CM), TNF-α-activated (RAWTNF−α − CM), ITLN-1-treated (RAWITLN−1 − CM), or TNF-α + ITLN-1-treated (RAWTNF−α +ITLN−1 − CM) RAW264.7 macrophages were collected and subjected to centrifugation at 2,000 × g for 10 min to remove dead cells and cellular debris. The supernatant was then collected and stored at − 80 °C or used for downstream experiments.

Cell counting kit-8 assay

Cell proliferation in BPH-1 cells was assessed by the Cell Counting Kit-8 (CCK-8; Cat. No. C0005, TargetMol) following to the manufacturer’s protocol. To evaluate the effect of omentin-1 on BPH-1 cells, the cells were firstly seeded in a 96-well plate at a density of 2 × 103 cells per well, and cultured for an additional 48 h. The cells were then subjected to PBS or ITLN-1 protein (300 ng mL−1) treatment. To assess the effect of the conditional medium (CM) of RAW264.7 cells intervened by ITLN-1 on BPH-1 cells, we treated BPH-1 cells with the following medium: Veh, RAWVeh − CM, RAWTNF−α − CM, RAWITLN−1 − CM, or RAWTNF−α +ITLN−1 − CM, respectively. After incubation at 37 °C for 48 h, the medium in each well was replaced with 90 µL fresh medium and 10 µL CCK-8 reagent, followed by incubation at 37 °C for 2 h. Finally, the absorbance was measured using a microplate reader (Varioskan LUX, Thermo Scientific) with a wavelength of 450 nm. The obtained OD value was used to calculate the final results.

EdU cell imaging assay

The cell proliferation in BPH-1 cells was also assessed by Yefluor 594 Click-iT EdU Imaging Kits (Cat. No. 40276ES60, Yeasen). For EdU imaging assay, BPH-1 cells were seeded into a 24-well plate slide and treated them with different conditions, including: Veh, RAWVeh − CM, RAWTNF−α − CM, RAWITLN−1 − CM, or RAWTNF−α +ITLN−1 − CM, respectively. Subsequently, the cells were incubated with the EdU reagent according to the manufacturer's instructions. The EdU reagent is a thymidine analogue that incorporates into the DNA of proliferating cells and can be detected by fluorescence microscopy. After a 2-h incubation, the cells were fixed in 4% paraformaldehyde and stained with 4′,6-diamidino-2-phenylindole (DAPI; Cat. No. H-1200, Vectorlabs) to visualize nuclei. Finally, the fluorescent images were acquired using a Zeiss ApoTome fluorescence microscope. The percentage of EdU positive cells was quantified for different groups.

Animals and treatments

To ensure the welfare of the animals, they were housed in a controlled environment with appropriate temperature (22 ± 3 °C), humidity (50 ± 20%), and lighting (12/12 h light-darkness cycle) conditions. Additionally, all animals had access to sufficient food and water sources throughout the experiment.

To evaluate the prostate states under omentin-1 deficiency, 5-month-old and 10-month-old C57BL/6 male Itln-1−/− mice and their WT littermates were used for further analyses. The Itln-1−/− mice were constructed by Cyagen Biosciences. The depletion of Itln-1 was verified by RT-PCR on collected small intestines from both Itln-1−/− mice and their WT littermates.

To investigate the therapeutic effect of omentin-1 on BPH, the 2-month-old male C57BL/6 WT mice were used to induce experimental BPH models by continuous intraperitoneal injection of testosterone propionate (TP, dissolved in 100 μL of corn oil; Cat. No. C805618, Macklin) at the dosage of 5 mg per Kg body weight each day, or equal volume of corn oil as a control. Mice were randomly divided into the following 6 groups, including control, corn oil, TP, TP + Ad-Itln-1, TP + Ad-GFP, TP + Fina groups (n = 8 per group). Ad-Itln-1 or Ad-GFP at the dose of 1 × 108 plaque-forming units (PFU) were intravenously injected into the tail vein of mice for twice a week. Body weight of mice was recorded weekly to adjust the TP dosage. After four weeks treatment, all mice were sacrificed to collect blood, prostate, and small intestine samples immediately after euthanasia. The serum samples were obtained by centrifugation at 1500 rpm for 15 min and stored at − 80 °C for further analyses. The prostate index was calculated by dividing the prostate wet weight (mg) by the body weight (g) of the mice. Histological analysis of the prostate tissues was performed after immersing them in 4% PFA or freezing them at − 80 °C.

Histomorphometry and immunohistochemical analyses

Prostate tissues were embedded in paraffin after dehydration in an increased gradient ethanol solution. We then processed 5 μm-thick sections of the prostate and performed histomorphometric and immunohistochemical analysis to gain the information of BPH pathogenesis. For histomorphological analysis, hematoxylin–eosin (H&E; Cat. No. G1005, Servicebio) staining was conducted to examine the samples. For immunohistochemical analysis, the sections were incubated with primary antibody against TNF-α (1:200 dilution; Cat. No. 17590, Proteintech) overnight at 4 °C. Subsequently, the slices were incubated with HRP-conjugated secondary antibody (Cat. No. 511203, ZenBio) for 1 h at room temperature. Histomorphometric and immunohistochemical analysis were observed under an optical microscope (Olympus CX31). Positive staining cells or relative staining area were measured in three random visual fields per section using Image-Pro Plus 6.0 software.

ELISA analysis

The levels of prostatic acid phosphatase (PAP), TNF-α, IL-2, and IL-6were evaluated using a mouse PAP ELISA kit (Cat. No. JM-02750M2, JingMei Biotechnology),TNF-α ELISA kit (Cat. No. 70-EK282/4-96, Elabscience), IL-2 ELISA kit (Cat. No. 70-EK202/2-96, Elabscience) and IL-6 ELISA kit (Cat. No. 70-EK206/3-96, Elabscience). All ELISA assays were performed according to the manufacturers’ instructions with strict quality control measures in place.

Western blot

Tissue grinding is performed in RIPA buffer containing 1% protease inhibitor. After a short sonication, the tissue homogenate is centrifuged at 3000×g for 20 min (4 °C). Tissue extracts were obtained by collecting the supernatant. Protein concentration was determined using BCA Protein Quantification Kit (Cat. No. E-BC-K318-M, Elabscience). The target proteins were detected by immunoblotting using ITLN-1 antibody (Cat. No. 11770-1-A, Proteintech). The intensity of the bands was quantified using imageJ software.

qRT-PCR

Total RNAs were isolated from tissues using TRIzol reagent (Cat. No. AG21102, AG), and the concentrations of the RNAs were measured using Varioskan LUX (Thermo Scientific). The All-in-One cDNA Synthesis SuperMix (Cat. No. E0 47, Novoprotein) was used to synthesize cDNA following the manufacture’s protocol. The qRT-PCR analysis was performed on the FTC-3000 real-time PCR system (Funglyn Biotech) using 2 × SYBR Green qRT-PCR Master Mix (Cat. No. 21202, Bimake). Relative gene expression was calculated by the 2–△△CT method, with Gapdh serving as the reference housekeeping gene.

The primers used for qRT-PCR were designed based on the gene sequences and were as follows: mus musculus Gapdh, forward, 5ʹ-CACCATGGAGAAGGCCGGGG-3′, reverse, 5′-GACGGACACATTGGGGGTAG-3′; Mus musculus Itln-1, forward, 5′-TTTCCTGCGCACGAAGAA-3′, reverse, 5′-TCATGTCACAGAAGGTCT-3′.

Statistical analysis

Data were presented as mean ± standard deviation (SD) and statistically analyzed using GraphPad Prism 7.0 software. The sample size (n) for each statistical analysis is specified in the figure legends. Unpaired two-tailed Student’s t-test was used to compare two groups, and one-way analysis of variance (ANOVA) was applied to compare multiple groups, and two-way ANOVA was used when two independent variables, in combination, affect a dependent variable. The difference was considered statistically significant at P < 0.05, P < 0.01, and P < 0.001. NS, not significant.