Samples collection
The tissues of 30 BPH, 16 PCa and adjacent normal control were collected from the First Affiliated Hospital of Guangxi Medical University. BPH tissues were obtained after the Transurethral Resection of Plasma in severe prostatic hyperplasia patients. The PCa tissues were obtained by surgical resection. 94 cases of serum were included in this study by dividing into three groups: Category III, Category IV, and healthy control. 16 cases of EPS fluidswere collected in 1.5 ml autoclaved tubes directly from the urethra after the patients’ prostates were massaged via rectum. All patients had not receivedany antimicrobial treatment before this evaluation. All specimens in this study were anonymously handled according to ethical and legal standards.
Rat model of nonbacterial prostatitis
A total of 30 4-month-old male Sprague–Dawley rats, weighing 250–300 g, were obtained from Guilin Medical University Animal Experiment Center. The experimental animals were divided into 2 groups: saline group and xiaozhiling group. Following abdominal surgery, the prostate was exposed and injected with 0.1 ml of normal saline or an equal volume of xiaozhiling respectively as described(22). One month later, rats were anesthetized with intraperitoneal injection of pentobarbital sodium, and prostate tissue was excised by abdominal surgery.
Multiple Reaction Monitoring (MRM)
Serum samples were prepared for mass spectrometry as described(23). Samples were lyophilized and redissolved in 2% ACN containing 0.1% formic acid, and peaked with 50 fmol of peptide mixture of β-galactosidase, as a relative internal standard peptide for LC-MS/MS analysis as described(24).
MRM experiments were performed on 4000 QTRAP mass spectrometer (Applied Biosystems) interfaced with a 2-D nanoLC (Eksigent) was used to perform LC-MS/MS analysis. MRM data on the 4000 QTRAP mass spectrometer were acquired with NanoSpray II source. The optimal acquisition parameters were as follows: ion spray voltage (2300 V), curtain gas (30 p.s.i.), nebulizer gas (16 p.s.i.), interface heater temperature (150 ℃), declustering potential (100). The resolution parameters of the first and the third quadrupoles were set as “unit”. In the MRM runs, the scan time was maintained at 50 ms for each transition, and the pause between transition scans was set to 5 ms. Result files (wiff and wiff.scan) were imported into peak area integration software, MultiQuant (Applied Biosystems, version 1.1) to extract the peak areas of transitions and to normalize using the peak area of internal standard peptide for the β-galactosidase peptide (VDEDQPFPAVPK, IDPNAWVER, GDFQFNISR) to adjust for variations between runs, as described.
Cell lines and cell cultures
The human BPH cell line BPH-1 and PCa cell lines 22RV1, VCaP, DU145, PC-3 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in high-glucose Dulbecco’s Modified Eagle Medium /F12 (DMEM/F12; Gibco Company, USA), or Roswell Park Memorial Institute (RPMI-1640, Gibco Company, USA), supplemented with 10% fetal bovine serum (FBS; Gibco Company, USA) and 1% penicillin/streptomycin, respectly. All cells were cultured at 37°C with 5% CO 2.
Real time quantitative polymerase chain reaction analysis (RT-qPCR)
Total tissues RNA was isolated using the Trizol reagent (Invitrogen, USA) according to the protocol described by the manufacturer. The cDNA was obtained following reverse transcription with PrimeScriptTM RT reagent Kit (Takara, Shiga, Japan). The annealing temperature of RT-qPCR and PCR cycle number were 58-60˚C and 40 cycles for SOD3, CP, DSG2, RBP4, and CFP. The primer sequences used were listed in Supplementary Table S1. Following the QR = 2−ΔCt , we got the relative expression levels of mRNA in each sample(25).
Immunohistochemistry and Hematoxylin-eosin staining
After fixed, the rat and human prostate tissues were embedded in paraffin, respectively. The sections were dewaxed with xylene and the antigen was repaired with citrate buffer in the condition of hyperbaric for 15 minutes. After blocked with 0.3% hydrogen peroxide and goat serum, the sections were incubated with the primary antibody: anti-SOD3 (1:1000, Proteintech) at 4°C for 12 hours. After washing with PBS solution, the sections were incubated with secondary antibody ( 1:2000, Proteintech) at room temperature for 30 minutes. The Hematoxylin-eosin staining (HE) was done directly after dewaxed with xylene.
Western blot analysis
Total proteins of tissues were extracted using radio immunoprecipitation assay (RIPA) buffer. After detecting the concentrations, a total protein of 20 μg were separated by electrophoresis on 10% Tris-HCl gels, transferred to polyvinylidene difluoride membranes, and blocked in 5% nonfat milk powder. Following the incubation with primary antibody: anti-SOD3 (1:1000, Proteintech) at 4°C for 12 hours, a horseradish peroxidase–conjugated secondary antibody (1:6000, Proteintech) were performed at RT for 1 hour. The bands were scanned and analyzed on the ChemiDoc XRS+ System (BioRad) at the end. Grayscale analysis were performed on Quantity One software.
Docking calculations
All protein files required were downloaded from the RCSB PDB website (https://www.rcsb.org/). The protein processing prior to docking was done using SYBYL-X 2.0 software. Protein-protein docking and image processing of the complexes were performed on HEX 8.0.0 software (http://hex.loria.fr). The docking condition is that the correlation type is 'Shape + Electro' and the final search value is 30. Furthermore, we predicted the hot spots of between SOD3 and its interactomes using the KFC Server (Knowledge-based FADE and Contacts) online(26) .
Data collection of SOD3 and its interactors
Further the potential interacting proteins of SOD3 were obtained from the our previous study, that were also observed overexpression in the serum of CNP. The online Search Tool for the Retrieval of Interacting Genes (STRING, https://string-db.org/)(27) was also used to search for potential interacting proteins of SOD3. For further detecting the function of SOD3 and its potential interactors, gene ontology (GO) analyses was done. FunRich3.1.3 was used to detect which biological process or component that SOD3 and its interacting proteins mainly involved in based on GO analyses.
Statistical analysis
Quantitative variables were expressed as mean±SD, and were analyzed by ANOVA. Statistical significance was assumed when p < 0.05. Receiver operating characteristic (ROC) curves were conducted and used to estimate the diagnostic value of the markers. The best cut-off value for SOD3 was defined as the point with maximum Youden index (sensitivity+specificity-1) on the ROC curve.