We purchased male Sprague-Dawley rats from the Experimental Animal Center of Anhui Medical University for experiments. Prostate antigen preparation. Healthy male NOD/LtJ (NOD/ShiLtJ, 4 weeks old) mice. All mice, CNP Model Animal Research Center, Nanjing University (Nanjing).We domesticated them for a week before the experiment. All operations were performed under anesthesia. Chloral hydrate was used that the purpose is to reduce pain. Before the experiment began, all the mice underwent a week of acclimatization to the laboratory environment. All animals were kept in a dedicated pathogen free research animal facility at Anhui Medical University. In short, prostate homogenates were obtained from Sprague - Dawley rats and emulsified with superclear and equal amounts of full Freund adjuvant. The mice were immunized on day 0 and day 28 respectively according to the experimental plan. Then the experimental mice were injected with VUF11027(5mg/kg/ day, DMSO as a solvent) through the tail vein for two consecutive weeks. The mice were killed on day 50.
Prostate tissue was taken, fixed with 10% formalin solution, paraffin embedded, sliced, dewaxed to water, stained with hematoxylin dye, then dyed with eosin dye, and finally dehydrated and sealed. The degree of inflammation was quantified on a 4-point scale from 0 to 3 [17], with the following criteria: 0, no inflammation; 1, mild inflammation, and clear monocytes around the blood vessels; 2, moderate inflammation, moderate perivascular monocytes surrounding; 3. Severe inflammation, bleeding, and a large number of monocytes in parenchyma with obvious infiltration around blood vessels.
IHC
Paraffin embedded tissue sections were dewaxed to water, heated at PH = 6.0, 0.01M sodium citrate solution at 100° for 15 minutes for antigen repair, and then put into 3% hydrogen peroxide solution to block endogenous peroxidase, and washed with PBS buffer three times for five minutes each time. Then it was sealed with 10% fetal bovine serum. The corresponding primary resistance was added and incubated at 4 degrees overnight. After washing with PBS for three times, the secondary antibody was added, incubated at room temperature for 2 hours, tested with freshly configured DAB chromogenic solution, and finally restained the nucleus with hematoxylin. The expression level was quantitatively analyzed using image J software
Masson staining
Paraffin-embedded tissue sections were dewaxed to water, immersed in 2.5% potassium dichromate mordant overnight, and rinsed. The slices are then put into Weigert iron hematoxylin dye for 1min, then rinsed, and hematoxylin differentiation solution for 10sec. After rinsing with running water, the slices are placed into ponceau acid fuchsin dye solution for 10min. After rinsing with distilled water, the slices are placed into 1% phosphomolybdic acid solution for 3min. After rinsing and drying slightly, the slices are placed into 2.5% aniline blue solution for 1min and then rinsed and differentiated with 1% acetic acid. Dehydration with anhydrous ethanol three times, xylene transparent, neutral resin seal. image J software was used to quantitatively analyze the proportion of collagen fibers.
Sirius Red staining
Paraffin-embedded tissue sections were dewaxed to water, immersed in Sirius red staining for 10 minutes, dehydrated with anhydrous ethanol three times, xylene transparent section for 5 minutes, and sealed with neutral resin. image J software was used to quantitatively analyze the proportion of collagen fibers.
Immunofluorescence
The paraffin sections were dewaxed to water, followed by antigen repair, sealing, adding primary antibody (CXCR7, ab72100, Abcam), and incubated in a wet box at 4° overnight. After adding the secondary antibodies (IL17A, ab79056, Abcam), the cells were incubated for 90 minutes away from light, DAPI was re-dyed in the nucleus, and the cells were incubated at dark room temperature. The co-localization of CXCR7 and IL17A was confirmed by Olympus FV3000 confocal laser scanning microscope.
Western Blotting Assays
The protein extract was separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. Incubate with 5% milk for 2 hours, then incubate with the corresponding primary antibody at 4°C overnight. GAPDH was used as loading control. TBST washing membrane was incubated with the corresponding secondary antibody (anti-rabbit or anti-mouse IgG, 1:50,000, Elabscience) for 2 hours, and then washed with TBST. The membranes were washed, incubated with secondary antibodies for 2 h, and visualized using an EZ-ECL Kit (Biological Industries, Israel).
Mouse Spleen Dissociation and Flow Cytometry
The proportion of Th17 and Treg cells was measured by flow cytometry. The splenic single-cell suspension was prepared according to our previous method [18]. Fitc-labeled CD4(BD Pharmingen, 557307,USA) and APC-labeled CD3e(BD Pharmingen, 553066,USA) were incubated with surface antibodies to APC-labeled CD25(BD Pharmingen, 553075,USA). After washing PBS again, Add 1 ml per tube containing phorbol 12myristate 13-acetate (PMA, MultiSciences, China), lonomycin (MultiSciences, China) 1640 medium (Gibco, USA) from monenin (MultiSciences, China) and Monenin (Multisciences, China) were incubated at 37℃ for 4 h and washed again. The cells were then sequentially immobilized and infiltrated, and incubated at 4 ° C for 60 min with intracellular antibodies, including PE-labeled IL-17A(BD Pharmingen, 559502, USA) and PE-labeled FoxP3(BD Pharmingen, 560401, USA). After washing again, analysis was performed with FACS Calibur flow cytometry (Beckman Coulter, USA). The resulting data is processed by FlowJo software (Tree Star, Ashland).
mRNA Sequencing Experimental
TRIzol reagent was used to extract the total RNA of the sample. Smarter amplification kit was used to reverse transcribed the qualified total RNA and amplified the cDNA. The qualified cDNA was constructed by Tn5 transposition enzyme method: First, when the cDNA was randomly interrupted by transposase, the interrupted fragment was added with sequencing splices. Then, part of the sequencing splices were used as primers to amplify and enrich the library. After the amplified library was purified by magnetic beads and qualified by Agilent 2100 Bioanalyzer, Sequencing with Illumina Nova6000 other sequencers produced a double end number of 150bp. DESeq2 package was used to analyze the differentially expressed genes, which were consistent with P-value <. Genes with a threshold of 0.05 and |LogFC2| > 1 were defined as differentially expressed genes (DEGs).
Untargeted Metabolomics Analysis
Weigh the sample into a 1.5mL centrifuge tube, add two small steel balls, methanol-water (V:V = 4:1, containing L-2-chlorophenylalanine, 4 µg/mL); Put it in the refrigerator at -40℃ for 2 min, and then put it into the grinding machine (60 Hz, 2 min). Ultrasonic extraction in ice water bath for 10 min; Stand at -40℃ for 30 min; Centrifuge at low temperature for 10 min (12000 rpm, 4℃), and put the supernatant into a glass derived bottle; Use a centrifugal concentrator to dry the sample. Methoxyamine hydrochloride pyridine solution (15 mg/mL) was added to a glass derived vial and incubated at 37°C for 60 min for oxime reaction. Remove samples after add 50 ul BSTFA derivatization reagent and 20 mu L n-hexane, add 10 kinds of internal standard 10 ul, 60 min at 70 ℃ reaction; After the samples were removed, they were placed at room temperature for 30 min for GC-MS analysis. The analytical instrument of the experiment was GMS-7890B /5977A (Aglient, USA).
Weigh the sample into a 1.5mL EP tube, add two small steel balls, and add methanol-water (V:V = 4:1, containing L-2-chlorophenylalanine, 4 µg/mL); After pre-cooling for 2 min in the − 40℃ refrigerator, put it into the grinder for grinding (60 Hz, 2 min); Ultrasonic extraction in ice water bath for 10 min and standing at -40℃ for 2 hours; Centrifuge for 10 min (12000 rpm, 4℃), the supernatant absorbed by syringe was filtered by 0.22 µm organic phase pinhole filter, transferred to LC sample vial and stored at -80℃ until LC-MS analysis. Quality control sample (QC) is prepared by mixing the extract of all samples in equal volume. The analytical instrument for the experiment was ACQUITY UPLC I-Class system (Waters Corporation, USA).