2.1 In vitro study

Human RWPE-1 cells (ATCC, VA), which are human prostate epithelial cells, were cultured in dulbecco's modified eagle's medium (DMEM) with 10% fetal bovine serum (FBS). LPS (Solarbio) was employed to induce inflammation in RWPE-1 cells.²³ To determine the effect of Li-ESWT on inflammatory response, the LPS-induced RWPE-1 cells were treated with a focused extracorporeal shock wave medical device. Cells were randomly divided into three groups: control group, LPS group, and Li-ESWT group. Specifically, the Li-ESWT probe was kept in contact with the bottom of the culture flask containing adherent cells. Li-ESWT was performed after the cells adherence of greater than 95%, and only one treatment per generation. On the other hand, the cells in the control group received a sham treatment. The Li-ESWT group of cells [200 pulses (0.09 mJ/mm²)] was cultured in DMEM-high glucose 10% FBS culture medium for 3 h, followed by co-cultured with LPS for 6 h. After 12 h, the cells were harvested and counted, and then stored at −80℃.

2.2 Animal experiments

All animal experimental procedures were approved by the Institute of the Animal Experiment Ethics Committee at Lanzhou University Second Hospital (Approval No. D2019-177) and were performed following the Guidelines for the Care and Use of Laboratory Animals (NIH publication No. 85–23, National Academy Press, Washington, DC, USA, revised 1996). Fifty-two-month-old male Sprague-Dawley (SD) rats (180–220 g) were obtained from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All the animals were raised under the specific pathogen-free (SPF) environmental conditions at 23 ± 2°C and 55 ± 5% humidity. Besides, the rats had free access to food and water and were given one week to adapt to the new environment before the experiment.

2.3 Establishment of the animal model

Prostate extracts were obtained from prostate tissue of twenty SD rats and were homogenized in a glass homogenizer with TritonX-100 (Solarbio). The homogenate was centrifuged at 10,000 g for 30 min at 4°C, and then, the supernatant was collected as prostate antigens (PAgs) and stored at −80℃.

Twenty-seven rats were randomly divided into the control group, EAP group, or Li-ESWT group (9 rats each). The rat model of EAP was established as previously described.²⁴ Equal volume of PAgs was emulsified with complete Freund's adjuvant (CFA), followed by multipoint intradermal injection into the EAP group and Li-ESWT group rats. Each rat was intradermally immunized with 1.0 ml of an isovolumetric mixture at different locations: abdomen, bilateral shoulder area, and bilateral hip area. Each rat in the control group was injected with isovolumetric saline. The immunization was given four times, at days 0, 7, 14, and 28. On day 30, rats in the Li-ESWT group were treated with Li-ESWT, whereas rats in the control or EAP groups were treated with sham operation only.

2.4 Von Frey filament in behavioral testing

Referred hyperalgesia and tactile allodynia were quantified using the Von Frey filaments (North Coast Medical) applied to the pubic region or the scrotal base. Six sizes of Von Frey filaments were used on days 0, 10, 20, 30, 40, and 50 according to the protocol.²⁵ Stimulation was performed when the rats remained quiet with the abdomen and scrotum resting on the bottom of the cage. Since the animal model cannot express its discomfort directly, this experiment assesses the experimental results through a series of behaviors such as sudden abdominal contraction, immediate licking or grasping the stimulation site, and jumping, all of which were defined as positive responses.^{25, 26}

2.5 The test of Li-ESWT on animal models

Rats were anesthetized with 1% sodium pentobarbital, exposed in a supine position and the abdominal hair was shaved. The shock wave applicator was placed on the pubic region. Rats in the Li-ESWT group received transcutaneous Li-ESWT therapy with an energy level of 0.20 mJ/mm² and a frequency of 120 pulses/min for a total of 200 pulses. The Li-ESWT was administered every two days for 4 weeks. After treatment, the rats were anesthetized and sacrificed to collect their prostate tissues in each group. The tissues were stored at −80°C for subsequent experiments.

2.6 Histology and immunohistochemistry

The collected prostates from each group were stored in 4% paraformaldehyde/0.1 mol/L PBS (pH 7.2) fixative, washed with water and dehydrated in gradient ethanol, xylene transparent, wax immersion, and embedding treatment at room temperature. The prostate tissues were then sliced for later use. Pathological sections were stained using hematoxylin and eosin (H&E) staining, and local pathological changes of the prostate were observed under the microscope.

The prostate tissue was treated with 1% H₂O₂ to block endogenous peroxidase and then heated to 97°C for 15 min in the antigen recovery solution. The samples were incubated with rat anti-rabbit TNF-α (bs-2081R; Bioss), anti-rabbit IL-1β antibody (bs-0812R; Bioss), anti-rabbit IL-6 antibody (bs-0782R; Bioss), anti-rabbit substance P (SP) antibody (bs-0065R, Bioss), or anti-rabbit COX-2 antibody (ab182422, Bio-Rad) at room temperature for 60 min, and then washed with 1× phosphate-buffered solution (PBS). The sections were then incubated with secondary antibody (Sigma), and then, the samples were developed using 3,3-Diaminobenzidine (DAB) (Sigma) to stain the sections with hematoxylin solution Counterstain.

2.7 Evaluation of oxidative stress

The intracellular ROS levels of the control group, LPS group, and the Li-ESWT group were determined by fluorescent dichlorofluorescein (DCF) assay with ROS Assay Kit (Jiancheng Bio). ROS generation was measured using 2,7-dichlorofluorescin diacetate (DCFH-DA) as described by Wang and Joseph with some modifications.²⁷ About 1 × 10⁴ RWPE-1 cells were inoculated in a culture flask. After being harvested, the cells were treated with DCFH-DA (10 μmol/L) for 20 min at 37℃ in the dark, and then washed twice with PBS. Intracellular ROS, as indicated by the fluorescence intensity of DCF was observed using a fluorescence microscope.

The prepared prostate tissue and RWPE-1 cells were homogenized for protein extraction, and then, the protein concentration was determined. We then used assay kits to detect superoxide dismutase (SOD) activity, malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), NADPH, or NADP⁺ in the prostate or cell samples. All operations were performed via following the manufacturer's instructions (Comin Bio).

2.8 Western blotting assay

The RIPA lysis buffer was used to lyse prostate tissue, followed by quantification of protein content using a BCA kit. The protein samples were then loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The blot was blocked in 5% skimmed milk powder in Tris hydrochloride buffer and incubated with monoclonal anti-AKT (GeneTex), anti-phospho AKT (GeneTex), anti-FOXO1 (Abcam), anti-phospho FOXO1 (Abcam), anti-CAT (GeneTex), anti-phospholipid or anti-GAPDH (Abcam) antibody, overnight at 4°C. The membranes were washed with TBST three times and were incubated with secondary goat anti-rabbit antibodies for 1 h at room temperature. After washing, the blots were observed by using the ECL chemiluminescence method for protein expression, and ImageJ software was used to analyze the optical density of the protein bands.

2.9 Statistical analysis

All data were collected and analyzed by SPSS v.25.0 software (IBM Corp). The differences among the treatments were evaluated using the Student's t test in behavioral testing. The rest data were subjected to ANOVA analysis. All the data are presented as mean ± SD, and a p-value <0.05 was considered statistically significant. Data descriptions were performed using GraphPad Prism v.5.0 (GraphPad Software Inc)^™.

3.1 Li-ESWT ameliorated the autoimmune-induced inflammation in the prostates

The morphological structure of prostate tissue is shown in Figure 1A. In the EAP group, a large number of inflammatory cells infiltrated in the prostate, and edema, hyperemia, fibrosis, and destruction of glandular epithelial cells were observed in the stroma. Compared with the Li-ESWT group, inflammatory cells infiltration in the prostate was significantly reduced, acini were relatively normal, and glandular epithelial cells were mildly hyperplastic, without injury or atrophy.

3.2 Li-ESWT ameliorated inflammation in EAP rats

Compared with the control group, the levels of TNF-α, IL-6, and IL-1β were significantly increased in the EAP group. However, after Li-ESWT treatment, the expression of these measures was significantly reduced (Figure 1B-D). Thus, Li-ESWT may reduce the prostate inflammation induced by autoimmunity.

3.3 Li-ESWT inhibits the decrease of pain threshold and hypersensitivity in the pelvic area

Tactile allodynic behavioral responses were measured at 0, 10, 20, 30, 40, and 50 days for rats in each group. During day 10 to 50, significant allodynic responses were detected in rats in the EAP and Li-ESWT groups. However, from day 40 onwards, the tactile allodynia responses in the Li-ESWT group were significantly improved compared with that in the EAP group (Figure 2A-D). Furthermore, while COX-2 and SP positive inflammatory cells were significantly increased in tissues suffering from prostatitis, their expression was significantly reduced by Li-ESWT treatment (Figure 1E-F).

3.4 Li-ESWT improves the oxidative stress in CP/CPPS rat prostate and LPS-induced RWPE-1 cells

We evaluated the antioxidant activity via using antioxidants markers (such as SOD, GSH, and NAPDH). Oxidation products (such as MDA, GSSG, and NADP⁺) play a key role in monitoring and indicating the degree of peroxidation damage. As shown in Figure 3, there was a decreased level of SOD, GSH, NAPDH, GSH/GSSG, and NAPDH/NADP⁺ in the model group both prostate tissues (Figure 3A-H) and cells (Figure 3I-P). However, the activities of MDA, GSSG, and NADP⁺ were instead significantly increased. Compared with the model group, SOD, GSH, GSH/GSSG, and NAPDH/NADP⁺ were increased in the Li-ESWT group. In contrast, the levels of MDA, GSSG, and NADP⁺ were significantly decreased.

3.5 Li-ESWT improved LPS-induced intracellular ROS generation

The ability of LPS to induce oxidative stress was determined by examining the level of ROS in RWPE-1 cells. Fluorescence microscope images (Figure 4A) and quantitative data (Figure 4B) showed that the Li-ESWT group had less production of intracellular ROS than the LPS group.

3.6 Li-ESWT ameliorated autoimmune-induced prostatitis in rats through the AKT/FOXO1 signaling pathway

The dysregulation of AKT/FOXO1 signaling is often correlated with the production of oxidative stress and inflammation. The immunoblot assay showed an upregulation of p-AKT and p-FOXO1, while a downregulation of CAT expression in the EAP group compared with the control group (Figure 5A-D). Interestingly, Li-ESWT treatment could significantly reduce the expression of p-AKT and p-FOXO1, whereas increase of CAT expression. Therefore, the effect of Li-ESWT on autoimmune-induced prostatitis in rats might be related to the PI3 K/Akt/FoxO1 signaling pathway.