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Journal of Traditional Chinese Medicine logoLink to Journal of Traditional Chinese Medicine
. 2023 Jun 8;43(5):963–972. doi: 10.19852/j.cnki.jtcm.20230608.001

Electroacupuncture stimulating Zhongji (CV3), Guanyuan (CV4), and bilateral Dahe (KI12) attenuates inflammation in rats with chronic nonbacterial prostatitis induced by estradiol through inhibiting toll-like receptor 4 pathway

Zhihao LI 1, Wenjun HAN 3, Xiuling SONG 4, Yan LI 1,✉, Yuelai CHEN 2,✉
PMCID: PMC10465839  PMID: 37679984

Abstract

OBJECTIVE

To investigate the anti-inflammatory effect of electroacupuncture (EA) on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), also known as chronic nonbacterial prostatitis (CNP), and explore its underlying mechanism.

METHODS

A CNP rat was established by surgical castration combined with 17-β estradiol injection in male Sprague-Dawley rats for thirty consecutive days. The CNP rats received EA treatment once a day for eight days. Chronic pelvic pain was evaluated by mechanical withdrawal threshold measurement. The histological change was assessed by hematoxylin-eosin staining. The inflammatory cytokines in prostates were determined by enzyme-linked immunosorbent assays. The expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), inhibitors of kappa‐B alpha (IκBα), and nuclear factor-kappa B (NF-κB) were detected by Western blotting. The nuclear translocation of NF-κB and the location of TLR4 were observed with immunofluorescence staining.

RESULTS

The results showed that EA decreased the prostate index, upregulated the mechanical withdrawal threshold, restored the histomorphology of the prostate, reduced the inflammatory factor levels, inhibited NF-κB p65 nuclear translocation, and downregulated the expression levels of critical proteins involved in the TLR4/NF-κB signaling pathway in prostates.

CONCLUSIONS

Our findings suggested that EA could relieve pelvic pain and attenuate prostatic inflammation in estradiol-induced CNP rats. The underlying mechanism may be related to the inhibition of the TLR4/NF-κB signaling pathway.

Keywords: chronic nonbacterial prostatitis, electroacupuncture, morphology, anti-inflammatory agents, toll-like receptors

1. INTRODUCTION

Chronic nonbacterial prostatitis (CNP) is a common disorder in adult men and is classified as type III prostatitis (chronic prostatitis/chronic pelvic pain syndrome; CP/CPPS).1 The main symptoms of CNP include pelvic pain or discomfort, frequency of urination, urgency, and dysuria, which have a considerable impact on the life and health of patients.2 The prevalence rate ranges from 2.2% to 9.7% around the world and 4.5% in China.3,4

Although the pathogenesis of CNP remains unclear, increasing evidence has shown that immune dysfunction and chronic inflammation play a significant role in CNP.5 Toll-like receptors (TLRs) are important receptors of the innate immune system that initiates inflammatory cascades by recognizing structurally conserved molecules derived from microbes. TLR4, as a member of the TLR protein family, plays a key role in the inflammatory response by activating myeloid differentiation factor 88 (MyD88).6 MyD88 is an essential adapter protein of nuclear factor-kappa B (NF-κB), which is the critical step of various inflammatory responses. As a nuclear transcription factor, NF‐κB binds to its inhibitory protein, the inhibitors of kappa‐B (Iκ‐B), and exists in the cytoplasm in a resting state. Inhibitors of kappa-B alpha (IκBα), as a member of the Iκ-B protein family, inhibits the activation of NF‐κB and prevents NF‐κB from entering the nucleus. The activation of MyD88 increases the phosphorylation level of IκBα and decreases the inhibitory function on NF‐κB, which results in the activation of NF‐κB. In the active state, NF-κB is phosphorylated and enters the nucleus, leading to the release of various inflammatory cytokines,7 such as interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), which then result in inflammation.8 Several studies have demonstrated that TLR4 is increased in estradiol-induced CNP rats and downregulated after treatment.9 As TLR4 agonists, lipopolysaccharides could accentuate inflammation in the prostates of a CNP model.10 This evidence suggests that TLR4 might be an intervention target for CNP.

There are several therapies for CNP including alpha-blockers, anti-inflammatory agents, physiotherapy, and neuroleptics.11 However, these medicines have limited effects and some side effects, including hypotension, gastrointestinal dysfunction, and decreased libido.12,13 Electroacupuncture (EA) is a widely used therapy in clinical practice based on Traditional Chinese Medicine (TCM) theory and modern technology and is characterized by low costs, few side effects, and controllable parameters.14 As a predominant therapy for pain management, EA has been widely used in various chronic diseases, including chronic pelvic pain in women, chronic low back pain, and knee osteoarthritis.15,⇓-17 Currently, acupuncture therapy has been included in the recommendations and is increasingly being applied to the treatment of CP/CPPS.18,19 Clinical studies have shown that EA is an effective intervention for CNP, especially in relieving pain symptoms and improving quality of life.20 Our research group has been committed to clinical research on urinary system diseases for many years and demonstrated the significant effect of EA on CNP patients.21,22 Recent studies showed that EA exerted protective properties on prostate morphological changes and decreased inflammation cytokines in CNP rats.23 However, few studies have investigated the mechanism underlying the anti-inflammatory effect of EA on CNP. In the present study, we investigated the anti-inflammatory effect of EA and the activities of the TLR4/NF-κB signaling pathway to clarify whether there is a correlation between the anti-inflammatory effect and EA on CNP. We hope this study will provide a theoretical basis for the treatment of CNP.

2. MATERIALS AND METHODS

2.1. Animals

A total of 27 male Sprague-Dawley rats weighing (300 ± 20) g were purchased from Vital River Laboratory Animal Technology Company (Beijing, China). All rats were maintained under standard laboratory conditions with a 12-h light/12-h dark cycle and free access to food and water. The room temperature was maintained at 23 ℃-26 ℃ and the relative humidity was between 45% and 50%. All experiments were conducted following the Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Institutional Animal Care Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM210326001).

2.2. Instruments and reagents

17-β estradiol (Sigma-Aldrich Co., St. Louis, MO, USA), hydrogenated soybean oil (Zhejiang Tianyushan Medicinal oil Co. Ltd., Quzhou, China), phosphate buffer saline, radio-immunoprecipitation assay (RIPA) lysis buffer, phosphatase protease inhibitor, bicinchoninic acid (BCA) assay kit (Beyotime Biotechnology, Shanghai, China). IL-1β, IL‐6, and TNF-α enzyme-linked immunosorbent assay kits (MultiSciences Biotech Co., Ltd., Hangzhou, China). Primary antibodies: TLR4 (Abcam, Cambridge, UK), MyD88 (Abcam, Cambridge, UK), inhibitor of NF-κB (IκB-α) (Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-IκBα (Cell Signaling Technology, Inc., Danvers, MA, USA), NF‐κB (Cell Signaling Technology, Inc., Danvers, MA, USA), phospho‐NF‐κB (Cell Signaling Technology, Inc., Danvers, MA, USA), and β-Actin (Abcam, Cambridge, UK), Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit immunoglobulin G (IgG) (Abcam, Cambridge, UK), 4,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Technology, Inc., Danvers, MA, USA). Disposable sterile acupuncture needles (0.25 mm × 25 mm), SDZ-V Hwato electroacupuncture apparatus (Suzhou Medical Appliances Co. Ltd., Suzhou, China), von Frey filaments (Danmic Global, Inc., Campbell, CA, USA), Electrophoresis system (Liuyi Instrument Factory Co. Ltd., Beijing, China), Clinx ChemiScope Mini-Series Western Blot Imaging System (CLINX Science Instruments Co., Ltd., Shanghai, China), JA 31002 electronic balance (Shanghai Jing Tian Electronic Instrument Co., Ltd., Shanghai, China), Leica DM 2000 microscope (Leica, Inc., Wetzlar, Germany), Fluorescence microscope (BX43, Olympus, Inc., Tokyo, Japan), Image-Pro Plus 6.0 (Media Cybernetics, Inc., Silver Springs, MD, USA).

2.3. Experimental procedures

After one week of acclimation, a total of 27 rats were randomly assigned to the control, model, and EA group (n = 9 per group) according to the method of random number table. The rats in the model group and EA group received surgical castration and subcutaneous injection of estradiol to establish the CNP models, and rats in the control group received a sham operation procedure as a control. After thirty days of establishing the model, one rat from each of the three groups was used for prostate hematoxylin-eosin (HE) staining to confirm that the model was established. Then, we hold rats in the supine position in a restrainer, while the rats in the EA group were administered EA treatment every day for eight days. After eight days of EA treatment, all rats from three groups were euthanized with an overdose of pentobarbital sodium. The prostates were quickly removed and dissected on ice. The wet weight of the prostates was measured using an electronic balance. Then all the prostates were cut in half. Part of the prostate tissue was fixed in the 4% formaldehyde solution for histological staining. The other part of the prostate tissue was stored at −80 °C until protein extraction for Western blotting and enzyme‐linked immunosorbent assays (ELISAs).

2.4. Establishing the chronic nonbacterial prostatitis rat models

In this study, we followed a standard protocol as previously described.24 Briefly, rats were anesthetized with 3% pentobarbital sodium (50 mg/kg, i.p.). Then, the abdominal skin was disinfected and opened, bilateral testes were gently separated and removed after ligation. The abdominal skin stump was sutured with absorbable sutures. Throughout the surgery process, a heating blanket was used to maintain the rats’ body temperature. The surgical procedures were performed at a biosafety laboratory. After the surgery, penicillin was intramuscularly injected once a day (250 000 U/kg, 0.1 mL for each rat) for 3 consecutive days to prevent infection. Postoperative conditions were observed every day after surgery. From the first day after surgery, a subcutaneous injection of 17-β estradiol with a dose of 0.25 mg·kg-1·d-1, dissolved in hydrogenated soybean oil, was conducted for thirty consecutive days. The rats in the control group received sham operations that involved exposing the testes without removing them and performing subcutaneous hydrogenated soybean oil injections in the same volume for thirty consecutive days. Then one rat selected at random from each group was used for hematoxylin-eosin (HE) staining on prostates. The obvious inflammatory cell infiltration with congestion and edema in the prostate tissue from the model group and the EA group would be identified as the CNP rats established successfully.

2.5. EA treatment

After establishing the CNP model, the rats in the EA group were subjected to EA treatment every day for eight days. Zhongji (CV3), Guanyuan (CV4), and bilateral Dahe (KI12) acupoints were used for EA treatment. Zhongji (CV3) is located on the ventral midline at approximately the upper 4/5 and lower 1/5 of the umbilicus and pubic symphysis. Guanyuan (CV4) is located on the ventral midline at approximately the upper 3/5 and lower 2/5 of the umbilicus and pubic symphysis, and Dahe (KI12) is located 0.5 cun [≈ 5 mm] besides Guanyuan (CV4).25 The rats were fixed in the designed restrainer in a supine position. Then a disposable sterile acupuncture needle was inserted into the acupoints 5 mm vertically and connected to an electroacupuncture apparatus for 20 min. The stimulating intensity was 2 mA, and the frequency was 4/20 Hz. The lower abdomen of the rats shook gently, but the rats did not cry out. All the treatments were performed at the same time every day, once a day for eight consecutive days.

2.6. Assessment of chronic pelvic pain

The rats were tested for allodynia on Days 0, 8, 15, 22, 30, 32, 34, 36, and 38 as previously described by Zeng et al.26 First, the rats were placed in Plexiglas cubicles with a stainless steel wire grid floor for 30 min to acclimatize. Then, a series of von Frey filaments from 0.4 to 26 g were applied one by one to the rat scrotal skins for 1 to 2 s each time. Different areas within this region were stimulated to avoid desensitization. The following behaviors were considered positive responses: sharp retraction of the abdomen, immediate licking or scratching of the area of filament stimulation, and jumping. The bending force of the filament to which the animal responded was taken as the baseline threshold of mechanical stimulus. The measurement was conducted five times with a 5‐min interval. The five measurements were averaged to obtain the final data. Behavioral testing was performed in a blind manner with no knowledge of grouping.

2.7. HE staining

The prostates were removed and fixed in 4% formaldehyde solution for 24 h. Then, the prostate was dehydrated in ethanol, embedded in paraffin, and cut into slices of 5 μm by a microtome. The sections were stained with HE and observed using a Leica DM 2000 microscope.

2.8. ELISA

The prostate tissue was homogenized in ice-cold RIPA lysis buffer. The expression of IL-1β, IL‐6, and TNF-α was determined by ELISA kits according to the manufacturer’s instructions.

2.9. Western blotting

The prostates were homogenized and lysed in RIPA lysis buffer with the phosphatase protease inhibitor plus phenylmethanesulfonyl fluoride. The supernatant was obtained after centrifugation at 12 000 × g for 10 min. The protein concentrations were determined by a BCA assay kit. The samples were denatured at 99 ℃ for 15 min. Protein (20 µg) from each sample was separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% nonfat milk for 1 h at room temperature and incubated with the following primary antibodies overnight at 4 ℃: TLR4, MyD88, IκBα, phospho-IκBα, NF‐κB, phospho‐NF‐κB, and β-Actin (1: 1000). The membranes were washed 3 times with Tris-HCl buffer and incubated with the secondary antibody for 1 h at room temperature. The blots were visualized with enhanced chemiluminescent Plus Western Blot Detection Reagents and photographed with Clinx ChemiScope Mini-Series Western Blot Imaging System. The images were analyzed using Image-Pro Plus 6.0.

2.10. Immunofluorescence staining

The collected prostates were fixed, dehydrated, and then cut into coronal sections at thicknesses of 10 μm. The prostate slices were blocked with 5% goat serum for 1 h and incubated overnight at 4 ℃ with primary antibodies against TLR4 (1:200) and p65 (1: 400). Then, Alexa Fluor 488-conjugated AffiniPure goat anti-rabbit IgG (1:400) was incubated with the prostate slices at room temperature for 1 h. Finally, the cell nuclei were stained with DAPI for 10 min at room temperature. The prostate slices were observed under a fluorescence microscope. Images were analyzed with Image J software.

2.11. Statistical analysis

The data were analyzed using the Statistical Package for Social Science software (SPSS version 25.0, IBM Corp., Armonk, NY, USA). Continuous data is represented by mean ± standard deviation ($\bar{x}±s$). The mechanical withdrawal threshold was analyzed by repeated-measures analysis of variance with the Sidak test for multiple comparisons, and all other data were analyzed by one-way analysis of variance with post-hoc multiple comparisons using Tukey's test to assess differences between groups. P < 0.05 indicates a statistically difference.

3. RESULTS

3.1. Model identification

After thirty days of model establishment, one rat from each group were sacrificed and the prostates were removed for model evaluation. Multifocal inflammatory cell infiltration, glandular atrophy, interstitial edema, and decreased luminal secretion in prostates were observed in the CNP group and EA group, while no significant pathological change was observed in the control group. These results showed that the model was successfully established (supplement Figure 1).

Figure 1. Effects of EA on mechanical withdrawal threshold in CNP rats.

Figure 1

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Control group: treated with sham operation and hydrogenated soybean oil; model group: treated with surgical castration and 17-β estradiol; EA group: treated with surgical castration, 17-β estradiol and EA for eight days. CNP: chronic nonbacterial prostatitis; EA: electroacupuncture. Data are shown as the mean ± standard deviation (repeated-measures analysis of variance and Sidak test), n = 8 rats/group. aP < 0.05 compared with the control group; bP < 0.01 compared with the control group; cP < 0.05 compared with the model group; dP < 0.01 compared with the model group.

3.2. EA regulated the prostate index

The prostates were weighed on Day 38. The prostate index was calculated by the following equation:27

Prostate index = weight of prostate (g)/body weight (g) × 1000.

In contrast with the control group, the prostate weight was significantly decreased in the model group (P < 0.01). After EA treatment, the prostate weight was decreased compared with the model group (P < 0.05). Furthermore, rats in the model group had a lower prostate index than that of the rats in the control group (P < 0.01), while rats in the EA group had a lower prostate index than that of the rats in the model group (P < 0.01) (Table 1).

Table 1.

Prostate weight and prostate index of CNP rats in each group ($\bar{x}±s$)

Group n Prostate weight (g) Prostate index
Control 8 1.14±0.10 2.36±0.22
Model 8 0.61±0.07a 1.71±0.28a
EA 8 0.45±0.07b 1.28±0.32c
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Notes: control: treated with sham operation and hydrogenated soybean oil; model: treated with surgical castration and 17-β estradiol; EA: treated with surgical castration, 17-β estradiol and EA for eight days. CNP: chronic nonbacterial prostatitis; EA: electroacupuncture. Data are shown as the mean ± standard deviation (one-way analysis of variance and post hoc Tukey’s test). aP < 0.01, compared with the control group; bP < 0.05, compared with the model group; cP < 0.01, compared with the model group.

3.3. EA reduced mechanical allodynia in CNP rats

To detect the effect of EA on pelvic pain, we evaluated the mechanical tactile sensitivity of the rats on Days 0, 8, 15, 22, 30, 32, 34, 36, and 38. As shown in Figure 1, rats in the model group had a significantly reduced mechanical withdrawal threshold compared with that of the control group from Day 8 (P < 0.05) to Day 38 (P < 0.01). Compared with the model group, EA increased the mechanical withdrawal threshold from Day 34 (P < 0.05) to Day 38 (P < 0.01).

3.4. EA alleviated histological inflammation of the prostate

To investigate the morphology of the prostate, HE staining was performed to observe histological changes in different groups. The results showed that a normal appearance of the glandular epithelium and stroma with little inflammatory cell infiltration was present in the prostate of rats from the control group, which was consistent with previous studies28 (Figure 2A). In contrast, glandular atrophy, decreased luminal secretion, severe congestion and edema of the prostatic stroma, destruction of glandular epithelial tissue, and diffuse inflammatory cell infiltration within the prostate were observed in the model group (Figure 2B). However, we found that the histomorphology lesion of prostates was repaired after EA treatment. Mild congestion of the prostatic stroma and slight invasion of inflammatory cells in local tissue were observed in the EA group, while there was still edema in the prostatic stroma (Figure 2C).

Figure 2. Representative histological images of prostates from each group.

Figure 2

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A-C: Prostate sections from all groups were stained with HE for morphological changes (× 100). A1-C1: selection part of images of A-C (× 200). A, A1: Control group; B, B1: Model group; C, C1: EA group. Control group: treated with sham operation and hydrogenated soybean oil; model group: treated with surgical castration and 17-β estradiol; EA group: treated with surgical castration, 17-β estradiol and EA for eight days. EA: electroacupuncture. The red arrow points to the blood vessel (normal structure); the black arrow points to the infiltration of inflammatory cells; the blue arrow points to interstitial edema; and the green arrow points to congestion in the prostates.

3.5. EA decreased inflammatory cytokine levels

We detected the levels of inflammatory cytokines in prostate tissue homogenates by ELISAs. The concen-trations of IL-1β, IL-6, and TNF-α in the prostates from the model group were remarkably increased in comparison with that in the control group (P < 0.01). In contrast to the model group, EA decreased the expression of IL-1β, IL-6 (P < 0.01), and TNF-α (P < 0.05) (Table 2).

Table 2.

Level of inflammatory factors in the prostates (pg/mL, $\bar{x}±s$)

Group n IL-1β IL-6 TNF-α
Control 8 253.4±25.5 15.7±7.5 6.6±1.2
Model 8 504.2±29.3a 64.9±5.6a 34.8±8.6a
EA 8 396.6±28.8b 33.8±3.6b 19.7±3.6c
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Notes: control: treated with sham operation and hydrogenated soybean oil; model: treated with surgical castration and 17-β estradiol; EA: treated with surgical castration, 17-β estradiol and EA for eight days. EA: electroacupuncture; IL-1β: interleukin-1 beta; IL-6: interleukin-6; TNF-α: tumor necrosis factor-alpha. Data are shown as the mean ± standard deviation (one-way analysis of variance and post hoc Tukey’s test). aP < 0.01, compared with the control group; bP < 0.01, compared with the model group; cP < 0.05, compared with the model group.

3.6. Effects of EA on the TLR4/NF-κB signaling pathway

To understand the underlying anti‐inflammatory mechanism of EA on CNP, the expression levels of critical proteins involved in the TLR4/ NF-κB signaling pathway were evaluated by Western blotting, including TLR4, MyD88, IκBα, p-IκBα, NF-κB p65, and p-NF-κB p65. Compared with the control group, the expression of TLR4 and MyD88 was significantly increased in the model group and EA group (P < 0.01). While the expression of TLR4 and MyD88 was lower in the EA group than that of the model group (P < 0.05) (Figure 3A, 3B). To evaluate the degree of activation of IκBα and NF-κB p65, the expression ratio of p-IκBα/IκBα and the ratio of p-NF-κB p65/NF-κB p65 were analyzed. The ratio of p-IκBα/IκBα was significantly increased in the model group when compared with that of the control group (P < 0.01), while the ratio of p-IκBα/IκBα was decreased in the EA group in comparison with that of the model group (P < 0.05) (Figure 3C). Similarly, the ratio of p-NF-κB p65/NF-κB p65 was also higher in the model group than that of the control group (P < 0.05), while EA reversed this elevation (P < 0.05) (Figure 3D).

Figure 3. TLR4, MyD88, IκB-α, and NF-κB expression in prostates from each group.

Figure 3

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A: Western blotting bands for TLR4; B: Western blotting bands for MyD88; C: Western blotting bands for IκB-α and p-IκBα; D: Western blotting bands for NF-κB p65 and p-p65. Control: treated with sham operation and hydrogenated soybean oil; model: treated with surgical castration and 17-β estradiol; EA: treated with surgical castration, 17-β estradiol and EA for eight days. EA: electroacupuncture; TLR4: toll-like receptor 4; MyD88: myeloid differentiation factor 88; IκBα: inhibitors of kappa‐B alpha; NF-κB: nuclear factor-kappa B. Data are shown as the mean ± standard deviation (one-way analysis of variance and post hoc Tukey’s test), n = 8 rats/group. aP < 0.01, compared with the control group; bP < 0.05, compared with the model group.

3.7. EA decreased TLR4 expression and nuclear transaction of NF-κB p65

The representative photographs of TLR4 immun-ofluorescence showed that TLR4 was mainly expressed at the prostatic interstitium in the model group, while TLR4 was expressed at the prostatic epithelium in the control group (Figure 4A). The mean fluorescence intensity of TLR4 was higher in the model group than that of the control group (P < 0.01). In contrast with the model group, the mean fluorescence intensity of TLR4 was decreased after EA treatment, which was consistent with the Western blotting results (P < 0.05) (Figure 4B). To further determine whether NF-κB was inhibited by EA, the localization of NF-κB p65 was observed with immunofluorescence staining. The results showed that NF-κB p65 primarily existed in the cytoplasm in the control group, while mostly transferred to the cell nucleus and colocalized with the nucleus in the model group. However, the nuclear translocation of NF-κB p65 was reduced in the EA group (Figure 4C).

Figure 4. Level of TLR4 expression and nuclear translocation of NF-κB.

Figure 4

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A: prostate sections were stained with immunofluorescent staining of TLR4 (× 200). A1-A3: control group, A4-A6: model group, A7-A9: EA group. A1, A4, A7: nuclei were stained with DAPI in blue; A2, A5. A8: TLR4 were stained in red; A3, A6, A9: immunofluorescent staining of TLR4 (red) with the DAPI (blue). B: the bar graph shows the quantitative analysis of the mean fluorescence intensity of TLR4. aP < 0.01 compared with the control group; bP < 0.05, compared with the model group. C: prostate sections were stained with immunofluorescent staining of NF-κB (× 200). C1-C4: control group, C5-C8: model group, C9-C12: EA group. C1, C5, C9: nuclei were stained with DAPI in blue; C2, C6. C10: p65 were stained in green; C3, C7, C11: immunofluorescent staining of NF-κB p65 (green) with the DAPI (blue); C4, C8. C12: Selection part of images of C3, C7, C11 (× 400). The arrow indicates the nuclear translocation of NF-κB p65. EA: electroacupuncture; TLR4: toll-like receptor 4; DAPI: 4',6-Diamidino-2-Phenylindole, Dihydrochloride; NF-κB: nuclear factor-kappa B. Data are shown as the mean ± standard deviation (one-way analysis of variance and post hoc Tukey’s test), n = 8 rats/group.

4. DISCUSSION

CNP is a common disease among men around the world, and it leads to substantial psychological and economic burdens among patients.29 Since the etiology and pathogenesis of nonbacterial prostatitis remain unclear, the current treatments are still mainly symptomatic treatments, such as treatments to relieve pain and discomfort and improve urination symptoms and quality of life.11 However, unsatisfactory efficacy and side effects, such as hypotension, gastrointestinal dysfunction, and decreased libido, greatly burden CNP patients.13 Thus, safe, economical, and effective treatments are needed. In recent years, EA has been gradually accepted by physicians and patients around the world. Studies have shown that EA decreases the expression of inflammatory cytokines in expressed prostatic secretions from patients with CNP and results in a significant improvement in symptoms.30 Therefore, EA is a potential treatment for CNP.

Zhongji (CV3), Guanyuan (CV4), and Dahe (KI12) are acupoint prescriptions for CP/CPPS based on our long-term clinical practice.22 And these acupoints are also generally applied in CP/CPPS. A recent Meta-analysis revealed that Zhongji (CV3) and Guanyuan (CV4) are the most frequently chosen acupoints for CP/CPPS.31 According to the TCM theory, the etiology of CP/CPPS mainly includes deficiency of kidney and damp-heat retention. The therapeutic principle is to tonify the kidney and dredge the waterway. Zhongji (CV3) is the front-mu acupoint of the small intestine, and Guanyuan (CV4) is the front-mu acupoint of the bladder, while the small intestine and bladder are in charge of the water metabolism in the body. Also, both Zhongji (CV3) and Guanyuan (CV4) belong to the conception vessel and have the effect of warming yang to promote diuresis and promoting Qi and blood circulation in the lower energizer. In addition, bilateral Dahe (KI12) were used to strengthen the local stimulation and promote blood circulation. Dahe (KI12) belongs to the kidney meridian and can tonify the kidney and dredge the bladder, as the kidney and bladder are in charge of the prostate function in TCM. Besides, all these acupoints are located in the lower abdomen near the prostate. According to the selection principle of local and adjacent acupoints, Zhongji (CV3), Guanyuan (CV4), and Dahe (KI12) were used for the treatment of CP/CPPS. According to the theory of Western Medicine, all these acupoints are located in the lower abdomen on the same spinal segmental nerve area as the reproductive system.32 It is believed that these acupoints could improve tissue healing and relieve local pain through axon reflexes, dichotomizing nerve fibers, and local endorphins.33

Estradiol-induced prostatitis is a commonly used rodent model for studying the histological and pathological features of CNP,33,34 as the pathological changes in this model are similar to those seen in human CP.24 In addition, the success rate of the model establishment is high, while the cost is low. Moreover, the functions of the bladder and other pelvic organs are not influenced in this model.35 Thus, we applied an estradiol-induced CNP rat model in this study.

The estradiol-induced CNP rats were characterized by body weight loss and decreased prostate weight. The results of this study showed that our CNP model rats fit these characteristics. We found that EA could decrease the prostate weight and prostate index, which indicates that EA may benefit the prostate to some extent. Chronic pelvic pain is one of the common symptoms of CNP. Chronic pain is often persistent and poorly treated, which is a crucial point of focus in acupuncture analgesia.36 This study indicated the effect of EA on alleviating chronic pelvic pain in CNP rats. It is known that EA could play an analgesic role in various ways. Studies showed that chronic inflammation is associated with the pain symptoms in patients in whom CP/CPPS have been identified.37 Given that EA alleviated the chronic inflammation in CNP rats, we supposed that the analgesic effects of EA occur as a result of the anti-inflammatory effects.

The main pathological features of estradiol-induced CNP rats are gland atrophy and inflammatory infiltration.38 In this study, we successfully established a rat model of CNP with diffuse inflammatory cell infiltration, glandular atrophy, and severe congestion and edema of the prostatic stroma. The results also showed that EA significantly reduced the extent and range of inflammatory cell infiltration and congestion. However, the structural alterations of gland atrophy did not seem to change after EA treatment. This result suggested that EA may not reverse the structural changes that have taken place. However, we plan to explore whether EA pre-treatment could restore gland atrophy in CNP rats.

To further observe the anti-inflammatory effect of EA, the levels of inflammatory cytokines were evaluated. IL-1β, IL-6, and TNF-α are multifunctional inflammatory cytokines that, as crucial mediators in the inflammatory response, could result in the recruitment of immune cells and the promotion of other inflammatory factors to aggravate inflammation. All these inflammatory factors contribute to the inflammation of prostates.39 A previous study demonstrated that EA decreased the level of TNF-α in the prostates of CNP rats.40 Our results were consistent with those of that study and further showed the same effect on IL-1β and IL-6. To further clarify the mechanism underlying this effect, we observed the effect of EA on inflammatory pathways.

A previous study showed that EA modulated the inflammatory reaction in colitis by suppressing the TLR4/NF-κB signaling pathway.41 Another study reported that EA decreased joint symptoms and eased paw tissue inflammation in adjuvant arthritis rats by inhibiting the expression of TLR4, MyD88, and NF-κB.42 However, few studies have focused on the role of the TLR4 signaling pathway in CNP, and whether there is a relationship between the TLR4 signaling pathway and the anti-inflammation effect of EA remains unclear. Thus, we investigated the underlying mechanism of the anti-inflammatory effect of EA in this study. First, we observed that the expression of TLR4 was markedly enhanced in CNP rats, which is in agreement with the previous studies.43 Furthermore, we further found the up-regulated expressions of other molecules involved in the TLR4/NF-κB signaling pathway, including MyD88, phosphorylated iκB-α, and NF-κB p65. The current study revealed that EA treatment decreased the expression levels of TLR4 and MyD88 and inhibited IκBα and NF-κB p65 phosphorylation in CNP rats. With an in-depth study of the mechanism of acupuncture therapy, it was determined that the inhibition of NF-κB plays an important role in EA for inflammatory diseases.44 Since the activation of NF-κB was verified by its entry into the nucleus, we observed the location of NF-κB p65 by immunofluorescence staining to further determine whether NF-κB was inhibited by EA. The results showed that nuclear translocation of NF-κB p65 was obvious in the prostates of CNP rats. However, EA treatment prevented NF‐κB p65 from entering the nucleus, which indicated that the NF-κB signaling pathway was inhibited by EA treatment. Taken together, these data indicate that regulating the TLR4/NF-κB pathway may take part in the effect of EA against inflammation in CNP.

Previous studies have confirmed the clinical and anti-inflammatory effects of EA on CP/CPPS. However, few studies have focused on the mechanism underlying the anti-inflammatory effects. In this study, we observed the effect of EA on the TLR4 signaling pathway, an inflammatory signaling pathway, on CNP rats and elucidated the possible mechanism underlying the anti-inflammatory effect, which has rarely been studied before. The results of this study would provide a certain scientific basis for the clinical application of EA on CP/CPPS. However, we provide evidence of an association, not causality, since we did not directly verify the relationship between the anti-inflammation effect of EA and the TLR4 pathway. The exact mechanism of the inhibition of the TLR4 pathway mediated by EA needs to be further clarified. Moreover, it is not clear whether EA could further influence adaptive immunity in CNP, since other molecules or pathways may also involve in the effect of EA. Besides, as the main symptoms of CNP, other behavioral indicators, such as reduced urine output or drinking consumption, and biochemical indicators should be investigated in the future study to further verify the anti-inflammatory effect of EA.

In summary, our findings indicates the effect of EA on relieving chronic pelvic pain, restoring the histomorphology of the prostate, and attenuating inflammation in estradiol-induced CNP rats. The mechanism may be associated with inhibition of the TLR4/MyD88/NF-κB signaling pathway.

5. ACKNOWLEDGEMENTS

We thank ZHANG Guangtao at the Department of Pathology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine for technical support. And we sincerely appreciate other colleagues in the laboratory for their help and effort.

6. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.com.

Contributor Information

Yan LI, Email: 0001liyan@163.com.

Yuelai CHEN, Email: chenyuelai@163.com.

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