Protective effect of astaxanthin on chronic prostatitis/chronic pelvic pain syndrome in rat through modulating NF-κB signaling pathway

Abstract

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common male urological disease characterized by chronic pelvic pain and various discomforts. Astaxanthin (AST) has multiple functions, including anti-inflammatory property, but it is unclear whether AST plays a key role in CP/CPPS and how it works. This study aimed to investigate the protective effect of AST on CP/CPPS in rats and the underlying mechanism.

Methods: A CP/CPPS rat model was induced by intraprostatic injection of carrageenan and the blood specimens and prostates were harvested for further research after oral administration of AST for 4 weeks.

Results: Tactile allodynia test showed that AST ameliorated chronic pelvic pain in a dose-depended manner. In addition, histological evaluation indicated that AST alleviated CP/CPPS rat prostate histological inflammation. Meanwhile, AST suppressed the expression of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Besides, AST inhibited the activities of prostaglandin E2 (PGE2) and cyclooxygenase 2 (COX2). Furthermore, AST decreased the activation of the nuclear factor-κB (NF-κB) signaling pathway.

Conclusions: Our study has shown that AST exerts an anti-inflammatory and protective effect against CP/CPPS and the function is mediated at least through the suppression of NF-κB signaling pathway. These results provide evidence of AST as the potential agents for the treatment of CP/CPPS.

Keywords: Astaxanthin (AST); NF-κB signaling pathway; anti-inflammation; chronic pelvic pain syndrome (CPPS); chronic prostatitis (CP).

Figure 1

Schematic illustration of animal experimental design. Forty-eight SD rats were randomly divided into four groups (normal: normal group; model: chronic prostatitis model group; AST 40 mg: experimental group treated with 40 mg/kg/d AST; AST 80 mg: experimental group treated with 80 mg/kg/d AST). All rats were acclimatized for 1 week before inducing chronic prostatitis with injection of carrageenan. From the secondary day, rats received different treatments for another four weeks. AST, astaxanthin; SD, Sprague-Dawley.

Figure 2

The surgical procedures of chronic prostatitis/chronic pelvic pain syndrome rat model. (A) Anesthetized rat was sterilized in the lower abdomen and cut through a longitudinal median incision with a length of approximately 1 cm. (B) Both prostate ventral lobes under the bladder were exposed. (C) 100 µL 1% carrageenan was injected into prostate gently using a 1-mL syringe. (D) The abdominal cavity was sutured layer by layer with absorbable suture.

Figure 3

AST ameliorated chronic pelvic pain. The positive responses frequencies of pelvic pain of rats in different groups were measured by the von Frey test. The force of the von Frey filaments was 6 g. Data were shown as mean ± SD (n=12). ^#, P<0.05 vs. normal and *, P<0.05 vs. model. AST, astaxanthin; SD, standard deviation.

Figure 4

AST alleviated CP/CPPS rat prostate histological damage. After fixation, prostate tissue specimens were treated by dehydration, clearing, and paraffin wax immersion. The paraffin-embedded specimens were cut into 5 µm sections and then stained with H&E. (A) No abnormal histological changes were observed in the normal group. (B) The model group had different degree of inflammatory cell infiltration in the mesenchyme. (C,D) In AST treated groups, the infiltration of inflammatory cells and vacuoles were decreased in a dose-depended manner. (E) Inflammation scores for each prostate specimens were analyzed. Magnification is 400×. Data were shown as mean ± SD (n=6). *, P<0.05. AST, astaxanthin; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome; H&E, hematoxylin and eosin; SD, standard deviation.

Figure 5

AST showed anti-inflammatory activity by inhibiting inflammatory cytokines expression in prostate tissue. (A-D) A significant enhancement in the levels of IL-1β, IL-6, IL-8 and TNF-α was found in model rats vs. normal rats. While in AST treated groups, AST downregulated inflammatory cytokines including IL-1β, IL-6, IL-8 and TNF-α. Data were shown as mean ± SD (n=6). *, P<0.05. AST, astaxanthin; IL, interleukin; TNF-α, tumor necrosis factor-α; SD, standard deviation.

Figure 6

AST inhibited the expression of PGE2 in CP/CPPS rat prostate tissue and serum. (A) AST decreased the expression of PGE2 in CP/CPPS rat prostate tissue. (B) AST decreased the expression of PGE2 in CP/CPPS rat serum. Data were presented as mean ± SD (n=6). *, P<0.05. AST, astaxanthin; PGE2, prostaglandin E2; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome; SD, standard deviation.

Figure 7

AST decreased the expression of COX2 in prostate tissue. Representative immunohistochemical staining of COX2 in the prostate tissue of different groups of rats. (A) Immunohistochemical staining of COX2 in the normal group. (B) Immunohistochemical staining of COX2 in the model group. (C,D) In AST treated groups, the levels of COX2 were decreased in a dose-depended manner after AST administration. (E) The AOD was measured by ImageJ software. Magnification is 100×. Data were shown as mean ± SD (n=6). *, P<0.05. AST, astaxanthin; COX2, cyclooxygenase 2; AOD, average optical density; SD, standard deviation.

Figure 8

AST suppressed the activation of the NF-κB signaling pathway. (A) AST suppressed the phosphorylation of NF-κB and increased the phosphorylation of I-κBα as shown by Western blotting. (B) Relative density analysis of each band compared with corresponding GAPDH band. Data were shown as mean ± SD (n=3). *, P<0.05. AST, astaxanthin; SD, standard deviation; NF-κB, nuclear factor-κB.

Figure 9

Schematic of proposed mechanisms of anti-inflammatory effect for AST in CP/CPPS rat model. Created with BioRender.com. As depicted, AST might be an efficacious treatment to alleviate pain and inflammation in CP/CPPS, possibly due to decreased activation of NF-κB signaling pathway. AST, astaxanthin; IL, interleukin; TNF-α, tumor necrosis factor-α; COX2, cyclooxygenase 2; NF-κB, nuclear factor-κB; CP/CPPS, chronic prostatitis/chronic pelvic pain syndrome.