Astaxanthin provides antioxidant protection in rats with chronic nonbacterial prostatitis by regulating the MAPK signaling pathway

Introduction

The term “prostatitis” is used to describe a set of syndromes encompassing bacterial infection, genitourinary and/or pelvic pain, and/or lower urinary tract symptoms (1,2). The majority (90–95%) of symptomatic cases are diagnosed as chronic nonbacterial prostatitis (CNP) (3,4). The etiology of CNP remains uncertain, but may involve bacterial infection, oxidative stress, and immune system, neurological, and endocrine system dysfunction (5). CNP has been treated with anti-inflammatory and antimicrobial agents, alpha blockers, and phytotherapy, among other means (6). Due to its heterogeneity and multifactorial etiology, multimodal therapeutic approaches appear to be more effective than single treatments, but optimal clinical efficacy has not been achieved. Thus, effective alternatives for the treatment of CNP need to be identified and applied.

Astaxanthin (AST), a lipid-soluble xanthophyll carotenoid, is a natural extract derived from fungi, bacteria, yeast, microalgae, and other marine and freshwater microorganisms (7). It is ubiquitous in marine environments and present in aquatic species such as salmon, trout, and krill. Natural AST is derived from Haematococcus pluvialis using single- and dual-stage methods (8). Relative to synthetic AST, it significantly reduces oxidative and free-radical stress, protecting biological membranes and fatty acids. The molecular structure of AST consists of polar ionone rings at both ends and a nonpolar zone in the middle, enabling effective free radical scavenging. Due to its chemical structure (Figure 1), AST has the greatest oxygen radical absorbance capacity and about 10-fold more radical inhibitory activity than related antioxidants, such as lycopene and β-carotene (9,10). In addition, AST exhibits anti-apoptotic, anti-inflammatory, and anti-proliferative activities (11,12).

Figure 1 Chemical structure of astaxanthin.

In this study, we explored the antioxidant and anti-inflammatory functions of AST in a rat model of CNP, and the molecular signaling pathways involved. With this work, we seek to provide an innovative perspective to guide the development of dietary immune modulators and the use of AST in the treatment of CNP. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-743/rc).

Methods

Materials

AST (#B25542) was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Olive oil (#O108686) and carrageenan (#C121014) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Anti-p38 antibody (#PAB46195), anti-extracellular signal-regulated kinase (ERK) antibody (#PAB43936), anti-c-Jun N-terminal kinase (JNK) antibody (#PAB47296), and anti-nerve growth factor (NGF) antibody (#MAB48171) were obtained from Wuhan Bioswamp Biotechnology Co., Ltd. (Wuhan, China). Anti-phosphorylated mitogen-activated protein kinase (p-MAPK) family antibody (#9910T) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; #7074) were obtained from Cell Signaling Technology (Beverly, MA, USA). Total superoxide dismutase (T-SOD) detection (#A001-1-2) and glutathione peroxidase (GSH-PX) assay (#A005-1-2) kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Tumor necrosis factor-α (TNF-α; #RA20035), interleukin (IL)-1β (#RA20035), and IL-6 (#RA20035) enzyme-linked immunosorbent assay (ELISA) kits were obtained from Wuhan Bioswamp Biotechnology Co., Ltd.

Animals

Forty-eight 6-week-old male Sprague-Dawley rats (weighing 220–250 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China), and housed in groups of four under standard laboratory conditions (12/12-h light/dark cycle, 24±2 ℃, 50%±5% relative humidity). All animals were reared at Wuhan Myhalic Biotechnology Co., Ltd. (Wuhan, China). The animals were given 1 week to acclimate and had ad libitum access to food and water. Experiments were performed under a project license (No. HLK-20230323-001) granted by the Biomedical Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd., in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Experimental design and treatments

The rats were randomly allocated to normal control (untreated), CNP (intragastric olive oil treatment), 40 mg/kg/day AST, and 80 mg/kg/day AST groups (n=12 each). AST was dissolved in olive oil and administered intragastrically. As described previously, the CNP rat model was induced by intraprostatic λ-carrageenan injection (13). Briefly, after the induction of anesthesia with 3% pentobarbital sodium solution, the prostate was exposed via midline lower-abdominal incision, and 0.1 mL 1% λ-carrageenan saline solution (pure saline solution for normal controls) was injected into its both dorsal lobes. The incision was closed with layered sutures and disinfected. All experimental rats were rehoused individually post-surgery. Beginning 2 days after this procedure, as a therapeutic intervention following disease onset, the AST and olive oil treatments were administered for 4 weeks. Outcome measurements were determined by investigators who were blinded to the treatment assignments and the animal experiments.

Sample collection and prostate index calculation

At the end of the experiment, all rats were anesthetized, weighed, and sacrificed. Blood samples were collected from the hearts, and the prostates were dissected and weighed. A portion of each prostate was fixed in 4% formaldehyde for 1–2 days for histological examination, and the remainder of the prostate was stored at −80 ℃ for subsequent analysis. The prostate index (the ratio of the prostate weight to the total body weight) was calculated as a measure of prostate inflammation.

Measurement of antioxidant enzyme and inflammatory factor levels

T-SOD and GSH-PX assay kits were used according to the manufacturer’s instructions to measure SOD (a lipid peroxidation product and oxidative stress biomarker) and GSH-PX (an antioxidant enzyme) activities, respectively, in serum specimens and prostate supernatants. The prostate tissues were weighed and then collected in a 0.9% sodium chloride solution, maintaining a tissue weight-to-solution ratio of 100 mg per 1 mL. Commercial ELISA kits were used according to the manufacturer’s instructions to determine the TNF-α, IL-1β, and IL-6 concentrations in serum and prostate tissue samples.

Histological examination

The fixed prostate specimens were dehydrated and processed by paraffin wax immersion, then cut into 5-µm-thick sections and stained with hematoxylin and eosin (H&E). They were observed and photographed under a light microscope (E100; Nikon Corporation, Tokyo, Japan) to assess the success of CNP model establishment and the effects of AST treatment on prostate inflammation.

Immunohistochemical staining

As increased NGF expression is associated with intractable pain (14), including CNP-induced chronic pelvic pain, we performed an immunohistochemical analysis to assess NGF expression. The prostate sections were prepared as described above for H&E staining. After dewaxing, soaking, sectioning, and boiling for antigen repair, 3% H₂O₂ was used to inactivate endogenous peroxidase. The sections were then incubated overnight at 4 ℃ with anti-NGF primary antibodies (1:200 dilution). After rinsing with phosphate-buffered saline (PBS), the samples were further incubated with goat anti-rabbit IgG-labeled secondary antibodies for 20 min at 37 ℃. Staining was visualized under a light microscope (E100; Nikon Corporation).

Western blot

Prostate tissues were lysed in lysis buffer and analyzed by western blot as described previously (15). Briefly, total protein was extracted, and samples with ≥40 µg protein were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked by incubation in PBS containing 0.01% Tween-20 (PBST) and 2.5% bovine serum albumin at room temperature for 8 h, followed by incubation with anti-p38, anti-ERK, anti-JNK, and anti-p-MAPK family primary antibodies (all 1:1,000) and anti-β-actin in PBST overnight at 4 ℃. It was then washed four times with PBST and incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000) for 2 h. Autoradiograms were obtained and scanned, and band densities were quantified using Quantity One (Bio-Rad, Hercules, CA, USA). We focused on MAPK phosphorylation because these molecules regulate pro-inflammatory cytokine release during inflammatory responses.

Statistical analysis

The data are presented as mean ± standard deviation. The statistical analyses were performed using SPSS 19.0 (IBM Corporation, Armonk, NY, USA). Student’s t-test was used for between-group comparisons. The significance level was set to P<0.05.

Results

Effects of AST on the prostate weight and index

The body weight did not differ significantly between the CNP group and the AST-treated groups (Figure 2A). The wet prostate weight was significantly greater in the CNP group than in the normal control group (P<0.001; Figure 2B,2C), and the AST treatments significantly reduced this increase (P=0.004 in the 40 mg/kg/day AST group; P<0.001 in the 80 mg/kg/day AST group). The prostate index was significantly higher in the CNP group than in the normal control and AST treatment groups (all P<0.001; Figure 2D).

Figure 2 The effect of AST on the rat body weight, prostate weight, and prostate index. (A) Rat body weight, (B) prostate weight, (C) macroscopic photographs of prostates from the experimental groups, and (D) prostate index were measured. Data are shown as mean ± standard deviation (n=6/group). ^###, P<0.001 vs. normal control. **, P<0.01; ***, P<0.001 vs. CNP group. AST, astaxanthin; CNP, chronic nonbacterial prostatitis.

Effects of AST on prostate inflammation

No inflammatory change was observed in the normal control group (Figure 3A). The CNP group showed pronounced stromal tissue infiltration by inflammatory cells, partial basilar membrane destruction, and prostatic duct dilation or damage (Figure 3B). The AST treatments significantly counteracted these inflammatory changes, inhibiting inflammatory cell infiltration, interstitial hyperemia, and prostate histoarchitecture disruption (Figure 3C,3D).

Figure 3 The effect of AST on the histopathology in rat prostate tissue. (A) Normal control group; (B) CNP group; (C) 40 mg/kg/day AST group; (D) 80 mg/kg/day AST group. H&E staining; 200× (left) and 400× (right) magnification. AST, astaxanthin; CNP, chronic nonbacterial prostatitis; H&E, hematoxylin and eosin.

Effects of AST on antioxidant enzyme activities

Carrageenan stimulation reduced the serum and prostate SOD levels, and the AST treatments abolished this effect (all P<0.001; Figure 4A,4B). Similarly, carrageenan stimulation reduced the levels of GSH-PX in serum and prostate tissue, and the AST treatments dose-dependently increased these levels (all P<0.001; Figure 4C,4D).

Figure 4 AST enhanced the activity of SOD and GSH-PX in prostate tissue and serum. (A,B) T-SOD activity and (C,D) GSH-PX activity in rat prostate tissue and serum. Data are shown as mean ± standard deviation (n=6/group). ^###, P<0.001 vs. normal control group. ***, P<0.001 vs. CNP group. AST, astaxanthin; CNP, chronic nonbacterial prostatitis; GSH-PX, glutathione peroxidase; SOD, superoxide dismutase; T-SOD, total superoxide dismutase.

Effects of AST on prostate and serum inflammatory factor and prostate NGF expression

The expression of TNF-α, IL-1β, and IL-6 in serum and prostate tissue was significantly greater in the CNP group than in the normal control group, and the AST treatments significantly reduced this expression (all P<0.001; Figure 5). Prostate NGF expression was markedly upregulated in the CNP group relative to the normal control group, and the AST treatments significantly decreased this upregulation (Figure 6).

Figure 5 AST enhanced the activity of TNF-α, IL-1β, and IL-6 in prostate tissue and serum. Inflammatory factor levels in rat (A-C) prostate tissue and (D-F) serum. Data are shown as mean ± standard deviation (n=6/group). ^###, P<0.001 vs. normal control group. ***, P<0.001 vs. CNP group. AST, astaxanthin; CNP, chronic nonbacterial prostatitis; IL, interleukin; TNF, tumor necrosis factor.

Figure 6 AST decrease the expression of NGF in prostate tissue. Representative immunohistochemical images of NGF expression in rat prostate tissue. (A) Normal control group; (B) CNP group; (C) 40 mg/kg/day AST group; (D) 80 mg/kg/day AST group. Immunohistochemical staining; 200× (left) and 400× (right) magnification. AST, astaxanthin; CNP, chronic nonbacterial prostatitis; NGF, nerve growth factor.

Effect of AST on MAPK signaling pathway activity

Carrageenan stimulation induced MAPK phosphorylation, and AST administration significantly reversed this effect (Figure 7). Both AST treatments significantly reduced the protein expression of p-ERK (P=0.003 in the 40 mg/kg/day AST group; P<0.001 in the 80 mg/kg/day AST group), p-JNK (P=0.002 in the 40 mg/kg/day AST group; P<0.001 in the 80 mg/kg/day AST group), and p-p38 (P=0.049 in the 40 mg/kg/day AST group; P<0.001 in the 80 mg/kg/day AST group).

Figure 7 AST inhibited the MAPK signaling pathway. (A) Western blots of p38, ERK, and JNK signaling. (B) Band densities relative to β-actin. Data are shown as mean ± standard deviation (n=6/group). ^###, P<0.001 vs. normal control group. *, P<0.05; **, P<0.01; ***, P<0.001 vs. CNP group. AST, astaxanthin; CNP, chronic nonbacterial prostatitis; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; p-, phosphorylated-.

Discussion

Using the rat model of CNP, we previously demonstrated that AST strongly ameliorated the chronic pelvic pain and inflammatory prostate tissue damage induced by carrageenan stimulation (16). We demonstrated that the anti-inflammatory effects of AST depended in part on the inhibition of nuclear factor-κB (NF-κB) signaling and conferred protection against prostatitis (16). The present study further demonstrated that AST has antioxidative effects, potentially making major contributions to the control of inflammation, in the CNP model. AST reduced the augmentation of the prostate weight and index caused by CNP and upregulated SOD and GSH-PX activity, thereby inhibiting TNF-α, IL-1β, and IL-6 production in the rat prostate and serum and having an antioxidant effect. We also demonstrated the regulation of MAPK signaling underlay this effect.

We observed not only increases in the prostate weight and index in the CNP group, reflecting the increased number of inflammatory cells in the prostatic parenchyma that characterizes prostatitis (17), but also significantly increased prostate damage due to the infiltration of these cells. The ability of AST to reduce these effects is in line with the previous findings that AST effectively attenuates oxidative stress and inflammation induced by lipopolysaccharide (LPS) (18) and cigarette smoke (the latter in a sirtuin 1-dependent manner, in vitro and in vivo) (19). IL-6 may be a major target of AST’s anti-inflammatory activities (20). AST has been shown to significantly reduce the LPS-induced elevation of IL-6 and IL-1β messenger RNA (mRNA) expression and to inhibit the nuclear translocation of NF-κB p65 (21).

Antioxidants alleviate oxidative stress, the imbalance between reactive oxygen species (ROS) production and elimination that characterizes CNP (22). SOD and GSH-PX convert superoxide into hydrogen peroxide and then water, and their activity levels thus reflect antioxidant capacity. GSH-PX also reduces lipid hydroperoxides to alcohols (23). In the prostates of rats with λ-carrageenan-induced CNP, Poria cocos polysaccharides have been shown to regulate several protein levels, including that of SOD, to alleviate oxidative stress and thus CNP (24). Moreover, the SOD product has been shown to restore body weight and SOD level in mice with oxidative stress, and research has verified the oxidative stress-remediation effects of SOD and its therapeutic role against hepatic inflammation (25). In this study, AST treatment similarly restored SOD activity, alleviating oxidative stress, in the prostates of rats with CNP. High cellular glutathione concentrations and GSH-PX activity protect against ROS-induced damage (26). In the present study, AST also increased GSH-PX activity, attenuating oxidative stress, in rats with CNP. Similarly, tadalafil has been found to significantly decrease TNF-α and IL-6 expression and increase GSH expression, alleviating the inflammation and oxidative stress induced by LPS in a human prostate epithelial cell line (27). Quercetin has also been shown to increase GSH-PX expression, contributing to anti-inflammatory and antioxidant effects, in a rat model of chronic prostatitis (28).

The MAPK signaling pathway is involved in the regulation of inflammation and oxidative stress through a phosphatase activation cascade (29,30). TNF-α and ROS activate p38 MAPKs, ERKs, and JNKs, triggering the phosphorylation of signaling molecules such as NF-κB. In in-vivo studies, AST was found to suppress the LPS-induced elevation of inflammatory factor levels via NF-κB activation and MAPK phosphorylation (31), to alleviate neuropathic pain via MAPK and NF-κB pathway inhibition (32), and to protect against sepsis-induced cardiac dysfunction via the inhibition of MAPK and phosphatidylinositol 3-kinase/Akt signaling (33). Recently, it has been reported that AST can alleviate allergic rhinitis by mitigating inflammation and oxidative stress through the HMGB1/TLR4/NF-κB pathway (34), and AST also can regulate oxidative stress and inflammation induced by carrageenan in the brains of mice by enhancing the activity of antioxidant enzymes, inhibiting inflammatory cytokines, and activating the Wnt/β-catenin signaling pathway (35). Therefore, AST might exert anti-inflammatory and antioxidant effects through different mechanisms of action in various tissues. In the present study, AST significantly inhibited the expression of MAPK-related phosphorylated antibodies but had no effect on their total protein levels, indicating that it prevented MAPK activation, and not biosynthesis. Our findings indicate that the MAPK superfamily is involved in carrageenan-induced CNP development in rats, and that AST alleviates prostate injury at least in part through the reduction of MAPK pathway activation. Nonetheless, there are certain limitations in this study as we only validated the function of AST in a carrageenan-induced in vivo model of CNP. In the follow-up investigations, we will construct an in vitro cell model to further corroborate the conclusions of this study.

Conclusions

This study revealed and substantiated a correlation between AST and CNP. AST decreased the augmentation of the prostate weight and index caused by CNP, and increased SOD and GSH-PX activity. It inhibited TNF-α, IL-1β, and IL-6 production in the rat prostate and serum. Furthermore, we demonstrated that the regulation of the MAPK signaling pathway is involved in these processes. In aggregate, the results of this study suggest that AST provides antioxidant protection in a rat model of CNP through MAPK signaling pathway regulation.

Acknowledgments

We would like to thank Professor Yanqin Li (School of Pharmacy, Wuhan University) for her assistance with the animal experiments. We also thank Medjaden Inc. for scientific editing of this manuscript.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-743/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-743/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-743/prf

Funding: This work was supported by the National Scientific Foundation of China (No. 81872852) and the National Scientific Foundation of Hubei Province (Nos. 2020CFB709 and 2023AFC008).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-743/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. HLK-20230323-001) granted by the Biomedical Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd., in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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