Identification of proteomic markers of chronic prostatitis/chronic pelvic pain syndrome treated with melatonin using a tandem mass tag approach

Abstract

Background: Chronic prostatitis (CP)/chronic pelvic pain syndrome is the most common urological disorder in young and middle-aged men. A previous study showed that melatonin attenuates prostate inflammation through Sirt1-dependent suppression of the nonobese diabetic-like receptor thermal protein domain-associated protein 3 inflammasome in mouse models of experimental autoimmune prostatitis (EAP). However, the main differentially expressed proteins (DEPs) in melatonin-treated mice with EAP have not yet been fully identified.

Materials and Methods: Mouse models of EAP were established. The pathological morphology of the prostate tissues was observed using hematoxylin-eosin staining. Chronic pelvic pain sensitivity was assessed using suprapubic allodynia. Inflammation-related cytokines were detected using an enzyme-linked immunosorbent assay. These methods were used to validate the successful establishment of the EAP mouse model. Tandem mass tag proteomics was used to identify the proteomic markers in melatonin-treated EAP mice. Next, we visualized the DEPs using bioinformatic analyses. Finally, we measured the expression of mitochondrial creatine kinase 1 and gap junction β-1, which were identified by the tandem mass tag in all groups, using Western blotting to explore the key proteins involved in the anti-inflammatory effects of melatonin on EAP.

Results: We identified 5910 proteins, with quantitative information available for over 85% of the total. We found 53 DEPs in mice between the EAP and control groups and 22 DEPs between the EAP-Melatonin and EAP groups. Bioinformatic analysis suggested significant alterations in immunosuppression, inflammatory chemotaxis, and energy metabolism signaling in EAP mice treated with melatonin. These alterations were confirmed using Western blotting.

Conclusions: Melatonin effectively relieves CP/chronic pelvic pain syndrome-related symptoms in mice with EAP. Mitochondrial kinases are potential key proteins in the treatment of EAP with melatonin, and these biomarkers may provide direction for studying the molecular mechanisms of melatonin in the treatment of CP.

Keywords

Chronic prostatitis/chronic pelvic pain syndrome / Tandem mass tag / Proteomics / Melatonin

Acknowledgments

We thank the Center for Scientific Research of the First Affiliated Hospital of Anhui Medical University for their valuable assistance with the experiments.

Statement of ethics

All the experimental protocols for animal studies in this study were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study was reviewed and approved by the Animal Care and Use Center of Anhui Medical University, and all relevant animal experiments were performed in accordance with its guidelines (approval number: LLSC20211051).

Conflicts of interest statement

The authors declare no conflicts of interest.

Funding source

This study received funding from the National Natural Science Foundation of China 82170787, 81870519, and 81802827; Scientific Research Foundation of the Institute for Translational Medicine of Anhui Province (2017ZHYX02); Natural Science Foundation of Anhui Province (grant number, 2208085QH239); Natural Science Research in Colleges and Universities of Anhui Province (grant number, 2022AH051133); and Postgraduate Innovation Research and Practice Program of the Anhui Medical University (YJS20230077).

Author contributions

Xiaoling L, WM, Xiao L: Research design, methodology, software operation, preliminary draft writing and modification, result analysis;

RF, JM: Data integration and explanation;

LZ, HD: Data acquisition and analysis, technical guidance;

MZ: Method and writing-reviewing;

CY: Method, analysis and guidance for software operation;

LZ: Writing-reviewing and discussion analysis;

JC, CL: Writing-reviewing and editing.

Data availability

The anonymized and raw data used and/or collected during the current study are available from the corresponding author upon reasonable request.

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