Juglone alleviates pelvic pain and prostatic inflammation via inhibiting the activation of NLRP3 inflammasome and alleviating oxidative stress in EAP mice

Abstract

Background

Juglone, a naphthoquinone compound that occurs naturally, is present predominantly in the fruits, leaves, and roots of walnut plants. Although its antioxidant and anti-inflammatory effects have been demonstrated in various diseases, its therapeutic potential remains unexplored in patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS).

Purpose

Our objective was to investigate the therapeutic effectiveness of juglone in treating CP/CPPS and elucidate the potential mechanism involved.

Methods

To establish experimental autoimmune prostatitis (EAP) mouse models and macrophage pyroptosis models, the therapeutic impact of juglone on CP/CPPS was evaluated. Molecular docking analysis, a cellular thermal shift assay (CETSA), and consultation with the Human Protein Atlas database were conducted to further explore the target molecules involved in juglone treatment for CP/CPPS. In addition, we utilized immunohistochemistry, immunofluorescence, Western blotting, and flow cytometry to assess macrophage pyroptosis and related pathway protein expressions. The evaluation of oxidative stress (OxS) was conducted through malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) assays. BzATP, an agonist of the NLRP3 pyroptosis pathway, was utilized for recovery experiments both in vitro and in vivo.

Results

Administration of juglone to EAP model mice ameliorated prostatic inflammation, reduced pain symptoms, and decreased proinflammatory cytokine levels. Molecular docking analysis and CETSA, in conjunction with data from the Human Protein Atlas database, indicated that NLRP3, caspase-1, and GSDMD, along with their effects on macrophage pyroptosis, may serve as key targets for the effects of juglone. Furthermore, juglone inhibited the expression of these proteins. Assays of OxS demonstrated that the administration of juglone mitigated OxS in both animal and cellular experiments. These results were reversed with BzATP treatment.

Conclusion

In conclusion, juglone can alleviate EAP by suppressing the pyroptosis of macrophages mediated by NLRP3/GSDMD and alleviating OxS; therefore, juglone has the potential as a therapeutic for CP/CPPS. Furthermore, our studies confirmed that juglone can bind stably to NLRP3, caspase-1, and GSDMD. These findings validate the mechanism of action of juglone and offer valuable insights for the treatment of other diseases mediated by these proteins, such as inflammatory bowel disease, nonalcoholic steatohepatitis, and multiple sclerosis.