Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 May 13:16:882493.
doi: 10.3389/fnsys.2022.882493. eCollection 2022.

Bladder Oxidative Stress and HMGB1 Release Contribute to PAR4-Mediated Bladder Pain in Mice

Shaojing Ye  1 , Fei Ma  1 , Dlovan F D Mahmood  1 , Katherine L Meyer-Siegler  2 , Lin Leng  3 , Richard Bucala  3 , Pedro L Vera  1   4
Affiliations

Bladder Oxidative Stress and HMGB1 Release Contribute to PAR4-Mediated Bladder Pain in Mice

Shaojing Ye et al. Front Syst Neurosci. .

Abstract

Activation of intravesical PAR4 receptors leads to bladder hyperalgesia (BHA) through release of urothelial macrophage migration inhibitory factor (MIF) and urothelial high mobility group box-1 (HMGB1). MIF deficiency and/or MIF antagonism at the bladder block BHA in mice yet the mechanisms are not clear. Since oxidative stress and ERK phosphorylation are involved in MIF signaling we hypothesized that oxidative stress and/or ERK signaling, activated by MIF release, promote intravesical HMGB1 release to induce BHA. We induced BHA by intravesical PAR4 infusion in female C57BL/6 mice. Mechanical sensitivity was evaluated by measuring abdominal von Frey (VF) 50% thresholds before (baseline) and 24 h post-infusion. Intravesical pre-treatment (10 min infusion prior to PAR4) with N-acetylcysteine amide (NACA; reactive-oxygen species scavenger; 3 mg in 50 μl), FR180204 (selective ERK1/2 inhibitor; 200 μg in 50 μl), ethyl pyruvate (EP; HMGB1 release inhibitor; 600 μg in 50 μl), or diluent controls (50 μl) tested the effects of pre-treatment on PAR4-induced BHA. Intravesical fluid was collected after each treatment and HMGB1 concentration was measured using ELISA. Awake micturition parameters (volume and frequency) were assessed at the end of the experiments. Bladders were collected and examined for histological signs of edema and inflammation. Pre-treatment with PBS followed by PAR4 induced BHA in mice but PBS followed by scrambled peptide did not. Pre-treatment with NACA or EP partially blocked PAR4-induced BHA while FR180204 had no effect. A significant correlation between intravesical HMGB1 levels and 50% VF thresholds was observed. All PAR4 treated groups had increased levels of HMGB1 in the intravesical fluid compared to PBS-Scrambled group although not statistically significant. No significant effects were noted on awake micturition volume, micturition frequency or histological evidence of bladder edema or inflammation. Our results show that intravesical antagonism of bladder reactive-oxygen species accumulation was effective in reducing PAR4-induced bladder pain. The correlation between intravesical levels of HMGB1 and bladder pain indicates that released HMGB1 is pivotal to bladder pain. Thus, modulating events in the MIF signaling cascade triggered by PAR4 activation (including bladder oxidative stress and HMGB1 release) warrant further investigation as possible therapeutic strategies.

Keywords: HMGB1; MIF; PAR4; ROS; bladder pain.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Experimental protocol for induction of bladder hyperalgesia (BHA). Baseline 50% von Frey (VF) threshold was measured before administering intravesical pre-treatments to block protease activated receptor-4 (PAR4)-induced BHA as detailed in the Methods section. NACA, N-acetylcysteine amide; PAR4-AP, PAR4-Activating peptide; VSOP, voided stain on paper.
Figure 2
Figure 2
Effects of different treatments (N = 6/group) on protease activated receptor-4 (PAR4)-induced bladder hyperalgesia (BHA). (A) No difference in 50% von Frey (VF) threshold score from lower abdominal mechanical stimulation using VF monofilaments was observed at baseline across all treatment groups (F = 0. 092; ns). (B) Significant differences were observed in VF threshold 24 h after treatment F = 69.71, p = 1.34−15. Mice in the (PBS)-PAR4 (pain group) showed a profound decrease in VF threshold score when compared to the (PBS)- Scrambled group (No pain group). Pre-treatment with ethyl pyruvate nearly blocked while pre-treatment with N-acetylcysteine amide (NACA) partially blocked PAR4-induced decrease in VF 50% threshold. Pre-treatment with either methyl cellulose (Methcell) or FR180204 (FR) was not effective in blocking PAR4-induced BHA. ***p < 0.001; ns, not significant at p < 0.05.
Figure 3
Figure 3
von Frey (VF) threshold is negatively correlated with intravesical high mobility group box 1 (HMGB1) levels in the intravesical fluid. Scatter plot shows a significant negative correlation between intravesical HMGB1 level (x-axis) and 50% VF threshold score (y-axis) indicating that higher HMGB1 concentrations correlate with greater BHA. Treatment groups are color coded as indicated in the legend.
Figure 4
Figure 4
Representative sections of hematoxylin and eosin (H&E) stained bladder cross-sections from all groups (N = 6/group) with pre-treatment for 10 min followed by bladder protease activated receptor-4 (PAR4) or scrambled peptide infusion for 1 h. Arrows in panels (A,B) show edge of urothelium; lamina propria (LP) and smooth muscle (SM) are also indicated. (A) PBS pre-treatment-scrambled treatment; (B) PBS pre-treatment-PAR4 treatment; (C) N-acetylcysteine amide (NACA) pre-treatment-PAR4 treatment; (D) Methylcellulose (Methcell; solvent) pre-treatment-PAR4 treatment; (E) FR 180204 (FR) pre-treatment-PAR4 treatment; (F) Ethyl pyruvate (EP) pre-treatment-PAR4 treatment. No significant changes in edema (F = 1.151, ns) or inflammation (F = 0.994; ns) scores were noted in all groups when compared to control (A, Table 3; ns, not significant at p < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Agalave N. M., Svensson C. I. (2014). Extracellular high-mobility group box 1 protein (hmgb1) as a mediator of persistent pain. Mol. Med. 20, 569–578. 10.2119/molmed.2014.00176 - DOI - PMC - PubMed
    1. Akiyama Y., Hanno P. (2019). Phenotyping of interstitial cystitis/bladder pain syndrome. Int. J. Urol. 26(Suppl 1), 17–19. 10.1111/iju.13969 - DOI - PubMed
    1. Alam A., Haldar S., Thulasiram H. V., Kumar R., Goyal M., Iqbal M. S., et al. . (2012). Novel anti-inflammatory activity of epoxyazadiradione against macrophage migration inhibitory factor: inhibition of tautomerase and proinflammatory activities of macrophage migration inhibitory factor. J. Biol. Chem. 287, 24844–24861. 10.1074/jbc.M112.341321 - DOI - PMC - PubMed
    1. Alexander J. K., Cox G. M., Tian J.-B., Zha A. M., Wei P., Kigerl K. A., et al. . (2012). Macrophage migration inhibitory factor (MIF) is essential for inflammatory and neuropathic pain and enhances pain in response to stress. Exp. Neurol. 236, 351–362. 10.1016/j.expneurol.2012.04.018 - DOI - PMC - PubMed
    1. Berry S. H., Elliott M. N., Suttorp M., Bogart L. M., Stoto M. A., Eggers P., et al. . (2011). Prevalence of symptoms of bladder pain syndrome/interstitial cystitis among adult females in the united states. J. Urol. 186, 540–544. 10.1016/j.juro.2011.03.132 - DOI - PMC - PubMed