(F-230) Therapeutic potential of nanoceria pretreatment in preventing the urological chronic pelvic pain syndrome: Immunomodulation via reactive oxygen species scavenging and SerpinB2 downregulation.
Professor Professor – Graduate Institute of Biomedical Engineering, National Chung Hsing University, TW Taichung, United States
Introduction:: Urological chronic pelvic pain syndrome (UCPPS) manifests as pelvic pain with frequent urination and has a 10% prevalence rate without effective therapy. Nanoceria (cerium oxide nanoparticles [CNPs]) were synthesized in this study to achieve potential long-term pain relief, using a commonly used UCPPS mouse model with cyclophosphamide-induced cystitis. Transcriptome sequencing analysis revealed that serpin family B member 2 (SerpinB2) was the most upregulated marker in mouse bladder, and SerpinB2 was downregulated with CNP pretreatment. The transcriptome sequencing analysis results agreed with quantitative polymerase chain reaction and western blot analysis results for the expression of related mRNAs and proteins. Analysis of Gene Expression Omnibus (GEO) datasets revealed that SerpinB2 was a differentially upregulated gene in human UCPPS. In vitro SerpinB2 knockdown downregulated proinflammatory chemokine expression (chemokine receptor CXCR3 and C-X-C motif chemokine ligand 10) upon treatment with 4-hydroperoxycyclophosphamide. In conclusion, CNP pretreatment may prevent the development of UCPPS, and reactive oxygen species (ROS) scavenging and SerpinB2 downregulation may modulate the immune response in UCPPS.
Materials and Methods:: CNPs were synthesized using a slight modification of a previously described method.22 Briefly, equal volumes of 0.0375 mol/L Ce(NO3)36H2O (99.5%; Alfa Aesar, Ward Hill, MA, USA) and 0.5 mol/L hexamethylenetetramine (99.9%, Alfa Aesar) solutions were mixed and stirred in a temperaturecontrolled environment at approximately 24 ± 2C for 24 h. Then, the solution was centrifuged at 9000 rpm for 30 min to obtain CNP sediments. The sediments were washed twice with deionized water and ethanol (95%; Alfa Aesar) for the SEM measurement samples. The resulting CNPs were resuspended in PBS solution and sterilized in an autoclave at 121C for 15 min for in vitro and in vivo studies.
T24 human urothelial cells (BCRC no. 60062) were cultured in 90% McCoy's 5A Medium with 1.5 mM L-glutamine, 10% (v/v) fetal bovine serum, and 1% (v/v) penicillin/streptomycin. The cells were maintained at 37C in a humidified incubator containing 5% CO2. 52 In the present study, 4-HC was applied in vitro to induce T24 cells to produce oxidative stress and apoptosis. In an aqueous solution, 4-HC can rapidly hydrolyze to 4-hydroxycyclophosphamide, the same intermediate product resulting from the microsomal activation of CYP,53 therefore, 4-HC was used instead of CYP because of the need of CYP for microsomal activation. To assess the effects of CNP treatment in the UCPPS model established in vitro, T24 cells were subdivided into four groups: control, 4-HC, CNP pretreatment, and only CNP groups. The methods used in the water-soluble tetrazolium salt-1 and DCFDA cellular ROS detection assays are provided.
Results, Conclusions, and Discussions:: To the best of our knowledge, this is the first in vitro and in vivo study to investigate the therapeutic potential of CNPs in UCPPS. Our study showed that CNPs are well tolerated, without inducing toxicity in T24 cells and no serious adverse events were observed in vivo mice. Further, CNP usage in a multimodal analgesic strategy may provide analgesic effects without serious risk of side effects, warranting further exploration. The ROS scavenging and downregulation of CXCL10 in the bladder tissue may play an immunomodulatory role in the management of UCPPS, and the downregulation of SerpinB2 may be also associated with decreased CXCL10 expression in the CYP cystitis mouse model pretreated with CNP. Further work is needed to explore the duration of CNP pretreatment for protection against UCPPS development. Our work supports CNP administration as a potential preventative strategy for patients at higher risk for UCPPS.
Acknowledgements (Optional): : The authors are grateful to Miss Ping-Hsuan Chou and Associate Prof. Pei-Fang Su, Department of Statistics, College of Management, National Cheng Kung University, for providing statistical consulting services. WeiChih Lien carried out his thesis research under the auspices of the Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, and National Health Research Institutes.
