25a12f04e7114ebaca5def9d86062286 ijms-27-08093.pdf 662049cdeff1d06a6e60d7aa4a898f2a094545b1 ijms-27-08093.pdf 4def6f1caedb87bb67edd5df520cf01af86e10882a51bdb32498f0d8ac96c86f ijms-27-08093.pdf Title: Hydrogen-Rich Water Enhances Ibuprofen-Induced Antinociception and Modulates NOX Signaling and the NLRP3 Inflammasome in Chronic Inflammatory Pain Subject: Chronic inflammatory pain is sustained by persistent oxidative stress and inflammatory signaling that promote peripheral and central sensitization. Although nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, remain first-line therapies, their long-term use is limited by dose-dependent adverse effects. Therefore, strategies capable of enhancing NSAID efficacy while maintaining an acceptable safety profile are of considerable therapeutic interest. In this study, we investigated whether hydrogen-rich water (HRW) enhances the antinociceptive effects of ibuprofen in male C57BL/6J mice with complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain. Dose–response studies demonstrated that both HRW and ibuprofen significantly attenuated mechanical allodynia and thermal hyperalgesia. Notably, co-administration of a low dose of HRW with ibuprofen produced significantly greater antiallodynic and antihyperalgesic effects than either treatment alone. Molecular analyses further showed that the combined treatment reduced the paw expression of the oxidative stress marker 4-hydroxynonenal (4-HNE) and the inflammasome component NLRP3, as well as spinal NADPH oxidases (NOX), NOX1 and NOX4 expression, while maintaining the CFA-induced upregulation of heme oxygenase-1 (HO-1) and superoxide dismutase-1 (SOD-1) protein levels. In contrast, spinal NOX2 expression remained unchanged. These findings indicate that the enhanced antinociceptive effect of HRW plus ibuprofen is associated with coordinated modulation of oxidative stress and inflammatory pathways at peripheral and/or spinal levels. Collectively, our results support HRW as a promising adjuvant strategy for improving NSAID-mediated analgesia and provide a strong preclinical rationale for further mechanistic and translational investigation of this combination as a multimodal therapeutic approach for chronic inflammatory pain. Keywords: molecular hydrogen; ibuprofen; NSAIDs; chronic inflammatory pain; oxidative stress; NLRP3 inflammasome; NADPH oxidase Author: Giovanna Schiavoni, Weitao Wang, Sylmara Esther Negrini-Ferrari and Olga Pol Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Fri Sep 11 12:25:14 2026 CEST ModDate: Fri Sep 11 12:28:57 2026 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3897349 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MMFMTW+URWPalladioL-Ital Type 1 Custom yes yes yes 122 0 LRHRCY+VnURWPalladioL Type 1 Custom yes yes yes 123 0 XEQDIS+URWPalladioL-Bold Type 1 Custom yes yes yes 124 0 CHCNYM+URWPalladioL-Roma Type 1 Custom yes yes yes 125 0 PDYJSF+EURM10 Type 1 Builtin yes yes yes 177 0 QMDDXF+CMSY10 Type 1 Builtin yes yes yes 265 0 JHIDKG+PalatinoLinotype TrueType WinAnsi yes yes no 209 0 JHIDKG+PalatinoLinotype TrueType WinAnsi yes yes no 234 0 JHIDKG+PalatinoLinotype TrueType WinAnsi yes yes no 315 0 WNOOLF+PalatinoLinotype-Bold TrueType WinAnsi yes yes yes 375 0 FLCQHN+PalatinoLinotype-Roman TrueType WinAnsi yes yes yes 376 0 ZCAGBZ+PalatinoLinotype-Italic TrueType WinAnsi yes yes yes 377 0 TLAWVL+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 378 0 JHIDKG+PalatinoLinotype TrueType WinAnsi yes yes no 418 0 JHIDKG+PalatinoLinotype TrueType WinAnsi yes yes no 449 0 JHIDKG+PalatinoLinotype TrueType WinAnsi yes yes no 610 0 JHIDKC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 611 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-10-02 04:04:57 CEST RepresentationInformation: ijms-27-08093.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-10-01 05:20:45 CEST Size: 3897349 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 774 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: CFA-Induced Mechanical Allodynia and Thermal Hyperalgesia Item: Title: Dose–Response Effects of HRW and Ibuprofen on CFA-Induced Allodynia and Hyperalgesia Item: Title: Effects of Combined HRW and Ibuprofen Therapy on CFA-Induced Mechanical Allodynia and Thermal Hyperalgesia Item: Title: Impact of Combined HRW and Ibuprofen Therapy on Oxidative Stress and Inflammation in the Paw of CFA-Injected Mice Item: Title: Effects of HRW and Ibuprofen, Administered Alone or in Combination, on NOX Isoform Expression in the SC of CFA-Injected Mice Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Animals Item: Title: Induction of Inflammatory Pain Item: Title: Assessment of Nociceptive Responses Item: Title: Pharmacological Treatment Item: Title: Experimental Design Item: Title: Protein Extraction and Western Blot Analysis Item: Title: Statistical Analysis Item: Title: Conclusions Item: Title: References Info: Title: Hydrogen-Rich Water Enhances Ibuprofen-Induced Antinociception and Modulates NOX Signaling and the NLRP3 Inflammasome in Chronic Inflammatory Pain Author: Giovanna Schiavoni, Weitao Wang, Sylmara Esther Negrini-Ferrari and Olga Pol Subject: Chronic inflammatory pain is sustained by persistent oxidative stress and inflammatory signaling that promote peripheral and central sensitization. Although nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, remain first-line therapies, their long-term use is limited by dose-dependent adverse effects. Therefore, strategies capable of enhancing NSAID efficacy while maintaining an acceptable safety profile are of considerable therapeutic interest. In this study, we investigated whether hydrogen-rich water (HRW) enhances the antinociceptive effects of ibuprofen in male C57BL/6J mice with complete Freund’s adjuvant (CFA)-induced chronic inflammatory pain. Dose–response studies demonstrated that both HRW and ibuprofen significantly attenuated mechanical allodynia and thermal hyperalgesia. Notably, co-administration of a low dose of HRW with ibuprofen produced significantly greater antiallodynic and antihyperalgesic effects than either treatment alone. Molecular analyses further showed that the combined treatment reduced the paw expression of the oxidative stress marker 4-hydroxynonenal (4-HNE) and the inflammasome component NLRP3, as well as spinal NADPH oxidases (NOX), NOX1 and NOX4 expression, while maintaining the CFA-induced upregulation of heme oxygenase-1 (HO-1) and superoxide dismutase-1 (SOD-1) protein levels. In contrast, spinal NOX2 expression remained unchanged. These findings indicate that the enhanced antinociceptive effect of HRW plus ibuprofen is associated with coordinated modulation of oxidative stress and inflammatory pathways at peripheral and/or spinal levels. Collectively, our results support HRW as a promising adjuvant strategy for improving NSAID-mediated analgesia and provide a strong preclinical rationale for further mechanistic and translational investigation of this combination as a multimodal therapeutic approach for chronic inflammatory pain. 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