Sodium hydrosulfide alleviates osteoporosis by suppressing osteoclastogenesis via the inhibition of the NF-κB signaling

Sodium hydrosulfide alleviates osteoporosis by suppressing osteoclastogenesis via the inhibition of the NF-κB signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sodium hydrosulfide alleviates osteoporosis by suppressing osteoclastogenesis via the inhibition of the NF-κB signaling Wenqin Tong, Wenchao Fei, Ke Xu, Yang Hong, Yinghua Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2599716/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background: While current pharmacotherapies for osteoporosis (OP) such as bisphosphonates reduce fracture risk, their long-term use is limited by adverse effects including gastrointestinal toxicity, osteonecrosis of the jaw, and atypical femoral fractures. Hydrogen sulfide (H₂S), an endogenous gasotransmitter with anti-inflammatory properties, emerges as a potential alternative, but the role of its donor sodium hydrosulfide (NaHS) in osteoporosis remains unexplored. This study investigates NaHS-driven bone-protective mechanisms, emphasizing its ability to modulate the NF-κB/IκB-α axis—a pathway critical in osteoclastogenesis. Methods: Sham controls (n=6) and Ovariectomized (OVX) mice (osteoporosis model; 4 groups, n=6) received NaHS (0, 0.38, 0.75, 1.5 mg/kg/day) or vehicle for 4 weeks. Bone mineral density (BMD) was quantified by Micro-CT. Osteoclast differentiation was assessed in bone marrow-derived macrophages (BMMs) and RAW264.7 cells via TRAP staining. Molecular mechanisms were deciphered using ubiquitination assays, nuclear/cytoplasmic fractionation, and immunofluorescence. Results: NaHS treatment increased lumbar BMD and trabecular bone volume/tissue volume in OVX mice compared to untreated controls. In vitro, NaHS (0.15mM) suppressed RANKL-induced osteoclastogenesis (p<0.05). Mechanistically, NaHS was shown for the first time to stabilize IκB-α by inhibiting its ubiquitination, thereby blocking NF-κB p65 nuclear translocation. Conclusions: NaHS mitigates OVX-induced bone loss via dual inhibition of osteoclast differentiation (through IκB-α/NF-κB blockade) and bone resorption. Its capacity to circumvent the limitations of current therapies positions NaHS as a novel candidate for osteoporosis management. NaHS Osteoporosis Osteoclastogenesis NF-κB IkB-α Potential Therapeutics Full Text Additional Declarations The authors declare no competing interests. Supplementary Files supportingfig1.tif Supporting Figure.1. Effects of NaHS on the apoptosis of BMM cells. BMMs cells treated with a range of NaHS concentrations for 48 hours were stained with Annexin V-FITC and PI (A, B) used to determine the percentage of apoptotic cells(Q2+Q3). And the levels of indicated proteins were determined(C). All experiments were performed at least three times. *P<0.05, **P<0.01, ***P<0.005, compared with the control group. supportingfig2.tif Supporting Figure.2. NaHS is not toxic to the liver or kidney in small doses. (A) H&E (magnification ×40 and ×100) staining, respectively, of the liver. (B) H&E (magnification ×40 and ×100) staining, respectively, of the kidney. Scale bar =100 μm. supportingfig3.tif Supporting Figure.3. NaHS effects on RANKL-induced MAPK signaling pathway. RAW264.7 cells were pretreated with 0.6 μM of ORI for 2 h before the addition of RANKL, and the levels of indicated proteins were determined. All experiments were performed at least three times. *P<0.05, **P<0.01, ***P<0.005, compared with RANKL-treated experiments. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2599716","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":178770745,"identity":"f78dae8f-c9e3-4a87-a693-80c7ed2a224b","order_by":0,"name":"Wenqin Tong","email":"","orcid":"","institution":"Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Wenqin","middleName":"","lastName":"Tong","suffix":""},{"id":178770744,"identity":"665d3de3-60d0-4aef-a422-dfd9cd77bcab","order_by":1,"name":"Wenchao Fei","email":"","orcid":"","institution":"Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Wenchao","middleName":"","lastName":"Fei","suffix":""},{"id":178770746,"identity":"1371514a-a405-4bef-a2cd-d199ac754b79","order_by":2,"name":"Ke Xu","email":"","orcid":"","institution":"Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Xu","suffix":""},{"id":178770748,"identity":"0a370398-c539-485f-b727-58c449cc2a60","order_by":3,"name":"Yang Hong","email":"","orcid":"","institution":"Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Hong","suffix":""},{"id":178770749,"identity":"c2d018f0-df8a-4c2e-b6c2-f4a5e3dac116","order_by":4,"name":"Yinghua Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACCQbGA4kNDAz8zAeAbBA4QFgLA1iLZFsCKVoYgVoMjhGrRX52j8GBhzvs8oyP8Rje+LmDQY7vRgLj5wI8WhjnnDE4kHgmudjsGI+xZe8ZBmPJGwnM0jPwaGGWyAFqaWNO3Ha/x0yCt40hccONBDZmHjxa2CBa6hM3t/GYSf5tY6gnqIUHouVw4gY2HjNpoC0JBoS0SEikFQC1HE+ccYyt2Fq2TcJw5pmHzdL4tMjPSN748GdbdWJ/G/PGm2/bbOT5jicf/IxPC4atQAyKplEwCkbBKBgFFAEAIQVNsCsRjm4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2058-1191","institution":"Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Yinghua","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-02-17 16:14:02","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-2599716/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2599716/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89427067,"identity":"33b0b0d6-4e1b-4e92-95ff-0983dadc0eb9","added_by":"auto","created_at":"2025-08-19 20:41:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1773369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2599716/v2_covered_950785a8-b1ac-47e9-8ef6-4efab772181a.pdf"},{"id":89425177,"identity":"424ba63d-63af-4407-812f-374bb2f03188","added_by":"auto","created_at":"2025-08-19 20:17:43","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":267142,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Figure.1. Effects of NaHS on the apoptosis of BMM cells. BMMs cells treated with a range of NaHS concentrations for 48 hours were stained with Annexin V-FITC and PI (A, B) used to determine the percentage of apoptotic cells(Q2+Q3). And the levels of indicated proteins were determined(C). All experiments were performed at least three times. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.005, compared with the control group.\u003c/p\u003e","description":"","filename":"supportingfig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-2599716/v2/dc23b2c098b82ba072060bd2.tif"},{"id":89425179,"identity":"093589d6-b90f-4796-9a3f-c041b07e8c37","added_by":"auto","created_at":"2025-08-19 20:17:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3058052,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Figure.2. NaHS is not toxic to the liver or kidney in small doses. (A) H\u0026amp;E (magnification ×40 and ×100) staining, respectively, of the liver. (B) H\u0026amp;E (magnification ×40 and ×100) staining, respectively, of the kidney. Scale bar =100 μm.\u003c/p\u003e","description":"","filename":"supportingfig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-2599716/v2/53598803194e01a8eff72962.tif"},{"id":89426351,"identity":"34bd07ce-2252-4b2f-b857-8c9ba0a8b7fc","added_by":"auto","created_at":"2025-08-19 20:25:43","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":190996,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Figure.3. NaHS effects on RANKL-induced MAPK signaling pathway. RAW264.7 cells were pretreated with 0.6 μM of ORI for 2 h before the addition of RANKL, and the levels of indicated proteins were determined. All experiments were performed at least three times. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.005, compared with RANKL-treated experiments.\u003c/p\u003e","description":"","filename":"supportingfig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-2599716/v2/78a550fe9179e8b1ed47a9dc.tif"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eSodium hydrosulfide alleviates osteoporosis by suppressing osteoclastogenesis via the inhibition of the NF-κB signaling\u003c/strong\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NaHS, Osteoporosis, Osteoclastogenesis, NF-κB, IkB-α, Potential Therapeutics","lastPublishedDoi":"10.21203/rs.3.rs-2599716/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2599716/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003eWhile current pharmacotherapies for osteoporosis (OP) such as bisphosphonates reduce fracture risk, their long-term use is limited by adverse effects including gastrointestinal toxicity, osteonecrosis of the jaw, and atypical femoral fractures. Hydrogen sulfide (H₂S), an endogenous gasotransmitter with anti-inflammatory properties, emerges as a potential alternative, but the role of its donor sodium hydrosulfide (NaHS) in osteoporosis remains unexplored. This study investigates NaHS-driven bone-protective mechanisms, emphasizing its ability to modulate the NF-κB/IκB-α axis—a pathway critical in osteoclastogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Sham controls (n=6) and Ovariectomized (OVX) mice (osteoporosis model; 4 groups, n=6) received NaHS (0, 0.38, 0.75, 1.5 mg/kg/day) or vehicle for 4 weeks. Bone mineral density (BMD) was quantified by Micro-CT. Osteoclast differentiation was assessed in bone marrow-derived macrophages (BMMs) and RAW264.7 cells via TRAP staining. Molecular mechanisms were deciphered using ubiquitination assays, nuclear/cytoplasmic fractionation, and immunofluorescence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e NaHS treatment increased lumbar BMD and trabecular bone volume/tissue volume in OVX mice compared to untreated controls. In vitro, NaHS (0.15mM) suppressed RANKL-induced osteoclastogenesis (p\u0026lt;0.05). Mechanistically, NaHS was shown for the first time to stabilize IκB-α by inhibiting its ubiquitination, thereby blocking NF-κB p65 nuclear translocation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e NaHS mitigates OVX-induced bone loss via dual inhibition of osteoclast differentiation (through IκB-α/NF-κB blockade) and bone resorption. Its capacity to circumvent the limitations of current therapies positions NaHS as a novel candidate for osteoporosis management.\u003c/p\u003e","manuscriptTitle":"Sodium hydrosulfide alleviates osteoporosis by suppressing osteoclastogenesis via the inhibition of the NF-κB signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-08-19 20:17:38","doi":"10.21203/rs.3.rs-2599716/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2023-02-24 18:25:11","doi":"10.21203/rs.3.rs-2599716/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0549a56e-3674-48dc-8608-a027640799c6","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-04-04T14:20:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 20:17:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2599716","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2599716","identity":"rs-2599716","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}