{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,8,2]],"date-time":"2025-08-02T19:13:38Z","timestamp":1754162018728,"version":"3.41.2"},"reference-count":41,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2025,7,29]],"date-time":"2025-07-29T00:00:00Z","timestamp":1753747200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>Chronic pain is a distressful condition that impacts strongly on people\u2019s health, and, to date, no cure has been found. However, several strategies against pain have been proposed. Promising data regarding the usage of nonsteroidal anti-inflammatory drugs (NSAIDs) in combination with gabapentin in pain management laid the foundations for more complex approaches. A recently published study proposed a multimodal approach based on ketoprofen lysine salt (KLS) combined with gabapentin (GABA) in the context of chronic pain. Experiments on <jats:italic><jats:italic>in vitro<\/jats:italic><\/jats:italic> models showed supra-additive effects in modulating key pathways involved in neuropathic pain and gastric mucosal damage. Thus, the co-crystallization of ketoprofen, lysine, and gabapentin led to a new ternary drug-drug co-crystal (KLS-GABA co-crystal) to better take advantage of such effects. The new compound showed positive features in <jats:italic><jats:italic>in vitro<\/jats:italic><\/jats:italic> and <jats:italic><jats:italic>in vivo<\/jats:italic><\/jats:italic> pain models, particularly at the gastrointestinal level. To better understand the gastric impact of the co-crystal we chose to analyze proteomic fluctuations that occur in an <jats:italic><jats:italic>in vitro<\/jats:italic><\/jats:italic> model of gastric epithelium upon ethanol injury, aiming to observe the gastric effects of KLS-GABA co-crystal\u2019s administration in comparison with KLS or GABA alone or co-administered as in the multimodal approach. Thus, we performed a 2-dimensional gel electrophoresis (2DE) to compare proteomes from lysates of NCI-N87 cells, chosen as model of gastric epithelium. Among all the localized spots (n\u2009=\u2009117), the differentially abundant ones have been filtered and excised (n\u2009=\u200924) to perform mass spectrometry. A total of 414 non-redundant proteins have been found in the excised spots analyzed. A Gene Ontology-based enrichment analysis identified the proteins involved in biological processes, cellular components, and pathways. We then compared the 2DE findings with the western blot analysis confirming the differential proteomic fluctuations in the model. The methodology described here provides a broader picture of the effects of KLS, GABA, and KLS-GABA co-crystal administration in the ethanol-gastric injury model, identifying processes not revealed by other studies by showing proteomic changes and related mechanisms in detail, particularly via modulation of the oxidative stress-related GSTP1 which suggests the higher gastric tolerability of KLS-GABA co-crystal in the analyzed model highlighting its clinical reliability.<\/jats:p>","DOI":"10.1371\/journal.pone.0328496","type":"journal-article","created":{"date-parts":[[2025,7,29]],"date-time":"2025-07-29T17:34:10Z","timestamp":1753810450000},"page":"e0328496","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Evaluation of the gastrotolerability of ketoprofen, lysine, and gabapentin co-crystal administration in an in vitro model of gastric epithelium: a proteomic update"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9695-6274","authenticated-orcid":true,"given":"Francesco","family":"D\u2019Egidio","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laura","family":"Brandolini","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7005-3218","authenticated-orcid":true,"given":"Vanessa","family":"Castelli","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2114-1019","authenticated-orcid":true,"given":"Massimiliano","family":"Quintiliani","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8390-6614","authenticated-orcid":true,"given":"Andrea","family":"Aramini","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2737-7970","authenticated-orcid":true,"given":"Annamaria","family":"Cimini","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marcello","family":"Allegretti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3693-840X","authenticated-orcid":true,"given":"Michele","family":"d\u2019Angelo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,7,29]]},"reference":[{"issue":"4","key":"pone.0328496.ref001","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1097\/ACO.0b013e32834871df","article-title":"A brief comparison of the pathophysiology of inflammatory versus neuropathic pain","volume":"24","author":"Q Xu","year":"2011","journal-title":"Curr Opin Anaesthesiol"},{"issue":"5","key":"pone.0328496.ref002","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1523\/JNEUROSCI.1650-20.2020","article-title":"The Neuroimmunology of Chronic Pain: From Rodents to Humans","volume":"41","author":"PM Grace","year":"2021","journal-title":"J Neurosci"},{"key":"pone.0328496.ref003","doi-asserted-by":"crossref","first-page":"282","DOI":"10.1016\/j.brainresrev.2011.03.003","article-title":"Cellular and molecular insights into neuropathy-induced pain hypersensitivity for mechanism-based treatment approaches","volume":"67","author":"JV Berger","year":"2011","journal-title":"Brain Res Rev"},{"issue":"16","key":"pone.0328496.ref004","doi-asserted-by":"crossref","first-page":"3533","DOI":"10.3390\/jcm10163533","article-title":"Combination Therapy for Neuropathic Pain: A Review of Recent Evidence","volume":"10","author":"A Serrano Afonso","year":"2021","journal-title":"J Clin Med"},{"key":"pone.0328496.ref005","doi-asserted-by":"crossref","first-page":"2058738419838383","DOI":"10.1177\/2058738419838383","article-title":"The neuropathic pain: An overview of the current treatment and future therapeutic approaches","volume":"33","author":"E Cavalli","year":"2019","journal-title":"Int J Immunopathol Pharmacol"},{"issue":"5","key":"pone.0328496.ref006","doi-asserted-by":"crossref","first-page":"1263","DOI":"10.1097\/00000542-200211000-00033","article-title":"Gabapentin and pregabalin can interact synergistically with naproxen to produce antihyperalgesia","volume":"97","author":"RW Hurley","year":"2002","journal-title":"Anesthesiology"},{"issue":"24","key":"pone.0328496.ref007","doi-asserted-by":"crossref","first-page":"2283","DOI":"10.1016\/j.lfs.2006.07.025","article-title":"Examination of the interaction between peripheral diclofenac and gabapentin on the 5% formalin test in rats","volume":"79","author":"A Picazo","year":"2006","journal-title":"Life Sci"},{"issue":"1","key":"pone.0328496.ref008","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1213\/ANE.0b013e31824e5da3","article-title":"Gabapentin augments the antihyperalgesic effects of diclofenac sodium through spinal action in a rat postoperative pain model","volume":"115","author":"Y Narai","year":"2012","journal-title":"Anesth Analg"},{"key":"pone.0328496.ref009","doi-asserted-by":"crossref","first-page":"114845","DOI":"10.1016\/j.biopha.2023.114845","article-title":"Ketoprofen, lysine and gabapentin co-crystal magnifies synergistic efficacy and tolerability of the constituent drugs: Pre-clinical evidences towards an innovative therapeutic approach for neuroinflammatory pain","volume":"163","author":"A Aramini","year":"2023","journal-title":"Biomed Pharmacother"},{"key":"pone.0328496.ref010","doi-asserted-by":"crossref","first-page":"114147","DOI":"10.1016\/j.bcp.2020.114147","article-title":"Non-steroidal anti-inflammatory drugs (NSAIDs) and organ damage: A current perspective","volume":"180","author":"S Bindu","year":"2020","journal-title":"Biochem Pharmacol"},{"issue":"4","key":"pone.0328496.ref011","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1002\/jcp.24809","article-title":"Gastroprotective effects of L-lysine salification of ketoprofen in ethanol-injured gastric mucosa","volume":"230","author":"A Cimini","year":"2015","journal-title":"J Cell Physiol"},{"issue":"3","key":"pone.0328496.ref012","doi-asserted-by":"crossref","first-page":"2304","DOI":"10.1002\/jcp.26102","article-title":"Differential protein modulation by ketoprofen and ibuprofen underlines different cellular response by gastric epithelium","volume":"233","author":"L Brandolini","year":"2018","journal-title":"J Cell Physiol"},{"issue":"3","key":"pone.0328496.ref013","doi-asserted-by":"crossref","first-page":"750","DOI":"10.1002\/jcp.21358","article-title":"The anti-apoptosis protein, survivin, mediates gastric epithelial cell cytoprotection against ethanol-induced injury via activation of the p34(cdc2) cyclin-dependent kinase","volume":"215","author":"MK Jones","year":"2008","journal-title":"J Cell Physiol"},{"key":"pone.0328496.ref014","first-page":"117","article-title":"The effect of ethyl alcohol on peroxidation processes and activity of antioxidant enzymes in rat\u2019s gastric mucosa","volume":"32","author":"K Lutnicki","year":"1992","journal-title":"Arch Vet Pol"},{"issue":"47","key":"pone.0328496.ref015","doi-asserted-by":"crossref","first-page":"7450","DOI":"10.3748\/wjg.v11.i47.7450","article-title":"Increased susceptibility of ethanol-treated gastric mucosa to naproxen and its inhibition by DA-9601, an Artemisia asiatica extract","volume":"11","author":"TY Oh","year":"2005","journal-title":"World J Gastroenterol"},{"issue":"4","key":"pone.0328496.ref016","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1016\/S1056-8719(99)00038-6","article-title":"A new in vitro model for ethanol-induced gastric mucosal damage","volume":"41","author":"YM Al-Mulla Hummadi","year":"1999","journal-title":"J Pharmacol Toxicol Methods"},{"issue":"20","key":"pone.0328496.ref017","doi-asserted-by":"crossref","first-page":"5383","DOI":"10.1021\/ac025747h","article-title":"Empirical statistical model to estimate the accuracy of peptide identifications made by MS\/MS and database search","volume":"74","author":"A Keller","year":"2002","journal-title":"Anal Chem"},{"issue":"17","key":"pone.0328496.ref018","doi-asserted-by":"crossref","first-page":"4646","DOI":"10.1021\/ac0341261","article-title":"A statistical model for identifying proteins by tandem mass spectrometry","volume":"75","author":"AI Nesvizhskii","year":"2003","journal-title":"Anal Chem"},{"issue":"8","key":"pone.0328496.ref019","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1093\/bioinformatics\/btp101","article-title":"ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks","volume":"25","author":"G Bindea","year":"2009","journal-title":"Bioinformatics"},{"issue":"10","key":"pone.0328496.ref020","doi-asserted-by":"crossref","first-page":"2366","DOI":"10.1038\/nprot.2007.324","article-title":"Integration of biological networks and gene expression data using Cytoscape","volume":"2","author":"MS Cline","year":"2007","journal-title":"Nat Protoc"},{"issue":"5","key":"pone.0328496.ref021","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1038\/s41419-022-04964-w","article-title":"Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions","volume":"13","author":"L Brandolini","year":"2022","journal-title":"Cell Death Dis"},{"key":"pone.0328496.ref022","doi-asserted-by":"crossref","first-page":"112110","DOI":"10.1016\/j.biopha.2021.112110","article-title":"PDIA3: Structure, functions and its potential role in viral infections","volume":"143","author":"F Mahmood","year":"2021","journal-title":"Biomed Pharmacother"},{"issue":"1","key":"pone.0328496.ref023","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1615\/CritRevEukarGeneExpr.v17.i1.30","article-title":"Membrane receptors for vitamin D metabolites","volume":"17","author":"R Khanal","year":"2007","journal-title":"Crit Rev Eukaryot Gene Expr"},{"issue":"2","key":"pone.0328496.ref024","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/j.abb.2009.12.004","article-title":"Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line","volume":"494","author":"S Chichiarelli","year":"2010","journal-title":"Arch Biochem Biophys"},{"key":"pone.0328496.ref025","first-page":"2265","article-title":"Expression of protein disulfide isomerase A3 and its clinicopathological association in gastric cancer","volume":"41","author":"T Shimoda","year":"2019","journal-title":"Oncology Reports"},{"issue":"9","key":"pone.0328496.ref026","doi-asserted-by":"crossref","first-page":"2438","DOI":"10.2337\/db08-0604","article-title":"Increase in endoplasmic reticulum stress-related proteins and genes in adipose tissue of obese, insulin-resistant individuals","volume":"57","author":"G Boden","year":"2008","journal-title":"Diabetes"},{"issue":"6","key":"pone.0328496.ref027","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1083\/jcb.148.6.1123","article-title":"Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum","volume":"148","author":"ES Trombetta","year":"2000","journal-title":"J Cell Biol"},{"issue":"4","key":"pone.0328496.ref028","doi-asserted-by":"crossref","first-page":"539","DOI":"10.2478\/s11658-011-0022-z","article-title":"ERp57\/GRP58: a protein with multiple functions","volume":"16","author":"C Turano","year":"2011","journal-title":"Cell Mol Biol Lett"},{"issue":"1","key":"pone.0328496.ref029","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1002\/j.1460-2075.1989.tb03354.x","article-title":"Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid","volume":"8","author":"A Lefort","year":"1989","journal-title":"EMBO J"},{"issue":"5","key":"pone.0328496.ref030","first-page":"6231","article-title":"Overexpression of calcyphosine is associated with poor prognosis in esophageal squamous cell carcinoma","volume":"14","author":"F Li","year":"2017","journal-title":"Oncol Lett"},{"issue":"6","key":"pone.0328496.ref031","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1097\/01.jnen.0000240475.35414.c3","article-title":"Identification of novel biomarkers in pediatric primitive neuroectodermal tumors and ependymomas by proteome-wide analysis","volume":"66","author":"JM de Bont","year":"2007","journal-title":"J Neuropathol Exp Neurol"},{"issue":"1","key":"pone.0328496.ref032","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1111\/IGC.0b013e3181a9026d","article-title":"Proteomics-based approach identified differentially expressed proteins with potential roles in endometrial carcinoma","volume":"20","author":"Z Li","year":"2010","journal-title":"Int J Gynecol Cancer"},{"issue":"16","key":"pone.0328496.ref033","doi-asserted-by":"crossref","first-page":"2846","DOI":"10.1016\/j.ejca.2006.06.026","article-title":"Expression analysis of stage III serous ovarian adenocarcinoma distinguishes a sub-group of survivors","volume":"42","author":"K Partheen","year":"2006","journal-title":"Eur J Cancer"},{"key":"pone.0328496.ref034","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/j.jprot.2013.04.037","article-title":"Identification of proteomic signatures associated with lung cancer and COPD","volume":"89","author":"MD Pastor","year":"2013","journal-title":"J Proteomics"},{"key":"pone.0328496.ref035","doi-asserted-by":"crossref","first-page":"477","DOI":"10.2147\/OTT.S92226","article-title":"Abnormal expression of calcyphosine is associated with poor prognosis and cell biology function in colorectal cancer","author":"J Xu","year":"2016","journal-title":"OTT"},{"issue":"3","key":"pone.0328496.ref036","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1002\/elps.1150110303","article-title":"Use of two-dimensional gel electrophoresis and autoradiography as a tool in cell biology: the example of the thyroid and the liver","volume":"11","author":"R Lecocq","year":"1990","journal-title":"Electrophoresis"},{"issue":"2","key":"pone.0328496.ref037","doi-asserted-by":"crossref","first-page":"201","DOI":"10.3390\/antiox10020201","article-title":"The Impact of Oxidative Stress in Human Pathology: Focus on Gastrointestinal Disorders","volume":"10","author":"R Vona","year":"2021","journal-title":"Antioxidants (Basel)"},{"key":"pone.0328496.ref038","doi-asserted-by":"crossref","DOI":"10.5772\/23875","article-title":"Oxidative Stress Pathway Driven by Inflammation in Gastric Mucosa","volume-title":"Gastritis and Gastric Cancer - New Insights in Gastroprotection, Diagnosis and Treatments","author":"D Jasna","year":"2011"},{"issue":"4","key":"pone.0328496.ref039","article-title":"Effects of Non-steroidal Anti-inflammatory Drugs (NSAIDs) and Gastroprotective NSAIDs on the Gastrointestinal Tract: A Narrative Review","volume":"15","author":"R Sohail","year":"2023","journal-title":"Cureus"},{"key":"pone.0328496.ref040","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/j.biopha.2018.01.102","article-title":"Efficacy and safety of combined low doses of either diclofenac or celecoxib with gabapentin versus their single high dose in treatment of neuropathic pain in rats","volume":"100","author":"MA Ibrahim","year":"2018","journal-title":"Biomed Pharmacother"},{"key":"pone.0328496.ref041","doi-asserted-by":"crossref","first-page":"818513","DOI":"10.3389\/fcell.2022.818513","article-title":"Remifentanil Promotes PDIA3 Expression by Activating p38MAPK to Inhibit Intestinal Ischemia\/Reperfusion-Induced Oxidative and Endoplasmic Reticulum Stress","volume":"10","author":"J Shen","year":"2022","journal-title":"Front Cell Dev Biol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0328496","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,29]],"date-time":"2025-07-29T17:34:20Z","timestamp":1753810460000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0328496"}},"subtitle":[],"editor":[{"given":"Anisha","family":"DSouza","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2025,7,29]]},"references-count":41,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2025,7,29]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0328496","relation":{},"ISSN":["1932-6203"],"issn-type":[{"type":"electronic","value":"1932-6203"}],"subject":[],"published":{"date-parts":[[2025,7,29]]}}}