ad0a837082549ac5ee1786c67d1f9f8d ijms-27-04893-v3.pdf 3ecbaef6c5c2743d808b260593f51bd87ca0cae7 ijms-27-04893-v3.pdf 3703914eb08bf35969606bdf23dd3e21ca45aa795e54e3878e5030d30dfe80b8 ijms-27-04893-v3.pdf Title: Sex Hormone-Binding Globulin Prevents Carbon Tetrachloride-Induced Liver Fibrosis Development Subject: Circulating sex hormone-binding globulin (SHBG) concentrations are lower in individuals with metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), reflecting its potential role in metabolic liver dysfunction. Our prior studies demonstrated that SHBG can attenuate MASLD by limiting hepatic lipid deposition, partly through suppression of lipogenic pathways, in both cellular and animal models. In the present work, we have examined whether SHBG could protect against development of liver fibrosis. For this purpose, in vitro and in vivo studies were performed. In vitro, we used co-cultures of human hepatocellular carcinoma cell line (HepG2) and human hepatic stellate cell line (LX-2) cells transfected using an SHBG expression vector vs. vehicle and treated with transforming growth factor beta 1 (TGF-1). For in vivo studies we used wild-type and human SHBG transgenic mice developing liver fibrosis induced by carbon tetrachloride (CCl4). Our results clearly showed that SHBG overexpression reduced the TGF-1-induced expression in collagen in LX-2 cells. Moreover, SHBG overexpression reduced the CCl4 induced liver fibrosis in both male and female mice. Histological examination revealed that SHBG transgenic mice had reduced NAS score and decreased collagen accumulation, assessed by Sirious Red staining. In addition, human SHBG transgenic mice treated with CCl4 exhibited lower collagen 1A1 (Col1A1) protein levels when compared with wild-type CCl4 treated mice. Mechanistically, SHBG attenuated fibrosis primarily through modulation of the TGF-1/matrix metalloproteinases (MMPs)/tissue inhibitor metalloproteinases 1 (TIMP1) axis, characterized by reduced TGF-1 levels, increased metalloprotease activity, and decreased TIMP1 levels compared with wild-type CCl4 treated mice. Notably, female SHBG transgenic mice exhibited greater protection against fibrosis than males, indicating a sex-dependent effect likely mediated by differences in sex steroid signaling. Taken together, we demonstrate for the first time that SHBG protects against liver fibrosis by promoting collagen degradation via the TGF-1/MMPs/TIMP1 pathway. Further research is needed to elucidate the role of sex steroids in the regulation of MMPs and the observed sexual dimorphism. Keywords: TGF-1; metabolic dysfunction-associated steatohepatitis (MASH); sexual dimorphism collagen; liver; hepatic fibrosis; transgenic mice; metalloproteinases Author: Anna Álvarez-Guaita, Laura Briansó-Llort, Julia Cabrera-Serra, Lidia Fuertes-Rioja, Lorena Ramos-Pérez, María Teresa Salcedo-Allende, Cristina Hernández, Rafael Simó and David M. Selva Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Jun 9 10:28:46 2026 CEST ModDate: Tue Jun 9 11:42:53 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: 3693392 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- MGUQID+URWPalladioL-Ital Type 1 Custom yes yes yes 112 0 LEVXPH+EURM10 Type 1 Builtin yes yes yes 113 0 PRJLCO+URWPalladioL-Bold Type 1 Custom yes yes yes 114 0 ESNPPC+URWPalladioL-Roma Type 1 Custom yes yes yes 115 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes yes 208 0 MIMNPA+PalatinoLinotype TrueType WinAnsi yes yes no 200 0 FFDGWE+CMSY10 Type 1 Builtin yes yes yes 237 0 MIMNPA+PalatinoLinotype TrueType WinAnsi yes yes no 231 0 MIMNPC+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 251 0 MIMNPA+PalatinoLinotype TrueType WinAnsi yes yes no 252 0 MIMNNP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 253 0 MIMNPA+PalatinoLinotype TrueType WinAnsi yes yes no 263 0 MIMNNP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 264 0 MIMNPA+PalatinoLinotype TrueType WinAnsi yes yes no 311 0 IAGBJC+PalatinoLinotype TrueType WinAnsi yes yes no 349 0 IAGBJC+PalatinoLinotype TrueType WinAnsi yes yes no 355 0 IAGBJA+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 356 0 MIMNPA+PalatinoLinotype TrueType WinAnsi yes yes no 375 0 MIMNNP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 376 0 MIMNPA+PalatinoLinotype TrueType WinAnsi yes yes no 400 0 MIMNNP+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 401 0 SSXPJJ+VnURWPalladioL Type 1 Custom yes yes yes 486 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-07-09 03:57:23 CEST RepresentationInformation: ijms-27-04893-v3.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-07-08 10:45:47 CEST Size: 3693392 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 587 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: SHBG Overexpression Reduced TGF-1-Induced Increase in Collagen Expression in LX-2 Cells and HepG2 Cell Co-Cultures Item: Title: SHBG Protected Against Liver Fibrosis Induced by CCl4 Item: Title: SHBG Reduced Collagen and TGF-1 Protein Levels in Liver Fibrosis Induced by CCl4 Item: Title: SHBG Protection Against Liver Fibrosis Induced by CCl4 Is Related to an Increase in Metalloprotease Protein Levels Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Cell Culture Experiments Item: Title: Animals Item: Title: In Vivo Experiments Item: Title: Histology Item: Title: RNA Analysis Item: Title: Western Blot Analysis Item: Title: Statistical Analyses Item: Title: References Info: Title: Sex Hormone-Binding Globulin Prevents Carbon Tetrachloride-Induced Liver Fibrosis Development Author: Anna Álvarez-Guaita, Laura Briansó-Llort, Julia Cabrera-Serra, Lidia Fuertes-Rioja, Lorena Ramos-Pérez, María Teresa Salcedo-Allende, Cristina Hernández, Rafael Simó and David M. Selva Subject: Circulating sex hormone-binding globulin (SHBG) concentrations are lower in individuals with metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), reflecting its potential role in metabolic liver dysfunction. Our prior studies demonstrated that SHBG can attenuate MASLD by limiting hepatic lipid deposition, partly through suppression of lipogenic pathways, in both cellular and animal models. In the present work, we have examined whether SHBG could protect against development of liver fibrosis. For this purpose, in vitro and in vivo studies were performed. In vitro, we used co-cultures of human hepatocellular carcinoma cell line (HepG2) and human hepatic stellate cell line (LX-2) cells transfected using an SHBG expression vector vs. vehicle and treated with transforming growth factor beta 1 (TGF-1). For in vivo studies we used wild-type and human SHBG transgenic mice developing liver fibrosis induced by carbon tetrachloride (CCl4). Our results clearly showed that SHBG overexpression reduced the TGF-1-induced expression in collagen in LX-2 cells. Moreover, SHBG overexpression reduced the CCl4 induced liver fibrosis in both male and female mice. Histological examination revealed that SHBG transgenic mice had reduced NAS score and decreased collagen accumulation, assessed by Sirious Red staining. In addition, human SHBG transgenic mice treated with CCl4 exhibited lower collagen 1A1 (Col1A1) protein levels when compared with wild-type CCl4 treated mice. Mechanistically, SHBG attenuated fibrosis primarily through modulation of the TGF-1/matrix metalloproteinases (MMPs)/tissue inhibitor metalloproteinases 1 (TIMP1) axis, characterized by reduced TGF-1 levels, increased metalloprotease activity, and decreased TIMP1 levels compared with wild-type CCl4 treated mice. Notably, female SHBG transgenic mice exhibited greater protection against fibrosis than males, indicating a sex-dependent effect likely mediated by differences in sex steroid signaling. Taken together, we demonstrate for the first time that SHBG protects against liver fibrosis by promoting collagen degradation via the TGF-1/MMPs/TIMP1 pathway. Further research is needed to elucidate the role of sex steroids in the regulation of MMPs and the observed sexual dimorphism. 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