Human Epididymis Protein 4 as a Potential Biomarker for Crohn’s Disease-Associated Intestinal Fibrosis

Human Epididymis Protein 4 as a Potential Biomarker for Crohn’s Disease-Associated Intestinal Fibrosis | 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 Article Human Epididymis Protein 4 as a Potential Biomarker for Crohn’s Disease-Associated Intestinal Fibrosis Zihao Liu, Xiaoping Xu, Mingzhu Liu, Junfeng Zhou, Lvzhou Xia, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8881694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Human epididymis protein 4 (HE4) plays a crucial role in promoting extracellular matrix (ECM) deposition by inhibiting matrix metalloproteinase (MMP) activity. Crohn's disease (CD) often leads to intestinal fibrosis and obstruction due to chronic inflammation. This study evaluated HE4 expression in serum and intestinal tissues from CD patients, and the effects of anti-HE4 antibody in a TNBS-induced mouse model of intestinal fibrosis. HE4 levels were significantly elevated in both serum and intestinal tissues of patients with stricturing CD, correlating with increased intestinal fibrosis and wall thickness. Inhibition of HE4 expression in mice reduced serum levels of IL-6, TNF-α, and intestinal expression of key fibrotic markers, including α-SMA, COL1A1, Fibronectin. These results suggest that HE4 is a potential biomarker for CD-associated intestinal fibrosis and a promising therapeutic target to mitigate fibrosis progression in CD. Health sciences/Biomarkers Health sciences/Diseases Health sciences/Gastroenterology Health sciences/Medical research Human Epididymis Protein 4 Intestinal fibrosis Crohn's disease Inflammatory bowel disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction In recent years, the incidence of inflammatory bowel disease (IBD) in Asia has increased rapidly 1 . The pathogenesis of IBD is multifactorial, involving dietary patterns, genetic predisposition, immune dysregulation, environmental factors, and the intestinal microbiota 2 . Crohn’s disease (CD), a common subtype of IBD, is characterized pathologically by transmural inflammation that can extend through the muscularis propria to the serosal layer, often leading to complications such as intestinal stenosis and frequently requiring surgical resection of the affected intestinal segments 3 . Clinical observations indicate that intestinal obstruction is a frequent and severe complication, affecting approximately 39.9–48.2% of patients with a disease duration exceeding 20 years, many of whom eventually require surgical intervention or biologic therapy 4 , 5 . This fibrotic progression is currently attributed to an imbalance in the intestinal mucosal immune barrier, which triggers the release of various inflammatory cytokines that activate mesenchymal cells, leading to the excessive accumulation of extracellular matrix (ECM) components like fibrin 6 . The human epididymis protein 4 (HE4) gene encodes a secreted glycoprotein with four whey acidic protein (WAP) domains that exhibits protease inhibitory activity. The WAP domains of HE4 are highly conserved across species and may play significant roles in antimicrobial and anti-inflammatory responses 7 , 8 . Functionally, HE4 has been shown to promote organ fibrosis by inhibiting the activity of matrix metalloproteinases (MMPs) through its WAP domain, thereby favoring the deposition of ECM 9 , 10 . Recent studies have reported HE4 expression in various fibrotic conditions, including renal fibrosis, hepatic fibrosis, idiopathic pulmonary fibrosis, and ischemic cardiomyopathy 11 – 14 . In the context of CD, the persistent elevation of pro-inflammatory cytokines such as TNF-α and IL-6 establishes a microenvironment that facilitates fibrosis 15 . Specifically, TNF-α can promote the production of IL-6 and directly stimulate the TGF-β1 signaling pathway, triggering the phosphorylation of Smad proteins (such as Smad3), inducing the expression of fibrosis-related genes and reducing matrix degradation 16 . However, its specific expression and role in CD-associated intestinal fibrosis remain unclear. Therefore, this study aims to evaluate whether HE4 could serve as a potential biomarker and therapeutic target for CD-associated intestinal fibrosis. Materials and Methods Patient Enrollment and Sample Collection All patients with stenotic CD who underwent surgical treatment at our hospital were included in the experimental group (n = 20). Patients with intestinal obstruction who had normal tissue from both ends of the obstructed segment were included as controls(n = 8). The specimens were obtained from archived tissue samples in the Pathology Department of Hunan Provincial People's Hospital. A cohort of 51 IBD patients (38 CD and 13 ulcerative colitis (UC)) admitted to Hunan Provincial People's Hospital between September 2021 and September 2022 was enrolled. A control group (CTR) included five healthy adults undergoing routine health examinations at the same hospital during the same period. All sample collection was performed following approval by the Medical Ethics Committee of Hunan Provincial People's Hospital (Ethical Approval No. 2022 − 191). All methods were performed in accordance with the relevant guidelines and regulations, including the Declaration of Helsinki. Written informed consent was obtained from all participants. On the first day of hospitalization, 3 mL of venous blood was collected from each patient into a coagulation tube, and serum was obtained after centrifugation. The 38 CD patients were further subdivided into four groups based on clinical diagnosis, imaging or endoscopic evidence of intestinal stricture, and the use of biologic agents (Infliximab): (1) stenotic CD (n = 16), (2) stenotic CD + biologic agents (n = 10), (3) non-stenotic CD (n = 6), and (4) non-stenotic CD + biologic agents (n = 6). Clinical Data Collection Clinical data were retrieved from the electronic medical record system of Hunan Provincial People's Hospital. Recorded parameters included patient age, gender, and laboratory values: serum albumin (ALB), hemoglobin (Hb), red blood cell count (RBC), absolute lymphocyte count (ALC), erythrocyte sedimentation rate (ESR), neutrophil percentage (N%), and platelet count (PLT). The location of any stenotic intestinal segments was also recorded. Intestinal wall thickness was measured from imaging data by a specialist using the hospital’s medical imaging system. For CD patients, three measurements were taken from the most severely affected intestinal segment and averaged. For control subjects, the ascending, transverse, and descending colon wall thicknesses were measured and averaged 17 . TNBS-Induced Mouse Model A TNBS-induced chronic intestinal fibrosis model was established using male BALB/c mice (8 weeks old, 18–20 g, n = 24; from Hunan SJA Laboratory Animal Co., Ltd). The mice were randomly assigned to four groups (n = 6 per group): (1) Saline, (2) TNBS, (3) TNBS + IgG, and (4) TNBS+HE4. Chronic colitis was induced by rectal administration of TNBS following a modified protocol 18 . After the fourth TNBS enema, mice in the TNBS + IgG and TNBS + HE4 groups received intraperitoneal injections of anti-IgG neutralizing antibody or anti-HE4 neutralizing antibody, respectively, while the Saline and TNBS groups received equivalent volumes of saline (5 mg/kg) twice weekly(Figure 1 ) 19 . Treatments continued for 3 weeks (six injections). Three days after the final injection, mice were fasted for 6 hours and then euthanized by CO2 asphyxiation followed by cervical dislocation to ensure death. Blood samples were collected via cardiac puncture, and tissues were harvested immediately and either snap-frozen in liquid nitrogen or fixed in 4% paraformaldehyde for subsequent analysis. All animal procedures were approved by the Medical Ethics Committee of Hunan Provincial People's Hospital (Ethical Approval No. 2022-17) and all methods were performed in accordance with relevant institutional guidelines and regulations, as well as the ARRIVE guidelines (v2.0) for reporting animal research. Serum and Tissue Analysis Serum HE4 levels in human samples and IL-6 and TNF-α, levels in mouse serum were measured by ELISA. Intestinal tissue samples from both humans and mice were fixed, embedded, and subjected to Masson’s trichrome staining to assess fibronectin deposition. Immunohistochemistry (IHC) was used to detect expression of HE4, α-SMA, and collagen. Fresh mouse colon tissue samples were homogenized and protein extracts were analyzed by Western blot to quantify α-SMA, COL3A1, COL1A1, fibronectin, TGF-β1, and phosphorylated Smad3 (P-Smad3/Smad3) levels. Statistical analysis The data are expressed as mean ± standard deviation. Spearman's rank correlation analysis was used to evaluate the correlation. Grayscale values from Western blot analyses and the percentage of positive cells from immunohistochemistry were quantified using ImageJ software. In the Western blot analysis, GAPDH served as the internal reference protein. Each sample's grayscale value was measured in triplicate, and the mean of these measurements was utilized as the final value. The relative expression levels of the target protein were determined by normalizing its grayscale value to that of GAPDH. All graphical representations and statistical analyses were conducted using GraphPad Prism version 8.0. Comparisons of continuous variables between two groups were analyzed using Student's t-test, while categorical variables were compared using the Chi-square test, with statistical significance set at a p-value of less than 0.05. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Results HE4 Expression Is Elevated in IBD Patient Serum Serum HE4 levels were measured by ELISA in the control, CD, and UC groups. As shown in Fig. 2 , serum HE4 levels were significantly higher in the CD group (6123 ± 2292.2 pg/mL) and UC group (7482.4 ± 2861.6 pg/mL) than in the control group (3817.4 ± 771.0 pg/mL) (P < 0.05). The increase in the UC group was more significant (P 0.05) . Biologic Agents Reduce Serum HE4 Levels in Stenotic CD Patients We compared serum HE4 levels among the CD patient subgroups (Fig. 3 ). Stenotic CD patients not treated with biologic agents had higher serum HE4 levels (7397.3 ± 2363.5 pg/mL) than the control group (P < 0.01) and also higher than stenotic CD patients treated with biologics (4630.8 ± 1460.7 pg/mL, P 0.05 for all comparisons). Correlation Between Serum HE4 Levels and Clinical Indicators We analyzed the correlation between serum HE4 levels and clinical parameters (Fig. 4 ). Serum HE4 levels were positively correlated with the thickness of the most stenotic intestinal segment (R = 0.608, P < 0.001), ESR (R = 0.478, P < 0.001), percentage of neutrophil (R = 0.441, P < 0.001), platelet count (PLT) (R = 0.583, P < 0.001), and patient age (R = 0.439, P = 0.001). HE4 levels were negatively correlated with serum albumin (ALB) (R=-0.514, P < 0.001), hemoglobin (R=-0.422, P = 0.002), RBC count (R=-0.451, P 0.05 for all). Fibronectin and HE4 Expression in Stenotic CD Intestinal Tissue Masson’s trichrome staining and IHC were performed on intestinal tissue specimens (Fig. 5 ). Compared to controls, stenotic CD tissues showed extensive fibronectin deposition in the submucosa, rupture of the muscularis mucosa, and destruction of normal tissue architecture. HE4 expression was increased in the mucosal and submucosal layers. Chi-square test analysis revealed that HE4 expression was significantly higher in stenotic CD tissues compared to the CTR group (p < 0.05). Macroscopic Appearance of Mouse Colon Tissue The colon length and gross appearance of mice in each group were evaluated. The Saline and TNBS + HE4 groups had normal colon length and uniform coloration, with consistent wall thickness and no necrosis, distortion, or stenosis. In contrast, the TNBS and TNBS + IgG groups exhibited colons with localized edema and lumen stenosis, and the colonic walls appeared rigid, twisted, and deformed(Figure 6 ). However, differences in colon length and weight between groups were not statistically significant (P > 0.05). Inhibition of HE4 Expression Reduces Inflammatory Cytokine Release Serum IL-6 and TNF-α levels were measured by ELISA. IL-6 and TNF-α levels were significantly higher in the TNBS group than in the Saline group (P < 0.05). In the TNBS + IgG group, IL-6 levels were not significantly different from c Salines, but TNF-α levels remained elevated (P < 0.01). Both IL-6 and TNF-α levels were significantly lower in the TNBS + HE4 group compared to the TNBS and TNBS + IgG groups (P < 0.05), and were not significantly different from the Saline group(Figure 7 ). These results indicate that HE4 inhibition attenuated the release of inflammatory cytokines. Inhibition of HE4 Expression Ameliorates Chronic Intestinal Fibrosis in Mice Masson’s trichrome and IHC staining (Fig. 8 ) revealed that fibronectin deposition was significantly higher in the submucosa and muscularis propria of the TNBS and TNBS + IgG groups compared to the Saline and TNBS+HE4 groups, with disorganized tissue structure. Chi-square test analysis showed that the expression levels of α-SMA and collagen I were significantly increased in the TNBS and TNBS + IgG groups compared with the Saline and TNBS + HE4 groups (P < 0.0001). In these groups, α-SMA and collagen I expression were also significantly increased in the mucosal, submucosal, and muscular layers. By contrast, the TNBS+HE4 group showed normal colon tissue structure and low fibronectin expression in the submucosa, comparable to Salines. In this group, α-SMA and collagen I levels in all layers did not differ significantly from the Saline group. Western blot analysis (Fig. 9 ) confirmed that protein levels of α-SMA, COL3A1, COL1A1, fibronectin, TGF-β1, and phosphorylated Smad3 (P-Smad3/Smad3) were significantly higher in the TNBS group than in Salines, indicating successful induction of fibrosis. In the TNBS+HE4 group, levels of fibronectin, COL3A1, COL1A1, P-Smad3/Smad3, α-SMA, and TGF-β1 were significantly lower than in the TNBS + IgG group (P < 0.05 for each), demonstrating that anti-HE4 treatment alleviated intestinal fibrosis by suppressing inflammatory cytokine production and extracellular matrix deposition. Discussion Intestinal fibrosis is a complex and dynamic process. It is currently believed that, on the basis of genetic susceptibility 20 , the imbalance of the intestinal mucosal immune barrier triggers the release of various inflammatory cytokines, which further activate intestinal mesenchymal cells, leading to excessive deposition of ECM components such as fibrin, which in turn induces fibrosis and subsequently causes segmental intestinal stricture or even obstruction 6 . Intestinal obstruction is one of the most common complications of CD, occurring in 39.9–48.2% of patients with a disease duration of over 20 years, most of whom require surgical intervention or biologic agents 4 , 5 . HE4, a WAP-domain glycoprotein, can inhibit matrix metalloproteinases (MMPs) via its WAP domain, leading to extracellular matrix (ECM) deposition and subsequent organ fibrosis 9 , 10 . While HE4 expression is typically confined to the epididymis and selected normal tissues with negligible levels in the normal colon, evidence from Kemal et al. demonstrates significant HE4 overexpression within colonic epithelial cells during malignancy, notably in advanced-stage (III-IV) colorectal cancer 21 , 22 . Such a shift in expression patterns indicates that the transcription of HE4 in the intestinal epithelium is inducible and can be triggered by specific pathophysiological stimuli, reinforcing its role as a responsive mediator in intestinal pathology. Previous studies have implicated HE4 in cystic fibrosis, renal fibrosis, and liver fibrosis 9 , 23 , 24 . Therefore, we explored whether HE4 could serve as a biomarker for CD-associated intestinal fibrosis. Clinical evaluation of CD often relies on symptoms, laboratory tests, and imaging, where severe anemia, hypoalbuminemia, elevated neutrophil percentage, and ESR typically indicate active disease 25 . Previous studies have shown that combining HE4 with other biomarkers improves diagnostic accuracy for ovarian cancer 26 . In our analysis, serum HE4 levels correlated positively with intestinal wall thickness, ESR, neutrophil percentage, platelet count, and age, and negatively with albumin, hemoglobin, RBC count, and lymphocyte count. The particularly strong correlation between HE4 and wall thickness suggests that HE4 may be useful for early prediction of CD-associated intestinal fibrosis. In this study, we found that the stenotic CD patients without biologic therapy (specifically Infliximab) had significantly higher serum HE4 levels than the control group. In contrast, there were no significant differences between non-stenotic CD patients (with or without biologics) and controls. Moreover, serum HE4 levels in untreated stenotic CD patients were significantly higher than in biologic-treated stenotic CD patients, indicating that HE4 expression is elevated in CD and shows specific expression in stenotic CD, and that biologic therapy may reduce serum HE4 levels. Our findings align with current clinical practices where biologic agents targeting TNF-α, IL-6, and leukocyte adhesion molecules improve CD treatment outcomes by blocking key inflammatory pathways 27 , 28 . Infliximab, as a TNF-α inhibitor, plays a critical role in autoimmune diseases by suppressing inflammation. TNF-α has been confirmed to induce the upregulation of IL-6, a key pro-inflammatory cytokine involved in acute phase responses and chronic inflammation 29 . Considering that excessive IL-6 production has been implicated in hepatic, pulmonary, and renal fibrosis 30 , 31 , it is plausible that inflammatory factors contribute to HE4 expression and drive the progression of CD-associated intestinal fibrosis. Thus, we established a TNBS-induced chronic intestinal fibrosis model in mice and demonstrated that inhibition of HE4 expression via neutralizing antibodies ameliorates fibrosis. The TNBS group showed shortened, rigid, and narrowed colons, along with elevated serum IL-6 and TNF-α levels and increased collagen in colon tissue, indicating successful fibrosis modeling 18 . In contrast, the TNBS + HE4 group displayed reduced serum IL-6 and TNF-α levels compared to the TNBS + IgG group, as well as decreased expression of TGF-β1 and downstream P-Smad3/Smad3 in colon tissue, alongside reduced expression of fibrosis-associated proteins including α-SMA, COL3A1, COL1A1, and fibronectin. These results suggest that HE4 contributes to intestinal fibrosis via inflammation-mediated pathways and that HE4 inhibition mitigates fibrosis by suppressing inflammatory cytokine production and ECM deposition. The imbalance of the intestinal mucosal immune barrier in CD leads to the release of various inflammatory cytokines. Pro-inflammatory cytokines, including IL-6 and TNF-α, are persistently elevated in CD and contribute to sustained inflammatory responses and repeated cycles of intestinal injury and repair, thereby promoting a profibrotic microenvironment 15 . Accumulating evidence indicates that increased levels of IL-6 and TNF-α are associated with enhanced expression of TGF-β1, suggesting that these inflammatory cytokines may act as upstream modulators of fibrotic responses 32 . As a core cytokine in CD, TNF-α further triggers inflammatory responses and immune cell activation; it not only promotes IL-6 production but also directly activates the TGF-β1 signaling pathway. Both TNF-α and IL-6 can enhance the expression of profibrotic factors, including TGF-β1. Concurrently, IL-6, as another key inflammatory cytokine, can further stimulate TGF-β1 secretion to amplify the fibrotic process and enhance TGF-β1 activity. TGF-β1 is widely recognized as a central profibrotic mediator in CD; once activated, the TGF-β1 signaling pathway triggers a cascade of intracellular events, including the phosphorylation of Smad proteins (specifically Smad2 and Smad3). These phosphorylated Smad proteins subsequently translocate to the nucleus to induce the expression of fibrosis-related genes, which is closely associated with fibroblast activation, ECM deposition, and reduced matrix degradation 16 . Recent studies have identified HE4 as an important regulator of fibrotic remodeling through its inhibitory effects on matrix metalloproteinase activity, thereby favoring ECM accumulation 9 . In the context of chronic intestinal inflammation, increased TGF-β1 expression may contribute to HE4 upregulation, which in turn shifts intestinal tissue repair toward fibrosis 33 . Collectively, these findings suggest that elevated IL-6 and TNF-α levels, enhanced TGF-β1 signaling, and HE4-mediated impairment of ECM degradation are closely associated with the development of intestinal fibrosis in CD, highlighting HE4 as a potential biomarker and therapeutic target for fibrotic complications. The interaction between TNF-α, IL-6, HE4, and TGF-β1 amplifies the fibrotic process in CD, ultimately leading to the formation of strictures and intestinal obstruction. Conclusions This study demonstrated that HE4 is specifically upregulated in CD-associated intestinal fibrosis and correlates with disease severity markers. Preliminary mouse model experiments support a role for HE4 in promoting fibrosis through inflammatory pathways. Further large-scale clinical studies are required to validate whether HE4 can serve as an early biomarker for CD-associated intestinal fibrosis and potential therapeutic target. Declarations Author contributions L.Z.H., X.X.P. and conceived and designed the experiments; L.M.Z, Z.J.F. and X.L.Z. performed the experiments; and Z.Y.Y. contributed to the statistical data analysis; Z.M.H., L.S.P. and P.Y. wrote the manuscript; L.X.Q., W.Y.Z. and W.M.H. corrected the manuscript. All authors have read and approved the manuscript for publication. Funding This research was mainly supported by a grant from National Natural Science Foundation of China (81900466) and Science and Technology Project of Hunan Province(2020JJ5307). Data availability The data sets generated and analyzed during this study are available from the corresponding author on reasonable request. Ethical Approval and Consent to participate Subjects were recruited from Hunan Provincial People's Hospital. All participants provided informed consent, and no prison populations or their organs were used. This study was performed in line with the principles of the Declaration of Helsinki. And the study is reported in accordance with ARRIVE guidelines (v2.0). Approval was granted by the Medical Ethics Committee of Hunan Provincial People's Hospital (Ethical Approval No. 2022-191) ＆ (Ethical Approval No. 2022-17). Declaration of Competing Interest All the authors declare no conflict of interest References Ng, S. C. et al. Incidence and phenotype of inflammatory bowel disease based on results from the Asia-pacific Crohn's and colitis epidemiology study. Gastroenterology 145 , 158–165e152. 10.1053/j.gastro.2013.04.007 (2013). Butera, A. et al. CD147 Targeting by AC-73 Induces Autophagy and Reduces Intestinal Fibrosis Associated with TNBS Chronic Colitis. J. Crohn's colitis . 16 , 1751–1761. 10.1093/ecco-jcc/jjac084 (2022). Latella, G. et al. 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Hypoxia-induced HE4 in tubular epithelial cells promotes extracellular matrix accumulation and renal fibrosis via NF-κB. FASEB journal: official publication Federation Am. Soc. Experimental Biology . 34 , 2554–2567. 10.1096/fj.201901950R (2020). Additional Declarations No competing interests reported. Supplementary Files OriginalWBImages.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version 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-8881694","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":602087628,"identity":"a8943ac3-b749-4671-9413-eef0e7655d8d","order_by":0,"name":"Zihao Liu","email":"","orcid":"","institution":"The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zihao","middleName":"","lastName":"Liu","suffix":""},{"id":602087629,"identity":"609f8030-6e0a-4a71-950d-9a7cf7174239","order_by":1,"name":"Xiaoping Xu","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Xu","suffix":""},{"id":602087630,"identity":"e79a36b4-0a98-41ec-8a62-d4730d9fb582","order_by":2,"name":"Mingzhu Liu","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Mingzhu","middleName":"","lastName":"Liu","suffix":""},{"id":602087631,"identity":"a3f4a394-61a8-478c-a14d-9fac950098dd","order_by":3,"name":"Junfeng Zhou","email":"","orcid":"","institution":"The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University/Hunan Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Junfeng","middleName":"","lastName":"Zhou","suffix":""},{"id":602087632,"identity":"83316a8f-6a76-4301-bdd8-17af16c1ffc0","order_by":4,"name":"Lvzhou Xia","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lvzhou","middleName":"","lastName":"Xia","suffix":""},{"id":602087633,"identity":"b815eb91-2537-47eb-afc0-8c7df7dbf394","order_by":5,"name":"Yuyang Zhang","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuyang","middleName":"","lastName":"Zhang","suffix":""},{"id":602087634,"identity":"21ce8a44-ded7-4d00-8bc0-b10a257db773","order_by":6,"name":"Minhong Zou","email":"","orcid":"","institution":"Foshan Fosun Chancheng Hospital","correspondingAuthor":false,"prefix":"","firstName":"Minhong","middleName":"","lastName":"Zou","suffix":""},{"id":602087635,"identity":"3caa6775-7e43-4538-8e03-e3fdd7690689","order_by":7,"name":"Shipeng Lu","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Shipeng","middleName":"","lastName":"Lu","suffix":""},{"id":602087636,"identity":"9058a608-3910-427c-8fd8-b3256c00fb00","order_by":8,"name":"Ya Peng","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Peng","suffix":""},{"id":602087637,"identity":"2b62a7a5-3540-4f83-9ff8-9eb4e9205d99","order_by":9,"name":"Xiongquan Long","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiongquan","middleName":"","lastName":"Long","suffix":""},{"id":602087638,"identity":"b09c5e99-9606-4667-bfcd-12ac5f2ea030","order_by":10,"name":"Minghao Wu","email":"","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":false,"prefix":"","firstName":"Minghao","middleName":"","lastName":"Wu","suffix":""},{"id":602087639,"identity":"aee10f86-3106-408b-941b-b9fc349fc3ab","order_by":11,"name":"Yizhong Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACAyCWgDCZjyELEqWFLY1kLTxmxGkxl25/ePPnnsOJG86f+faYd8edxAb25m0SDDV3cGqxnHPG2JrnGVDLgbPbjXnPPEts4DlWJsFw7Bluh93IYZNmOHA4d8PB3m3SvG2HExskcswkGBsO49GS/kzyB0jLYZ5nEC3ybwhpSTCT4AFpOcbDBrWFh5CWHKBfDqTXzzzDZiY5t+2wcRtPWrFFwjG8Dnt488cBa2O+84efSbxtOyzbz354440PNbi1QEEzg8IBKJMNRCQQ0sDAUMcg30BY1SgYBaNgFIxQAABdgVxRJl7qyAAAAABJRU5ErkJggg==","orcid":"","institution":"Hunan Provincial People’s Hospital ,The First Affiliated Hospital of Hunan Normal University","correspondingAuthor":true,"prefix":"","firstName":"Yizhong","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-02-14 17:23:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8881694/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8881694/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104406131,"identity":"c3a38163-1814-4e71-9ffe-5b69c047cdbc","added_by":"auto","created_at":"2026-03-11 12:24:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":230553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of the TNBS-induced intestinal fibrosis mouse model.\u003c/strong\u003e\u003cbr\u003e\nAn experimental timeline depicting the induction of intestinal fibrosis in male C57BL/6 mice (approximately 8 weeks old). Prior to intrarectal administration, mice were presensitized by epicutaneous application of TNBS on a shaved area of the dorsal skin to assess potential allergic reactions. Subsequently, intestinal fibrosis was induced via intrarectal (\u003cem\u003ei.r.\u003c/em\u003e) administration of TNBS at varying concentrations and time points, combined with intraperitoneal (\u003cem\u003ei.p.\u003c/em\u003e) injections (5 mg/kg). Colonic tissue samples were harvested from the mice at 7 weeks post-modeling.\u003cem\u003eAbbreviations: i.r., intrarectal; i.p., intraperitoneal.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/dd7e01db134932668c88c5d5.png"},{"id":104337976,"identity":"85c224a8-7e3c-459c-b5fe-8181526621a6","added_by":"auto","created_at":"2026-03-10 16:17:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":254547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum HE4 concentration in human subjects measured by ELISA.\u003c/strong\u003e\u003cbr\u003e\n Serum levels of HE4 were quantified by ELISA in healthy controls (CTR), patients with Crohn's disease (CD), and patients with ulcerative colitis (UC). Blood samples were collected, centrifuged, and the supernatant was analyzed. Experiments were performed in at least triplicate. Data are presented as mean ± SD. Statistical significance was determined by Student’s t-test (*p \u0026lt; 0.05, **p \u0026lt; 0.01*).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/7f4f0b2114f8d045036576d5.png"},{"id":104337981,"identity":"d160911d-8fca-4f23-b05e-3d372764d9a2","added_by":"auto","created_at":"2026-03-10 16:17:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":244986,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum HE4 levels in CD patient subgroups with or without biologic therapy.\u003c/strong\u003e\u003cbr\u003e\n Serum HE4 concentrations were compared across five groups: healthy controls (CTR), stenotic CD , stenotic CD + biologic agents , non-stenotic CD, non-stenotic CD + biologic agents .Data are presented as mean ± SD. Differences were analyzed by Student’s t-test (*p \u0026lt; 0.05, **p \u0026lt; 0.01*).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/d5cfbc6340de47118b1c5eb8.png"},{"id":104337983,"identity":"430a74a7-0053-41f3-b9af-30a50814cfce","added_by":"auto","created_at":"2026-03-10 16:17:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":999255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation Between Serum HE4 Levels and Clinical Indicators\u003c/strong\u003e\u003cbr\u003e\n Spearman’s rank correlation analysis was performed to assess the association of serum HE4 levels with (a) intestinal wall thickness and (b–i) various clinical indicators. The correlation coefficient (R) ranges from −1 to +1, where values closer to ±1 indicate stronger monotonic relationships.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/bb98ff58b66423826f39022c.png"},{"id":104337984,"identity":"46e31da4-fad4-4587-9956-e49c2c521b4f","added_by":"auto","created_at":"2026-03-10 16:17:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1234784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological evaluation of human intestinal tissues.\u003c/strong\u003e\u003cbr\u003e\n(a) Representative images of Masson’s trichrome staining and immunohistochemical (IHC) staining for HE4 in human colon sections. (b) Quantitative analysis of IHC staining positivity was performed using ImageJ. Differences in positive rates between groups were compared by Chi-square test (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/90c3f2e9b5f93d9492e16b2a.png"},{"id":104337977,"identity":"6f4b8936-1802-47e3-9e9f-71072c619c7f","added_by":"auto","created_at":"2026-03-10 16:17:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1118299,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacroscopic Appearance of Mouse Colon Tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eColon tissues were collected after euthanasia, rinsed with cold physiological saline, and photographed. The TNBS and TNBS+IgG groups exhibited colons with localized edema and lumen stenosis, and the colonic walls appeared rigid, twisted, and deformed.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/8b8464ac58f32343b7c9927e.png"},{"id":104405643,"identity":"3cfcbccb-d68c-45c7-88c5-4aa1723f3a6d","added_by":"auto","created_at":"2026-03-11 12:23:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":186674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum cytokine levels in mice measured by ELISA.\u003c/strong\u003e\u003cbr\u003e\n Concentrations of (a) IL-6 and (b) TNF-α in mouse serum were determined by ELISA. Data are presented as mean ± SD. Statistical comparisons between groups were performed using Student’s t-test (*p \u0026lt; 0.05, **p \u0026lt; 0.01, **\u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/04bb4f92f18b7534f6e3a786.png"},{"id":104337979,"identity":"a5618d1c-a238-47fc-961f-76da0764e25b","added_by":"auto","created_at":"2026-03-10 16:17:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2468901,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/aafcf69774832408181df7fd.png"},{"id":104337982,"identity":"c7b308db-7d34-4297-b10b-cbc27f15ef80","added_by":"auto","created_at":"2026-03-10 16:17:20","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":877910,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u0026nbsp;\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/b936c36e241ecb8da54d6228.png"},{"id":107413835,"identity":"71c25d24-9e81-49f2-a3e6-8afd508f24f5","added_by":"auto","created_at":"2026-04-21 09:28:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9857727,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/b428d9d0-82dd-45e0-ba2d-b90d855e69eb.pdf"},{"id":104337985,"identity":"aac36fd4-6913-47ea-a141-e3abd48b9b6e","added_by":"auto","created_at":"2026-03-10 16:17:21","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":38832692,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalWBImages.zip","url":"https://assets-eu.researchsquare.com/files/rs-8881694/v1/52fdc497efa411617986a404.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Human Epididymis Protein 4 as a Potential Biomarker for Crohn’s Disease-Associated Intestinal Fibrosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, the incidence of inflammatory bowel disease (IBD) in Asia has increased rapidly\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The pathogenesis of IBD is multifactorial, involving dietary patterns, genetic predisposition, immune dysregulation, environmental factors, and the intestinal microbiota\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Crohn\u0026rsquo;s disease (CD), a common subtype of IBD, is characterized pathologically by transmural inflammation that can extend through the muscularis propria to the serosal layer, often leading to complications such as intestinal stenosis and frequently requiring surgical resection of the affected intestinal segments\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Clinical observations indicate that intestinal obstruction is a frequent and severe complication, affecting approximately 39.9\u0026ndash;48.2% of patients with a disease duration exceeding 20 years, many of whom eventually require surgical intervention or biologic therapy\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This fibrotic progression is currently attributed to an imbalance in the intestinal mucosal immune barrier, which triggers the release of various inflammatory cytokines that activate mesenchymal cells, leading to the excessive accumulation of extracellular matrix (ECM) components like fibrin\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eThe human epididymis protein 4 (HE4) gene encodes a secreted glycoprotein with four whey acidic protein (WAP) domains that exhibits protease inhibitory activity. The WAP domains of HE4 are highly conserved across species and may play significant roles in antimicrobial and anti-inflammatory responses \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Functionally, HE4 has been shown to promote organ fibrosis by inhibiting the activity of matrix metalloproteinases (MMPs) through its WAP domain, thereby favoring the deposition of ECM \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Recent studies have reported HE4 expression in various fibrotic conditions, including renal fibrosis, hepatic fibrosis, idiopathic pulmonary fibrosis, and ischemic cardiomyopathy\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the context of CD, the persistent elevation of pro-inflammatory cytokines such as TNF-α and IL-6 establishes a microenvironment that facilitates fibrosis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Specifically, TNF-α can promote the production of IL-6 and directly stimulate the TGF-β1 signaling pathway, triggering the phosphorylation of Smad proteins (such as Smad3), inducing the expression of fibrosis-related genes and reducing matrix degradation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, its specific expression and role in CD-associated intestinal fibrosis remain unclear. Therefore, this study aims to evaluate whether HE4 could serve as a potential biomarker and therapeutic target for CD-associated intestinal fibrosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient Enrollment and Sample Collection\u003c/h2\u003e \u003cp\u003eAll patients with stenotic CD who underwent surgical treatment at our hospital were included in the experimental group (n\u0026thinsp;=\u0026thinsp;20). Patients with intestinal obstruction who had normal tissue from both ends of the obstructed segment were included as controls(n\u0026thinsp;=\u0026thinsp;8). The specimens were obtained from archived tissue samples in the Pathology Department of Hunan Provincial People's Hospital.\u003c/p\u003e \u003cp\u003eA cohort of 51 IBD patients (38 CD and 13 ulcerative colitis (UC)) admitted to Hunan Provincial People's Hospital between September 2021 and September 2022 was enrolled. A control group (CTR) included five healthy adults undergoing routine health examinations at the same hospital during the same period. All sample collection was performed following approval by the Medical Ethics Committee of Hunan Provincial People's Hospital (Ethical Approval No. 2022\u0026thinsp;\u0026minus;\u0026thinsp;191). All methods were performed in accordance with the relevant guidelines and regulations, including the Declaration of Helsinki. Written informed consent was obtained from all participants. On the first day of hospitalization, 3 mL of venous blood was collected from each patient into a coagulation tube, and serum was obtained after centrifugation. The 38 CD patients were further subdivided into four groups based on clinical diagnosis, imaging or endoscopic evidence of intestinal stricture, and the use of biologic agents (Infliximab): (1) stenotic CD (n\u0026thinsp;=\u0026thinsp;16), (2) stenotic CD\u0026thinsp;+\u0026thinsp;biologic agents (n\u0026thinsp;=\u0026thinsp;10), (3) non-stenotic CD (n\u0026thinsp;=\u0026thinsp;6), and (4) non-stenotic CD\u0026thinsp;+\u0026thinsp;biologic agents (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClinical Data Collection\u003c/h3\u003e\n\u003cp\u003eClinical data were retrieved from the electronic medical record system of Hunan Provincial People's Hospital. Recorded parameters included patient age, gender, and laboratory values: serum albumin (ALB), hemoglobin (Hb), red blood cell count (RBC), absolute lymphocyte count (ALC), erythrocyte sedimentation rate (ESR), neutrophil percentage (N%), and platelet count (PLT). The location of any stenotic intestinal segments was also recorded. Intestinal wall thickness was measured from imaging data by a specialist using the hospital\u0026rsquo;s medical imaging system. For CD patients, three measurements were taken from the most severely affected intestinal segment and averaged. For control subjects, the ascending, transverse, and descending colon wall thicknesses were measured and averaged\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eTNBS-Induced Mouse Model\u003c/h3\u003e\n\u003cp\u003eA TNBS-induced chronic intestinal fibrosis model was established using male BALB/c mice (8 weeks old, 18\u0026ndash;20 g, n\u0026thinsp;=\u0026thinsp;24; from Hunan SJA Laboratory Animal Co., Ltd). The mice were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;6 per group): (1) Saline, (2) TNBS, (3) TNBS\u0026thinsp;+\u0026thinsp;IgG, and (4) TNBS+HE4. Chronic colitis was induced by rectal administration of TNBS following a modified protocol\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. After the fourth TNBS enema, mice in the TNBS\u0026thinsp;+\u0026thinsp;IgG and TNBS\u0026thinsp;+\u0026thinsp;HE4 groups received intraperitoneal injections of anti-IgG neutralizing antibody or anti-HE4 neutralizing antibody, respectively, while the Saline and TNBS groups received equivalent volumes of saline (5 mg/kg) twice weekly(Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e19\u003c/sup\u003e. Treatments continued for 3 weeks (six injections). Three days after the final injection, mice were fasted for 6 hours and then euthanized by CO2 asphyxiation followed by cervical dislocation to ensure death. Blood samples were collected via cardiac puncture, and tissues were harvested immediately and either snap-frozen in liquid nitrogen or fixed in 4% paraformaldehyde for subsequent analysis. All animal procedures were approved by the Medical Ethics Committee of Hunan Provincial People's Hospital (Ethical Approval No. 2022-17) and all methods were performed in accordance with relevant institutional guidelines and regulations, as well as the ARRIVE guidelines (v2.0) for reporting animal research.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSerum and Tissue Analysis\u003c/h3\u003e\n\u003cp\u003eSerum HE4 levels in human samples and IL-6 and TNF-α, levels in mouse serum were measured by ELISA. Intestinal tissue samples from both humans and mice were fixed, embedded, and subjected to Masson\u0026rsquo;s trichrome staining to assess fibronectin deposition. Immunohistochemistry (IHC) was used to detect expression of HE4, α-SMA, and collagen. Fresh mouse colon tissue samples were homogenized and protein extracts were analyzed by Western blot to quantify α-SMA, COL3A1, COL1A1, fibronectin, TGF-β1, and phosphorylated Smad3 (P-Smad3/Smad3) levels.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Spearman's rank correlation analysis was used to evaluate the correlation. Grayscale values from Western blot analyses and the percentage of positive cells from immunohistochemistry were quantified using ImageJ software. In the Western blot analysis, GAPDH served as the internal reference protein. Each sample's grayscale value was measured in triplicate, and the mean of these measurements was utilized as the final value. The relative expression levels of the target protein were determined by normalizing its grayscale value to that of GAPDH. All graphical representations and statistical analyses were conducted using GraphPad Prism version 8.0. Comparisons of continuous variables between two groups were analyzed using Student's t-test, while categorical variables were compared using the Chi-square test, with statistical significance set at a p-value of less than 0.05. Significance levels are indicated as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHE4 Expression Is Elevated in IBD Patient Serum\u003c/h2\u003e \u003cp\u003eSerum HE4 levels were measured by ELISA in the control, CD, and UC groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, serum HE4 levels were significantly higher in the CD group (6123\u0026thinsp;\u0026plusmn;\u0026thinsp;2292.2 pg/mL) and UC group (7482.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2861.6 pg/mL) than in the control group (3817.4\u0026thinsp;\u0026plusmn;\u0026thinsp;771.0 pg/mL) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The increase in the UC group was more significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas the difference between the CD and UC groups was not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiologic Agents Reduce Serum HE4 Levels in Stenotic CD Patients\u003c/h3\u003e\n\u003cp\u003eWe compared serum HE4 levels among the CD patient subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Stenotic CD patients not treated with biologic agents had higher serum HE4 levels (7397.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2363.5 pg/mL) than the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and also higher than stenotic CD patients treated with biologics (4630.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1460.7 pg/mL, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences were observed between the stenotic CD patients treated with biologics, the non-stenotic CD patients (with or without biologics), and the control group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all comparisons).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCorrelation Between Serum HE4 Levels and Clinical Indicators\u003c/h2\u003e \u003cp\u003eWe analyzed the correlation between serum HE4 levels and clinical parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Serum HE4 levels were positively correlated with the thickness of the most stenotic intestinal segment (R\u0026thinsp;=\u0026thinsp;0.608, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), ESR (R\u0026thinsp;=\u0026thinsp;0.478, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), percentage of neutrophil (R\u0026thinsp;=\u0026thinsp;0.441, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), platelet count (PLT) (R\u0026thinsp;=\u0026thinsp;0.583, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and patient age (R\u0026thinsp;=\u0026thinsp;0.439, P\u0026thinsp;=\u0026thinsp;0.001). HE4 levels were negatively correlated with serum albumin (ALB) (R=-0.514, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), hemoglobin (R=-0.422, P\u0026thinsp;=\u0026thinsp;0.002), RBC count (R=-0.451, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and absolute lymphocyte count (R=-0.364, P\u0026thinsp;=\u0026thinsp;0.009). No significant correlation was found between HE4 levels and patient gender or the location of stenosis (ileum, colon, or ileocolon) (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFibronectin and HE4 Expression in Stenotic CD Intestinal Tissue\u003c/h2\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining and IHC were performed on intestinal tissue specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Compared to controls, stenotic CD tissues showed extensive fibronectin deposition in the submucosa, rupture of the muscularis mucosa, and destruction of normal tissue architecture. HE4 expression was increased in the mucosal and submucosal layers. Chi-square test analysis revealed that HE4 expression was significantly higher in stenotic CD tissues compared to the CTR group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMacroscopic Appearance of Mouse Colon Tissue\u003c/h2\u003e \u003cp\u003eThe colon length and gross appearance of mice in each group were evaluated. The Saline and TNBS\u0026thinsp;+\u0026thinsp;HE4 groups had normal colon length and uniform coloration, with consistent wall thickness and no necrosis, distortion, or stenosis. In contrast, the TNBS and TNBS\u0026thinsp;+\u0026thinsp;IgG groups exhibited colons with localized edema and lumen stenosis, and the colonic walls appeared rigid, twisted, and deformed(Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, differences in colon length and weight between groups were not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of HE4 Expression Reduces Inflammatory Cytokine Release\u003c/h2\u003e \u003cp\u003eSerum IL-6 and TNF-α levels were measured by ELISA. IL-6 and TNF-α levels were significantly higher in the TNBS group than in the Saline group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the TNBS\u0026thinsp;+\u0026thinsp;IgG group, IL-6 levels were not significantly different from c Salines, but TNF-α levels remained elevated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Both IL-6 and TNF-α levels were significantly lower in the TNBS\u0026thinsp;+\u0026thinsp;HE4 group compared to the TNBS and TNBS\u0026thinsp;+\u0026thinsp;IgG groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and were not significantly different from the Saline group(Figure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results indicate that HE4 inhibition attenuated the release of inflammatory cytokines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of HE4 Expression Ameliorates Chronic Intestinal Fibrosis in Mice\u003c/h2\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome and IHC staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) revealed that fibronectin deposition was significantly higher in the submucosa and muscularis propria of the TNBS and TNBS\u0026thinsp;+\u0026thinsp;IgG groups compared to the Saline and TNBS+HE4 groups, with disorganized tissue structure. Chi-square test analysis showed that the expression levels of α-SMA and collagen I were significantly increased in the TNBS and TNBS\u0026thinsp;+\u0026thinsp;IgG groups compared with the Saline and TNBS\u0026thinsp;+\u0026thinsp;HE4 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In these groups, α-SMA and collagen I expression were also significantly increased in the mucosal, submucosal, and muscular layers. By contrast, the TNBS+HE4 group showed normal colon tissue structure and low fibronectin expression in the submucosa, comparable to Salines. In this group, α-SMA and collagen I levels in all layers did not differ significantly from the Saline group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWestern blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) confirmed that protein levels of α-SMA, COL3A1, COL1A1, fibronectin, TGF-β1, and phosphorylated Smad3 (P-Smad3/Smad3) were significantly higher in the TNBS group than in Salines, indicating successful induction of fibrosis. In the TNBS+HE4 group, levels of fibronectin, COL3A1, COL1A1, P-Smad3/Smad3, α-SMA, and TGF-β1 were significantly lower than in the TNBS\u0026thinsp;+\u0026thinsp;IgG group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for each), demonstrating that anti-HE4 treatment alleviated intestinal fibrosis by suppressing inflammatory cytokine production and extracellular matrix deposition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIntestinal fibrosis is a complex and dynamic process. It is currently believed that, on the basis of genetic susceptibility \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, the imbalance of the intestinal mucosal immune barrier triggers the release of various inflammatory cytokines, which further activate intestinal mesenchymal cells, leading to excessive deposition of ECM components such as fibrin, which in turn induces fibrosis and subsequently causes segmental intestinal stricture or even obstruction \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Intestinal obstruction is one of the most common complications of CD, occurring in 39.9\u0026ndash;48.2% of patients with a disease duration of over 20 years, most of whom require surgical intervention or biologic agents\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHE4, a WAP-domain glycoprotein, can inhibit matrix metalloproteinases (MMPs) via its WAP domain, leading to extracellular matrix (ECM) deposition and subsequent organ fibrosis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While HE4 expression is typically confined to the epididymis and selected normal tissues with negligible levels in the normal colon, evidence from Kemal et al. demonstrates significant HE4 overexpression within colonic epithelial cells during malignancy, notably in advanced-stage (III-IV) colorectal cancer \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Such a shift in expression patterns indicates that the transcription of HE4 in the intestinal epithelium is inducible and can be triggered by specific pathophysiological stimuli, reinforcing its role as a responsive mediator in intestinal pathology. Previous studies have implicated HE4 in cystic fibrosis, renal fibrosis, and liver fibrosis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, we explored whether HE4 could serve as a biomarker for CD-associated intestinal fibrosis.\u003c/p\u003e \u003cp\u003eClinical evaluation of CD often relies on symptoms, laboratory tests, and imaging, where severe anemia, hypoalbuminemia, elevated neutrophil percentage, and ESR typically indicate active disease\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that combining HE4 with other biomarkers improves diagnostic accuracy for ovarian cancer\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In our analysis, serum HE4 levels correlated positively with intestinal wall thickness, ESR, neutrophil percentage, platelet count, and age, and negatively with albumin, hemoglobin, RBC count, and lymphocyte count. The particularly strong correlation between HE4 and wall thickness suggests that HE4 may be useful for early prediction of CD-associated intestinal fibrosis.\u003c/p\u003e \u003cp\u003eIn this study, we found that the stenotic CD patients without biologic therapy (specifically Infliximab) had significantly higher serum HE4 levels than the control group. In contrast, there were no significant differences between non-stenotic CD patients (with or without biologics) and controls. Moreover, serum HE4 levels in untreated stenotic CD patients were significantly higher than in biologic-treated stenotic CD patients, indicating that HE4 expression is elevated in CD and shows specific expression in stenotic CD, and that biologic therapy may reduce serum HE4 levels. Our findings align with current clinical practices where biologic agents targeting TNF-α, IL-6, and leukocyte adhesion molecules improve CD treatment outcomes by blocking key inflammatory pathways \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Infliximab, as a TNF-α inhibitor, plays a critical role in autoimmune diseases by suppressing inflammation. TNF-α has been confirmed to induce the upregulation of IL-6, a key pro-inflammatory cytokine involved in acute phase responses and chronic inflammation \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Considering that excessive IL-6 production has been implicated in hepatic, pulmonary, and renal fibrosis \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, it is plausible that inflammatory factors contribute to HE4 expression and drive the progression of CD-associated intestinal fibrosis.\u003c/p\u003e \u003cp\u003eThus, we established a TNBS-induced chronic intestinal fibrosis model in mice and demonstrated that inhibition of HE4 expression via neutralizing antibodies ameliorates fibrosis. The TNBS group showed shortened, rigid, and narrowed colons, along with elevated serum IL-6 and TNF-α levels and increased collagen in colon tissue, indicating successful fibrosis modeling \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In contrast, the TNBS\u0026thinsp;+\u0026thinsp;HE4 group displayed reduced serum IL-6 and TNF-α levels compared to the TNBS\u0026thinsp;+\u0026thinsp;IgG group, as well as decreased expression of TGF-β1 and downstream P-Smad3/Smad3 in colon tissue, alongside reduced expression of fibrosis-associated proteins including α-SMA, COL3A1, COL1A1, and fibronectin. These results suggest that HE4 contributes to intestinal fibrosis via inflammation-mediated pathways and that HE4 inhibition mitigates fibrosis by suppressing inflammatory cytokine production and ECM deposition.\u003c/p\u003e \u003cp\u003eThe imbalance of the intestinal mucosal immune barrier in CD leads to the release of various inflammatory cytokines. Pro-inflammatory cytokines, including IL-6 and TNF-α, are persistently elevated in CD and contribute to sustained inflammatory responses and repeated cycles of intestinal injury and repair, thereby promoting a profibrotic microenvironment \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence indicates that increased levels of IL-6 and TNF-α are associated with enhanced expression of TGF-β1, suggesting that these inflammatory cytokines may act as upstream modulators of fibrotic responses \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. As a core cytokine in CD, TNF-α further triggers inflammatory responses and immune cell activation; it not only promotes IL-6 production but also directly activates the TGF-β1 signaling pathway. Both TNF-α and IL-6 can enhance the expression of profibrotic factors, including TGF-β1. Concurrently, IL-6, as another key inflammatory cytokine, can further stimulate TGF-β1 secretion to amplify the fibrotic process and enhance TGF-β1 activity. TGF-β1 is widely recognized as a central profibrotic mediator in CD; once activated, the TGF-β1 signaling pathway triggers a cascade of intracellular events, including the phosphorylation of Smad proteins (specifically Smad2 and Smad3). These phosphorylated Smad proteins subsequently translocate to the nucleus to induce the expression of fibrosis-related genes, which is closely associated with fibroblast activation, ECM deposition, and reduced matrix degradation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Recent studies have identified HE4 as an important regulator of fibrotic remodeling through its inhibitory effects on matrix metalloproteinase activity, thereby favoring ECM accumulation \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In the context of chronic intestinal inflammation, increased TGF-β1 expression may contribute to HE4 upregulation, which in turn shifts intestinal tissue repair toward fibrosis \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Collectively, these findings suggest that elevated IL-6 and TNF-α levels, enhanced TGF-β1 signaling, and HE4-mediated impairment of ECM degradation are closely associated with the development of intestinal fibrosis in CD, highlighting HE4 as a potential biomarker and therapeutic target for fibrotic complications. The interaction between TNF-α, IL-6, HE4, and TGF-β1 amplifies the fibrotic process in CD, ultimately leading to the formation of strictures and intestinal obstruction.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated that HE4 is specifically upregulated in CD-associated intestinal fibrosis and correlates with disease severity markers. Preliminary mouse model experiments support a role for HE4 in promoting fibrosis through inflammatory pathways. Further large-scale clinical studies are required to validate whether HE4 can serve as an early biomarker for CD-associated intestinal fibrosis and potential therapeutic target.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.Z.H., X.X.P. and conceived and designed the experiments; L.M.Z, Z.J.F. and X.L.Z. performed the experiments; and Z.Y.Y. contributed to the statistical data analysis; Z.M.H., L.S.P. and P.Y. wrote the manuscript; L.X.Q., W.Y.Z. and W.M.H. corrected the manuscript. All authors have read and approved the manuscript for publication.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was mainly supported by a grant from National Natural Science Foundation of China (81900466) and Science and Technology Project of Hunan Province(2020JJ5307).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data sets generated and analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubjects were recruited from Hunan Provincial People\u0026apos;s Hospital. All participants provided informed consent, and no prison populations or their organs were used. This study was performed in line with the principles of the Declaration of Helsinki. And the study is reported in accordance with ARRIVE guidelines (v2.0). Approval was granted by the Medical Ethics Committee of Hunan Provincial People\u0026apos;s Hospital (Ethical Approval No. 2022-191) ＆ (Ethical Approval No. 2022-17).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare no conflict of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNg, S. C. et al. 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Experimental Biology\u003c/em\u003e. \u003cb\u003e34\u003c/b\u003e, 2554\u0026ndash;2567. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1096/fj.201901950R\u003c/span\u003e\u003cspan address=\"10.1096/fj.201901950R\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"Human Epididymis Protein 4, Intestinal fibrosis, Crohn's disease, Inflammatory bowel disease","lastPublishedDoi":"10.21203/rs.3.rs-8881694/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8881694/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHuman epididymis protein 4 (HE4) plays a crucial role in promoting extracellular matrix (ECM) deposition by inhibiting matrix metalloproteinase (MMP) activity. Crohn's disease (CD) often leads to intestinal fibrosis and obstruction due to chronic inflammation. This study evaluated HE4 expression in serum and intestinal tissues from CD patients, and the effects of anti-HE4 antibody in a TNBS-induced mouse model of intestinal fibrosis. HE4 levels were significantly elevated in both serum and intestinal tissues of patients with stricturing CD, correlating with increased intestinal fibrosis and wall thickness. Inhibition of HE4 expression in mice reduced serum levels of IL-6, TNF-α, and intestinal expression of key fibrotic markers, including α-SMA, COL1A1, Fibronectin. These results suggest that HE4 is a potential biomarker for CD-associated intestinal fibrosis and a promising therapeutic target to mitigate fibrosis progression in CD.\u003c/p\u003e","manuscriptTitle":"Human Epididymis Protein 4 as a Potential Biomarker for Crohn’s Disease-Associated Intestinal Fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-10 16:17:15","doi":"10.21203/rs.3.rs-8881694/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":"907a9ee1-5809-43d4-99bb-3dbde3740310","owner":[],"postedDate":"March 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64077352,"name":"Health sciences/Biomarkers"},{"id":64077353,"name":"Health sciences/Diseases"},{"id":64077354,"name":"Health sciences/Gastroenterology"},{"id":64077355,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-04-21T09:26:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-10 16:17:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8881694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8881694","identity":"rs-8881694","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}