Alterations And Clinical Efficacy Accessment Of Syndecan-1 In Inflammatory Bowel Diseases

Background and AimsSyndecan-1 (Sdc1, CD138), which widely distributes on the surface of mammalian epithelial cells, is a predominant member of type Ⅰ transmembrane heparan sulfate proteoglycans. Its structure mainly consists of a short conserved cytoplasmic and transmembrane domain, and a long, variable ectodomain carrying heparan sulfate (HS) chains, which dock with many chemokine, growth factors, matrix components, etc. Sdcl plays important roles in inflammation, wound healing and tumor progression by controlling cell proliferation, differentiation, adhesion and migration. In physiological conditions, Sdc1 is one of the key components of intestinal mucosal barrier and its expression is closely associated with the barrier function. With the help of HS chains, Sdcl regulates and assists a variety of growth factors combining to their receptors on cells, activates downstream signaling pathway and maintains the regeneration of epithelial cells. It also enhances the expression of tight junctions (such as Occludin, Claudin, etc.), blocks the interplay between β1 integrin and pathogens, and prevents the destruction of ZO-1. Sdc1 would be destructed when accompanied by trauma, infection, ischemia, immune disorders, etc., and hence disturb its essential functions to intestinal barrier. Sdcl loss might amplify the response to IFN-γ and TNF-α, reduce the expression of tight junctions, and increase apoptosis, leading to some specific disorders, such as protein-losing enteropathy, secretory diarrhea and malnutrition. After Sdc1 knockdown, the growth factor-dependent proliferations of intestinal epithelial cells are decreased remarkably and the healing of impaired gut is serious delay.The pathogenesis of Inflammatory Bowel Diseases (IBD), including Crohn’s disease and Ulcerative Colitis, is still unknown. They seem to be caused by a combination of genetic predisposition, autoimmune, environmental abnormalities, and psychological disorders. Although many endoscopic, radiological and histological criteria have been recommended, the difficulties on diagnosis and treatment of these diseases are always challenging. In recent years, growing attentions have been focused on the relationships between Sdcl and IBD, but their interactions and the mechanisms are not yet clear. Generally, Sdcl binds with a variety of inflammatory cells, chemokine, growth factor and other bio-substances which involve in inflammatory process. The shedding of Sdcl ectodomain is accelerated after stimulations of specific proteases such as heparanase and matrix metalloproteinases (MMPs). It is also mediated by TNF-a, INF-y, thrombin, RANTES, EGF and TGF-β, etc., and involves several activated signal pathways, such as PKC, PTK, MAPK. As similar structure and function, soluble Sdcl may acts as an analog, or even an antagonist, to the membrane-anchored Sdcl. Excessive soluble Sdcl reduces the tolerance of epithelial cells to IFNy/TNFα, facilitates the formation of chemokine gradient to attract neutrophils, and promotes fibrosis by inhibiting epithelial repair.Due to special epitopes and polyanionic nature, Sdcl may assist bacteria, fungus and virus to adhesion and invasion. Most of Gram-positive bacteria present heparin-binding protein in the cell membrane. The bacteria exploit it to combine with Sdcl and then facilitate the process of endocytosis. Exogenous HS analogues (heparin) significantly block endocytosis in HT-29 cells with a high expression of Sdcl. The conservative RGD integrin binding motif replaced by RGSD motif, Foot-and-mouth disease virus, which is one of the human entero virus, obtains the ability to bind to cell surface HS and significantly improves its attack efficiency. Newborn mice deficient in syndecan-1 resist Pseudomonas aeruginosa and Staphylococcus aureus infection, but become susceptible when were given purified syndecan-1 ectodomains or heparin. Some pathogens enhance their colonization and damage through accelerating Sdcl ectodomains shedding by their virulence factors (such as LasA). These evidences indicate that Sdcl may have different roles in intestinal infection diseases from those IBD.Presumably, the abnormity expression and the shedding of Sdc I are important parts of intestinal inflammation. Despite the existing researches reveal the links between Sdcl and inflammatory factors, it is not clear whether there is a remarkable change of Sdcl when animals and humans are suffering from IBD. Their potential mechanism of regulation and overall roles are still awaiting assessment. Based on the above background, this study intended to measure the expression of Sdc1, analyze the internal relationship among Sdcl, regulatory factors and clinical features, and systematically evaluate the value of Sdcl and its analogues in the diagnosis and treatment. It is expected to provide theoretical foundations and clinical evidences for deepening the understanding and optimizing treatment strategies of IBD.Research Contents1. Induce animal enteritis model, measure the expression of Sdcl, related cytokines (TNF-a, TGF-β) and proteases (Heparanase, MMP-7) to confirm the alteration of Sdcl and potential mechanism of regulation in enteritis condition.2. Collect tissue and serum specimen from IBD patients, measure the expression of Sdcl and its protease, and evaluate their relevance with clinical features and value in observation and diagnosis.3. Systematically evaluate the value of Sdc1 and its analogue in the treatment of IBD through animal experiments and systematic review.Subjects and methods1. A total of 24 male NIH mice (weighting about 18-22 g) were obtained from the Animal Center of Southern Medical University (Guangzhou, China). Mice were divided randomly into the control group, acute enteritis group, chronic enteritis group, and heparin treatment group composed of 6 mice each and all were given standard rodent chow. In the control group, mice were given fresh distilled water and were killed on day 49. In acute enteritis group, mice were all given 5% DSS in distilled water for 7 days, and were killed on day 7. In chronic enteritis group, mice were all given 5% DSS in distilled water on day 1 to 7, day 22 to 28 and day 43 to 49, fresh distilled water on the other days, and killed on day 49. In the heparin treatment group, mice had the same DSS administration as the chronic enteritis group, and were given 0.5ug/g (weight) of low molecular weight heparin (LMWH) through subcutaneous injection on the Day 1 and 4 of each DSS cycle. Mice weight and stool characters were recorded and a disease activity index (DAI) was determined daily by an investigator blinded to the protocol.2. Mice were sacrificed after anesthesia. Blood was collected from the hearts and the colons were removed through a midline incision. For each tissue sample, a histological examination with H-E staining was given. Tissue expressions of Sdcl, Heparanase, Metalloproteinase-7 (MMP-7), Tumor Necrosis Factor a (TNF-a), Transforming Growth Factor β(TGF-β) were determined by Western blotting. Reverse transcription PCR (RT-PCR) was employed to detect the level of Sdcl mRNA and enzyme-linked immunosorbent assay (ELISA) were used to measure serum Sdc1 in each mouse.3. Hospitalized patients with Functional Bowel Disorders (FBD), Intestinal Tuberculosis (ITB) and Crohn’s Disease (CD) in Nanfang hospital from 2008 to 2010, were included. Clinical data, imagine, endoscopy and pathology records were recorded. Tissue specimens were obtained from normal mucosa (in FBD patients, parts of remission CD patients) or lesions (in ITB and CD patients) when patients underwent endoscopy or quickly after surgery, then washed twice with chilled phosphate buffered saline, immediately isolated and stored in liquid nitrogen and then at -80℃ in tissue bank until further use. Venous blood was collected after an overnight fast. Routine blood tests were soon analyzed by Department of Laboratory, Nanfang Hospital. Serum was obtained after 15 minutes,1800xg centrifugated and the aliquots of the serum were stored at-80℃ until analysis.4. For each tissue sample from patients, a histological examination with H-E staining was given. Paraffin biopsies and serum samples were studied by immunohistochemisty and ELISA to detect both SYD1 and HPA. RT-PCR was employed to detect the level of Sdcl mRNA.5. PubMed, Embase, Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI) and Wanfang data resource system were searched up to December 2012. Randomized controlled trials (RCTs) recruited subjects with inflammatory bowel disease and prescriptions of HS analogues (heparin) were eligible. Efficacy and safety of heparin were reevaluated, and dichotomous data were pooled to obtain odd ratio (OR) with a 95% confidence interval. Heterogeneity and publication bias were assessed by the inconsistency index statistic and funnel plot analysis, respectively.6. For experiments measuring Sdcl and others biomarkers, statistical analysis was performed with SPSS 13.0 software (Chicago, IL, USA). Descriptive statistics were calculated with means and standard deviation. When Data are normally distributed and the variances are equal, Independent samples T-test for comparing means between two groups and One-way ANOVA test for among three or more groups (with LSD method for post Hoc Multiple Comparisons) were used; when the variances are not equal, Satterthwaite’s approximate t test and Welch’s F test (with Dunnett’s T3 method for post Hoc Multiple Comparisons) were used respectively; but when data aren’t normally distributed, Mann-Whitney U test and Kruskal-Wallis H test were used respectively. The enumeration data were calculated with sample number and percentage and compared with the χ2 test. Comparisons of Ranked data were determined by Mann-Whitney U Test (between two independent groups) or Kruskal-Wallis H Test (among multiple independent groups). Spearman’s correlation analysis was employed to define associations. A P-value less than 0.05 was considered significant.The extracted data for systematic review and meta-analysis in Part 3 were analyzed using RevMan (Version 5.2, Cochrane Collaboration, Denmark) and Comprehensive Meta Analysis software (Version 2.2023, Englewood, NJ, USA). Binary outcomes were compared by odd ratio (OR) and continuous outcomes by weighted mean difference (WMD), and then presented in forest plots. The 95% confidence interval (CI) was used and p value< 0.05 was assumed to be showing a statistically significant difference. The statistical significance for heterogeneity was assessed by use of the Chi2-based Q statistic and the I2 statistic for the extent of heterogeneity. Heterogeneity was considered significant if p<0.1 and I2 was more than 50%. The fixed effect model was employed in pooling data where there was no evidence of heterogeneity and where there was evidence of heterogeneity, the random effects model was used instead. Sensitivity analyses were performed by excluding studies with unclear quality. Possible publication bias or other small study effects were evaluated using the Egger test.Results1. In two enteritis groups, significant symptoms (including weight loss, diarrhea, rectal bleeding) were observed. The DAI and histological score showed significant higher in these two groups than those in the control group.2. Compared to control group, protein level of Sdcl significantly decreased in colon tissue of enteritis groups (P=0.002), but their mRNA level of Sdcl was similar (P=0.615). The expression of Sdcl protein was much lower in chronic enteritis group than in acute enteritis group (IOD median,0.50 vs.0.70).3. Protein levels of MMP-7, Heparanase and TNF-a were much higher in the enteritis groups than the control group (P=0.003,0.007,0.002). No significant changes in the protein expression of TGF-P were detected (P=0.415).4. Eighty-nine patients, including 15 FBD patients,18 active ITB patients,56 CD patients (remission n=19, active n=37) had been enrolled. In CD group, Sdcl was significantly decreased in mucosa and increased in serum, while staining intensity and positive proportion of heparanase significantly elevated (all P<0.05).5. A remarkable alteration of Sdcl was acquired from CD group when the disease activity increasing. The reduction of Sdcl ectodomain negatively correlated with CDAT (P<0.05).6. The alterations of Sdcl and heparanase in CD group were significantly associated with clinical manifestations representing disease activity (melena/haemorrhage, fever, weight loss, etc.), and the severity of intestinal epithelial injury (rectal/perianal disorder, extensive bowel lesion, tissue inflammation, etc.) (P<0.05).7. Compared to the chronic enteritis group, higher content of Sdcl protein, DAI, and histological scores were shown in heparin treatment group (all P<0.01).8. The search identified 1863 citations, and 12 RCTs including 686 individuals were eligible. In general, heparin performed a non-inferior role to the conventional therapy when was used as first-line drug for UC (OR=2.50,95%CI:1.05-5.93, P=0.04). When was used as second-line drug, it could enhance the therapeutic effects of the existing conventional therapy (OR=2.51,95%CI:1.31-4.83, P=0.006). Specifically, heparin administrated significantly improved the conditions of UC patients with mild to moderate condition (OR=3.22,95% CI:1.29-8.05, P=0.01), but failed for those with moderate to severe condition (OR=0.72,95%CI:0.03-19.01, P=0.85). Heparin treatment was generally safe except that 1 trial reported 3 cases of severe intestinal bleeding.Conclusions:1. Sdcl shedding is activated in DSS-induced enteritis. It is positively correlated with the extent of intestinal inflammation and might be synergistically regulated by Sdc1 protease and proinflammatory mediators.2. Unique alterations of Sdcl and heparanase were showed in CD patients, which indicate they are potential markers for Crohn’s disease in evaluating its disease activity and differentiating it from ITB.3. Exogenous Sdcl analogue (heparin) helps alleviate Sdcl reducation, inflammation levels and intestinal injury. Current clinical evidences show heparin is reasonable effective and general safe for patients with IBD, but more well-designed RCTs should be conducted to fully confirm its practical value.