Mucin levels in glands of the colonic mucosa of rats with diversion colitis subjected to enemas containing sucralfate and n-acetylcysteine alone or in combination.

PURPOSE: To evaluate the tissue content of neutral and acidic mucins, sulfomucins and sialomucins in colonic glands devoid of intestinal transit after enemas containing sucralfate and n-acetylcysteine alone or in combination. METHODS: Sixty-four rats underwent intestinal transit bypass. A colonic segment was collected to compose the white group (without intervention). After derivation, the animals were divided into two groups according to whether enemas were performed daily for two or four weeks. Each group was subdivided into four subgroups according to the substance used: control group: saline 0.9%; sucralfate group (SCF): SCF 2 g/kg/day; n-acetylcysteine group (NAC): NAC 100 mg/kg/day; and SCF+NAC group: SCF 2 g/kg/day + NAC 100 mg/kg/day.Neutral and acidic mucins were stained by periodic acid-Schiff and alcian-blue techniques, respectively. The distinction between sulfomucins and sialomucin was made by the high alcian-blue iron diamine technique. The content of mucins in the colonic glands was measured by computerized morphometry. The inflammatory score was assessed using a validated scale. The results between the groups were compared by the Mann-Whitney's test, while the variation according to time by the Kruskal-Wallis' test (Dunn's post-test). A significance level of 5% was adopted. RESULTS: There was reduction in the inflammatory score regardless of the application of isolated or associated substances. Intervention with SCF+NAC increased the content of all mucin subtypes regardless of intervention time. CONCLUSIONS: The application of SCF+NAC reduced the inflammatory process of the colonic mucosa and increased the content of different types of mucins in the colonic glands of segments excluded from fecal transit.

Total flavonoids of Rhizoma Drynariae enhances CD31hiEmcnhi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis by activating PDGF­BB/VEGF/RUNX2/OSX signaling axis.

Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney­tonifying Traditional Chinese medicine Rhizoma Drynariae, can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31hiEmcnhi during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31hiEmcnhi vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co­immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31hiEmcnhi vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow­derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet­derived growth factor (PDGF)­BB, VEGF, runt­related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription­quantitative PCR. The in vivo assays demonstrated that TFRD markedly promoted CD31hiEmcnhi vessel formation during DO, whereas PDGF­BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF­BB. The protein and mRNA levels of PDGF­BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF­BB. Thus, TFRD could facilitate CD31hiEmcnhi vessel formation and subsequently enhance angiogenic­osteogenic coupling to regenerate bone defects during DO via the PDGF­BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31hiEmcnhi vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31hiEmcnhi vessels.

Tissue sulfomucin and sialomucin content in colon mucosa without intestinal transit subjected to intervention with Curcuma longa (curcumin).

PURPOSE:: To measure the tissue sulfomucin and sialomucin content of the colon mucosa without fecal flow, subjected to intervention with curcumin, and the influence of the concentration used and the intervention time. METHODS:: Thirty-six rats were subjected to proximal right colostomy and distal mucous fistula. They were divided into two groups according to whether sacrifice was performed two or four weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone; curcumin at 50 mg/kg/day or curcumin at 200 mg/kg/day. Acid mucins were diagnosed using the Alcian blue technique. The mucin content was quantified by means of computer-assisted image analysis. The significance level of 5% was used throughout (p < 0.05). RESULTS:: There were dose-related increases in the quantities of sulfomucins in the animals subjected to interventions with curcumin, both after two weeks (p < 0.00001) and after four weeks (p < 0.00001). There were increases in sialomucin quantity that were concentration-related (p < 0.00001) and time-related (p < 0.00001). CONCLUSION:: Curcumin enemas increase the quantity of acid mucins in the intestinal flow in the excluded colon, with dose and time dependency.

Tissue sulfomucin and sialomucin content in colon mucosa without intestinal transit subjected to intervention with Curcuma longa (curcumin)

Abstract Purpose: To measure the tissue sulfomucin and sialomucin content of the colon mucosa without fecal flow, subjected to intervention with curcumin, and the influence of the concentration used and the intervention time. Methods: Thirty-six rats were subjected to proximal right colostomy and distal mucous fistula. They were divided into two groups according to whether sacrifice was performed two or four weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone; curcumin at 50 mg/kg/day or curcumin at 200 mg/kg/day. Acid mucins were diagnosed using the Alcian blue technique. The mucin content was quantified by means of computer-assisted image analysis. The significance level of 5% was used throughout (p < 0.05). Results: There were dose-related increases in the quantities of sulfomucins in the animals subjected to interventions with curcumin, both after two weeks (p < 0.00001) and after four weeks (p < 0.00001). There were increases in sialomucin quantity that were concentration-related (p < 0.00001) and time-related (p < 0.00001). Conclusion: Curcumin enemas increase the quantity of acid mucins in the intestinal flow in the excluded colon, with dose and time dependency.

Tissue content of sulfomucins and sialomucins in the colonic mucosa, without fecal stream, undergoing daily intervention with sucralfate.

PURPOSE: To measure the content of acidic mucin, sialomucin, and sulfomucins in the colonic mucosa without fecal stream submit to intervention with sucralfate (SCF). METHODS: Thirty-six rats were submitted to a right colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, SCF at 1.0 g/kg/day or 2.0 g/kg/day. Colitis was diagnosed by histological analysis. Acid mucins were determined with the Alcian-Blue and sulfomucin and sialomucin by high iron diamine-alcian blue (HID-AB) techniques. The mucins were quantified by computer-assisted image analysis. Mann-Whitney and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). RESULTS: SCF enemas decreased the inflammation score and was related to the concentration used and time of the intervention. SCF at both concentrations increased the content of acid mucin, which was related to the concentration used and to the improvement in the inflammatory score. There was an increase in the content of sulfomucins and sialomucins in SCF groups. SCF increased sulfomucins from 2 weeks of intervention, which was not related to the dose or time of application. The increase in sialomucin content was related to the time and dose used in the intervention. CONCLUSION: Sucralfate increased the content of acidic mucins, primarily at the expense of sialomucin, which was affected by the dose and time of intervention.

Tissue content of sulfomucins and sialomucins in the colonic mucosa, without fecal stream, undergoing daily intervention with sucralfate

PURPOSE: To measure the content of acidic mucin, sialomucin, and sulfomucins in the colonic mucosa without fecal stream submit to intervention with sucralfate (SCF). METHODS: Thirty-six rats were submitted to a right colostomy and a distal mucous fistula and divided into two groups according to sacrifice to be performed two or four weeks. Each group was divided into three subgroups according daily application of enemas containing saline, SCF at 1.0 g/kg/day or 2.0 g/kg/day. Colitis was diagnosed by histological analysis. Acid mucins were determined with the Alcian-Blue and sulfomucin and sialomucin by high iron diamine-alcian blue (HID-AB) techniques. The mucins were quantified by computer-assisted image analysis. Mann-Whitney and ANOVA tests were used to analyze the results establishing the level of significance of 5% for both (p<0.05). RESULTS: SCF enemas decreased the inflammation score and was related to the concentration used and time of the intervention. SCF at both concentrations increased the content of acid mucin, which was related to the concentration used and to the improvement in the inflammatory score. There was an increase in the content of sulfomucins and sialomucins in SCF groups. SCF increased sulfomucins from 2 weeks of intervention, which was not related to the dose or time of application. The increase in sialomucin content was related to the time and dose used in the intervention. CONCLUSION: Sucralfate increased the content of acidic mucins, primarily at the expense of sialomucin, which was affected by the dose and time of intervention. .

Glycyrrhizic acid suppresses the development of precancerous lesions via regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of Wistar rats.

BACKGROUND: Colon carcinogenesis is a multistep process and it emanates from a series of molecular and histopathological alterations. Glycyrrhizic acid (GA) is a natural and major pentacyclic triterpenoid glycoside of licorice roots extracts. It has several pharmacological and biological properties such as anti-inflammatory, anti-viral, and anti-cancer. In the present study, we investigated the chemopreventive potential of GA against 1,2-dimethyhydrazine (DMH)-induced precancerous lesions i.e., aberrant crypt foci (ACF) and mucin depleted foci (MDF), and its role in regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of Wistar rats. METHODS: Animals were divided into 5 groups. In group III, IV and V, GA was administered at the dose of 15 mg/kg b. wt. orally while in group II, III and IV, DMH was administered subcutaneously in the groin at the dose of 20 mg/kg b.wt once a week for first 5 weeks and animals were euthanized after 9 weeks. RESULTS: GA supplementation suppressed the development of precancerous lesions and it also reduced the infiltration of mast cells, suppressed the immunostaining of Ki-67, NF-kB-p65, COX-2, iNOS and VEGF while enhanced the immunostaining of p53, connexin-43, caspase-9 and cleaved caspase-3. GA treatment significantly attenuated the level of TNF-α and it also reduced the depletion of the mucous layer as well as attenuated the shifting of sialomucin to sulphomucin. CONCLUSION: Our findings suggest that GA has strong chemopreventive potential against DMH-induced colon carcinogenesis but further studies are warranted to elucidate the precise mechanism of action of GA.

Butyrate enemas do not affect human colonic MUC2 and TFF3 expression.

INTRODUCTION: The colonic mucus layer plays an important role in the protection of the intestinal epithelium and mainly consists of mucin glycoproteins (primarily MUC2 in the colon) trefoil factor 3 (TFF3) and secretory IgA. Butyrate is a major end product of fermentation of dietary fibres and is associated with beneficial effects on colonic health. Earlier in-vitro and animal studies showed that butyrate modulates MUC2 and TFF3 expression and mucin secretion, although data from human studies are not yet available. METHODS: Sixteen healthy volunteers and 35 ulcerative colitis (UC) patients in clinical remission self-administered a 60 ml rectal enema containing 100 mmol/l butyrate or placebo once daily for 2 and 3 weeks, respectively. After each treatment, biopsies were taken from the distal sigmoid for quantitative RT-PCR and immunohistochemical analysis of MUC2 and TFF3. In addition, mucosal sections were stained with high iron diamine-alcian blue to distinguish between sialomucins and sulphomucins. To analyse total mucin secretion and secretory IgA concentrations, 24 h faeces were collected during the day before the endoscopic examination. RESULTS: The butyrate intervention did not significantly modulate the expression of MUC2 (fold change: 1.04 and 1.05 in healthy volunteers and ulcerative colitis patients, respectively) or TFF3 (fold change: 0.91 and 0.94 in healthy volunteers and UC patients, respectively). Furthermore, the percentage of sialomucins, mucus secretion and secretory IgA concentrations were not affected by the butyrate intervention in both the groups. CONCLUSION: Butyrate exposure in healthy volunteers and UC patients in remission did not affect the measured parameters of the colonic mucus layer.

Observations of high iron diamine-alcian blue stain in uterine cervical glandular lesions.

We studied epithelial glycoproteins in uterine cervical lesions, including glandular lesions, and investigated whether a more accurate diagnosis could be obtained using high iron diamine-alcian blue (HID-AB) stain and immunostaining with carcinoembryonic antigen (CEA). In addition, we examined the usefulness of preoperative diagnosis using biopsy specimens stained with HID-AB and CEA. Normal endocervical glands showed a predominance of sulfomucin, while adenocarcinoma predominantly showed sialomucin. CEA was highly detected in adenocarcinoma, but not in normal endocervix or in glandular dysplasia, suggesting that this stain has high specificity. The staining patterns of biopsy specimens and hysterectomy specimens were similar. Therefore HID-AB stain and CEA stain may be useful as a supplementary means of diagnosing uterine cervical lesions.

Ground squirrel model for cholelithiasis: role of epithelial glycoproteins.

The cholesterol-fed Richardson's ground squirrel (Spermophilus richardsonii) has proven to be an effective animal model in which to study factors that influence cholesterol gallstone formation and associated alterations in the gallbladder epithelium. Ground squirrels of either sex, fed a 2% cholesterol-enriched diet, exhibit cholesterol monohydrate crystal precipitation within 24 hours and macroscopically visible cholesterol stones by 3 weeks. Data on bile chemistry, biliary cholesterol precipitation, and various mucosal alterations occurring prior to, during, and after stone formation were collected using sampling intervals from 6 hours to 20 weeks on the diet. The results indicate that mucin hypersecretion appears to be more closely related to the initiation of nucleation than does either bile calcium of pH. Mucus hypersecretion begins within 18 hours of diet initiation and continues throughout the 20 week experimental period. Apical excrescences became more common and were larger in size during the early stages of cholelithiasis. Administration of aspirin during the experimental period demonstrated an inhibition of mucin synthesis and release. Gallstones were not formed in these aspirin-treated animals. A lectin-binding panel for 10 epithelial glycoprotein-related sugars indicated the mucin secreted by the gallbladder epithelium of 7 day experimental animals differed from that of controls. The most obvious difference was the abolition of WGA binding in the experimental animals, suggesting an absence of sialic acid expression in the mucin during the lithogenic process. Ultrastructural histochemistry indicated that both sulphomucin and sialomucin were present in the secretory granules and within the surface mucus layer of both experimental and control animals. Experimental animals, however, exhibited a significant predominance for sulphomucin. This pattern varies from that typically seen in other regions of the gastrointestinal tract where sialomucins predominate during pathologic processes.

Assessment of dysplasia, mucosal mucins, p53 protein expression, and DNA content in ulcerative colitis patients with colectomy and ileorectal anastomosis.

BACKGROUND: Patients with ileorectal anastomosis after colectomy for ulcerative colitis remain at risk of developing rectal malignancy. Detection of mucosal dysplasia has been used for regular screening but is difficult in inflammatory mucosa, prompting the search for complementary markers. METHODS: This prospective study aimed to assess the prevalence of dysplasia, the predominance of sialomucin, DNA aneuploidy, and p53 overexpression as possible predictors of colorectal tumourigenesis, in the rectal mucosa of an unselected group of 27 patients with ileorectal anastomosis performed for ulcerative colitis. Patients had neither neoplastic nor dysplastic lesions on the colectomy specimen and the retained rectum at the time of surgery. One biopsy specimen of each lateral rectal wall was studied, using routine histology, mucin histochemistry, DNA flow cytometry, and the streptavidin-biotin complex method with D07 monoclonal antibodies directed towards the p53 protein. RESULTS: Seventeen, seven, and three patients showed inflammatory lesions of inactive, moderate, and severe active colitis, respectively. Dysplasia, sialomucin predominance, DNA aneuploidy, and p53 overexpression were not detected. CONCLUSIONS: The risk of malignant transformation of the rectal mucosa after ileorectal anastomosis seemed to be low in this ulcerative colitis group without high-grade dysplasia or carcinoma in the previous colectomy specimen, carefully followed up endoscopically and histologically. It remains to be evaluated which of the methods studied above will optimize the histopathologic surveillance of the rectal mucosa of ulcerative colitis patients with ileorectal anastomosis.

Efficacy of differential mucin staining for predicting synchronous and metachronous colorectal carcinomas.

Despite the controversy regarding the significance and usefulness of histochemically differential mucin staining as a marker for colorectal neoplasms, some investigators have used this technique to help define those persons at risk for development of recurrent colorectal carcinoma. To further evaluate the efficacy of this method, we reviewed 85 surgical specimens of the colon and rectum using a high iron diamine-alcian blue staining technique. The group studied included 73 patients with synchronous or metachronous carcinomas and adenomas and 12 patients with no recurrence for more than 5 years who had undergone adequate follow-up, including physical examination; evaluation of serum chemistry findings; and colonoscopy, barium enema, or both. Evaluation of Dukes' staging (A and B1 versus B2 and C2) and distance of the resection margins from the tumor (less than 5 cm versus 5 cm or more) showed no correlation with the amount of sialomucin present. Resection margins from patients with either synchronous or metachronous carcinomas had significantly higher sialomucin ratios than the group without recurrence, whereas those with synchronous or metachronous adenomas did not. The false-negative rate was 4 percent (1 of 23 specimens) and the false-positive rate, 42 percent (5 of 12 specimens). We conclude that this method for evaluating resection margins of colorectal cancer specimens with differential mucin staining is highly reliable for predicting the population with synchronous and metachronous colorectal carcinomas.