Immunomodulatory effect of Hawthorn extract in an experimental stroke model

<p>Abstract</p> <p>Background</p> <p>Recently, we reported a neuroprotective effect for Hawthorn (<it>Crataegus </it><it>oxyacantha</it>) ethanolic extract in middle cerebral artery occlusion-(MCAO) induced stroke in rats. The present study sheds more light on the extract's mechanism of neuroprotection, especially its immunomodulatory effect.</p> <p>Methods</p> <p>After 15 days of treatment with Hawthorn extract [100 mg/kg, pretreatment (oral)], male Sprague Dawley rats underwent transient MCAO for 75 mins followed by reperfusion (either 3 or 24 hrs). We measured pro-inflammatory cytokines (IL-1β, TNF-α, IL-6), ICAM-1, IL-10 and pSTAT-3 expression in the brain by appropriate methods. We also looked at the cytotoxic T cell sub-population among leukocytes (FACS) and inflammatory cell activation and recruitment in brain (using a myeloperoxidase activity assay) after ischemia and reperfusion (I/R). Apoptosis (TUNEL), and Bcl-xL- and Foxp3- (T_regmarker) positive cells in the ipsilateral hemisphere of the brain were analyzed separately using immunofluorescence.</p> <p>Results</p> <p>Our results indicate that occlusion followed by 3 hrs of reperfusion increased pro-inflammatory cytokine and ICAM-1 gene expressions in the ipsilateral hemisphere, and that Hawthorn pre-treatment significantly (<it>p </it>≤ 0.01) lowered these levels. Furthermore, such pre-treatment was able to increase IL-10 levels and Foxp3-positive cells in brain after 24 hrs of reperfusion. The increase in cytotoxic T cell population in vehicle rats after 24 hrs of reperfusion was decreased by at least 40% with Hawthorn pretreatment. In addition, there was a decrease in inflammatory cell activation and infiltration in pretreated brain. Hawthorn pretreatment elevated pSTAT-3 levels in brain after I/R. We also observed an increase in Bcl-xL-positive cells, which in turn may have influenced the reduction in TUNEL-positive cells compared to vehicle-treated brain.</p> <p>Conclusions</p> <p>In summary, Hawthorn extract helped alleviate pro-inflammatory immune responses associated with I/R-induced injury, boosted IL-10 levels, and increased Foxp3-positive T_regsin the brain, which may have aided in suppression of activated inflammatory cells. Such treatment also minimizes apoptotic cell death by influencing STAT-3 phosphorylation and Bcl-xL expression in the brain. Taken together, the immunomodulatory effect of Hawthorn extract may play a critical role in the neuroprotection observed in this MCAO-induced stroke model.</p

Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium

BACKGROUND: Helicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry. METHODS: RUT and ELISA were down for H. pylori confirmation. Human gastric biopsy sections were stained using immunohistochemistry with MUC2, MUC5AC and MUC6 antibodies. RESULTS: MUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric glands. MUC2 was expressed in intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the progressive stages of H. pylori infected human gastric epithelium allows the identification of intestinal metaplasia, which is characterized by a decreased expression of the gastric mucins (MUC5AC and MUC6) and de novo expression of MUC2. CONCLUSION: In conclusion, our results suggest that there is altered expression of MUC5AC and MUC6 together with the aberrant expression of MUC2 in intestinal metaplasia, during the process of gastric carcinogenesis. The present study indicates that the MUC2 mucin expression pattern is a reliable marker of intestinal metaplasia, which appears in the context of H. pylori infected individuals

EVALUATING THE ANTIPROLIFERATIVE POTENTIAL OF METHONOLIC LEAF EXTRACT OF CASSIA NIGRICANS

Objective: It is well established that plants have always been useful source as anticancer compounds. This study was attempted to investigate the in vitro anti-cancer potential of methonolic extract of Cassia nigricans on breast cancer MCF-7 cell lines.Methods: The methanol extract of C. nigricans was screened for its anti-proliferative effect against MCF-7 (Breast cancer) cell lines using MCF-7 cells seeded 96 well plates.Results: Extract was exposed with MCF-7 cell lines for 24h and 72h at a range of increasing concentrations (0-500μg/ml) in order to obtain a dose-response graph and IC50 value. The C. nigricans extract showed cytotoxic effect in MCF-7cells with IC50 of 82.6μg/ml.Conclusion: The C. nigricans extract showed effective cytotoxic activity in a dose and time dependent manner. Future work will be interesting to know the chemical composition and also better understand the mechanism of action present in the extract for developing it as drug for therapeutic application.Â

Expression of peanut agglutinin-binding mucin-type glycoprotein in human esophageal squamous cell carcinoma as a marker

BACKGROUND: The TF (Thomson – Friedenreich) blood group antigen behaves as an onco-foetal carcinoma-associated antigen, showing increased expression in malignancies and its detection and quantification can be used in serologic diagnosis mainly in adenocarcinomas. This study was undertaken to analyze the sera and tissue level detectable mucin-type glycoprotein (TF-antigen) by Peanut agglutinin (PNA) and its diagnostic index in serum as well tissues of human esophageal squamous cell carcinoma as marker. RESULTS: We examined 100 patients for serological analysis by Enzyme Linked Lectin Assay (ELISA) and demonstrated a sensitivity of 87.5%, specificity of 90% and a positive predictive value of 95%. The immuno-histochemical localization of TF antigen by Fluorescence Antigen Technique (FAT) in 25 specimens of normal esophageal squamous epithelium specimens and 92 specimens with different grades of, allowed a quicker and more precise identification of its increased expression and this did not correlate with gender and tumor size. There was a positive correlation between membrane bound TF antigen expression with different histological progression, from well differentiated to poorly differentiated, determined by PNA binding. Specimens showed morphological changes and a pronounced increase in PNA binding in Golgi apparatus, secretory granules of the cytosol of well differentiated and an increased cell membrane labeling in moderately and poorly differentiated, when compared with ESCC and normal tissues. CONCLUSION: The authors propose that the expression of TF-antigen in human may play an important role during tumorigenesis establishing it as a chemically well-defined carcinoma-associated antigen. Identification of the circulating TF-antigen as a reactive form and as a cryptic form in the healthy individuals, using PNA-ELLA and Immunohistochemical analysis of TF antigen by FAT is positively correlated with the different histological grades as a simple and cost-effective method for the early diagnosis of ESCC. The present study reveals that, during tumorigenesis, an aberrant glycosylation takes place in Golgi apparatus leading to over secretion of TF antigen into the cytoplasm along with mucin granules and later into cell membrane. We suggest that the further characterization of TF antigen may unravel pathogenetic aspects of this silent disease

Up-Regulation of MUC2 and IL-1β Expression in Human Colonic Epithelial Cells by Shigella and Its Interaction with Mucins

BACKGROUND: The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis. METHODS: The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence. RESULTS: The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucin<guinea pig colonic mucin< Human colonic mucin. Invasion of Shigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells. CONCLUSIONS: Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells

Apoptosis Inducing Effect of Plumbagin on Colonic Cancer Cells Depends on Expression of COX-2

Plumbagin, a quinonoid found in the plants of the Plumbaginaceae, possesses medicinal properties. In this study we investigated the anti-proliferative and apoptotic activity of plumbagin by using two human colonic cancer cell lines, HT29 and HCT15. IC50 of Plumbagin for HCT15 and HT29 cells (22.5 µM and 62.5 µM, respectively) were significantly different. To study the response of cancer cells during treatment strategies, cells were treated with two different concentrations, 15 µM, 30 µM for HCT15 and 50 µM, 75 µM for HT29 cells. Though activation of NFκB, Caspases-3, elevated levels of TNF-α, cytosolic Cytochrome C were seen in both HCT15 cells HT29 treated with plumbagin, aberrant apoptosis with decreased level of pEGFR, pAkt, pGsk-3β, PCNA and Cyclin D1was observed only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells. This suggests that plumbagin induces apoptosis in both HCT15 cells and HT29 treated, whereas, proliferation was inhibited only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells, but not in 50 µM plumbagin treated HT29 cells. Expression of COX-2 was decreased in 75 µM plumbagin treated HT29 cells when compared to 50 µM plumbagin treated HT29 cells, whereas HCT15 cells lack COX. Hence the observed resistance to induction of apoptosis in 50 µM plumbagin treated HT29 cells are attributed to the expression of COX-2. In conclusion, plumbagin induces apoptosis in colonic cancer cells through TNF-α mediated pathway depending on expression of COX-2 expression

Isolation and Partial Characterisation of a Novel Lectin from Aegle marmelos Fruit and Its Effect on Adherence and Invasion of Shigellae to HT29 Cells

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 µg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host

Antioxidant effect of eugenol in rat intestine

1192-1195<span style="font-size:14.0pt;line-height: 115%;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" color:black;mso-ansi-language:en-in;mso-fareast-language:en-in;mso-bidi-language:="" hi"="" lang="EN-IN">The effect of eugenol on the antioxidant status of the rat intestine after short and long term (15 days and 90 days, respectively) oral administration of 1000 mg/kg.b.wt (a dosage which has been reported to be highly hepatoprotective)1 was studied. The level of lipid peroxidation products (TBARS) and the activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) were found to be near normal on eugenol treatment. The level of glutathione (GSH) did not show any change on 15 days of eugenol treatment, but it was increased significantly on 90 day eugenol treatment. The activit y of glutathione-S-transferases (GSTs) was increased significantly in both 15 day eugenol treated and 90-day eugenol treated groups. The results suggest that eugenol is nontoxic, protective and induces, glutathione-S-transferases (GSTs) and thereby it may facilitate the removal of toxic substances from the intestine.</span