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Objective:To evaluate immune response of murine peritoneal macrophage challenging by methicillin-resistant S.aureus(MRSA)after pretreatment with Pam3Csk4(TLR2 agonist).Methods: Murine peritoneal macrophage was pretreated with Pam3Csk4(1 μg/ml).Following pretreatment 12 h later,heat-killed MRSA( HK-MRSA) was added and incubated for another 2 or 6 hours.The protein and mRNA level of TNF-α, IL-6 and IL-1 were determined by ELISA and Q-PCR, respectively.To estimate phagocytosis of macrophage,HK-MRSA/MSSA labeled with FITC( FITC-HK-MRSA/MSSA) were added to well and incubated for 30 min.After washing 5 times with PBS,intracellular FITC-HK-MRSA was detected by flow cytometry.To estimate antimicrobal activity of macrophage,live MRSA and MSSA were added to well and incubated at indication time,the CFU of s.aureus was estimated via a 10-fold serial dilution on agar media.cDNA was further quantitative assessed using primers for mouse FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 by Q-PCR .Results: Compared with saline-pretreated cell, the protein and mRNA level of TNF-α, IL-6 and IL-1 were markely reduced, respectively.However, both the phagocytosis and antimicrobal activity to S.aureus were significantly increased in macrophages pretreated with Pam3Csk4.Further study found that the macrophages had higher FCR-Ⅰ,FCR-Ⅲ,CR-1,CR-3,iNOS and LL37 expression at 6 h and 12 h post-stimulation Pam3Csk4.Conclusion: The results suggest that Pam3Csk4 could activate murine antimicrobal activity of peritoneal macrophage challenging by methicillin-resistant Saureus via increasing opsonophagocytosis in depended antibodies, complements manners.The results suggest Pam3Csk4 probably be a novel immunotherapy candidate against MRSA.
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As an inhibitory receptor , T cell immunoglobulin mucin domain containing molecules-3 ( Tim-3 ) expresses high levels in the gastrointestinal tumor microenvironment .Tim-3 can promote T cell exhaustion , negatively regulate the anti-cancer immuni-ty of NK cell and induce polarization of macrophages to M 2.Tumor can escape from immunological surveillance through Tim-3.Tim-3 plays an important role in the development and transformation of gastrointestinal tumors .This article summarizes the role of Tim-3 in the gastrointestinal tumors from the aspects of gene polymorphism , T cells, NK cells and mononuclear macrophage .
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Aim To investigate the effect of triptolide ( TP) on human colorectal cancer HCT116 cell prolif-eration and explore the potential mechanism of TP in treating colon cancer. Methods MTT assay was used for estimating the survival rates of HCT116 cells ex-posed to different concentrations and different duration time of TP. Western blot ( WB ) was used for testing the expression of LC3-Ⅱ. MDC staining was employed to detect cell autophagy. Flow cytometry ( FCM) was applied to test the effects of TP on cell apoptosis. Re-sults TP could inhibit cell proliferation in HCT116 cells. The expression of LC3-Ⅱwas enhanced with the increase of the concentration of TP after exposed to dif-ferent concentrations of TP for 48 h and the duration time of TP after exposed to 40 nmol·L-1 TP,and the number of acid autophagic vacuoles in HCT116 cells had increased, which was observed by fluorescence mi-croscope. The HCT116 cells apoptotic rates in TP group had decreased compared to control group and de-creased significantly compared to TP +3-MA group. The HCT116 cells apoptotic rates in TP group had in-creased compared to control group and increased signif-icantly compared to TP+RAPA group. Conclusion TP could inhibit the proliferation of human colon canc-er HCT116 cells and induce apoptosis in HCT116 cells, which indicates that synergy may exist between autophagy and apoptosis.
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Objective To determine the effect of lipoxins (LX) A4 and its agonist (BML-111) on the survival of RAW264.7 macrophage cells and TLR4/NF-κB signaling pathway. Methods RAW264.7 cells were treated with different concentrations of LPS, then the effect of LX A4 and BML-111 on the survival rate of these cells was observed. Cytotoxicity were detected by CCK-8 method and RT-PCR was used to detect the TLR4 and TRAF6 mRNA. The protein levels of TLR4 and pNF-κB p65 in RAW264.7 were determined by Western Blot. Results The survival rates of macrophage treated with LPS for 6 h in 1 000 ng/mL LPS in LX A4 group and BML-111 group were significantly higher than that in control group (P 0.05). Conclusion LX A4 and BML-111 could inhibit the cytotoxicity of LPS on the RAW264.7 macrophage cells through the inhibition of the activation of TLR4/NF-κB signaling pathway, then reduce the inflammation. And this stable BML-111 may appear as another promising treatment for IBD disease.
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BACKGROUND: Different derivatives of chitosan with different molecular weights or degrees of deacetylation show different anti-tumor effects.OBJECTIVE: To study the inhibition effect of water-soluble chitosan and its derivatives, such as sulfonated chitosan, carboxymethyl chitosan and chitooligosaccharides for the growth of hepatocellular carcinoma cell line SMMC7721.DESIGN, TIME AND SETTING: Controlled experiments based on observation were carried out in Jiangxi Institute of Digestive Disease (Nanchang, Jiangxi, China) from January 2004 to December 2006.MATERIALS: Hepatoma cell line SMMC7721 was provided by Jiangxi Institute of Digestive Disease (China). 85.5% deacetylated chitooligosaccharides and 85% deacetylated water-soluble chitosan were produced by Jinan Haidebei Ocean Biological Engineering Co., Ltd (China); Carboxymethyl chitosan and 88.5% deacetylated chitosan were the products of Shanghai Qisheng Biological Products Co., Ltd (China).METHODS: Sulfonated chitosan was prepared using 88.5% deacetylated chitosan and chlorosulfonic acid-formamide, and then was detected with infrared spectroscopy in the Detection Analysis and Test Center, East China University of Science and Technology. SMMC7721 cells in the log phase were inoculated into 96-well culture plates, which were then added with water-soluble chitosan, sulfonated chitosan, carboxymethyl chitosan and chitooligosaccharides with the final concentrations of 25, 50, 100, 200, 400 and 800mg/L. This test was repeated for 3 times, while the control group was also set each time. After 72 hours of routine culture, MTT solution was added into each well and inoculated for another 4 hours. After the culture was terminated, dimethyl sulfoxid was added. The absorbance value of each well was measured at 490nm wavelength on a microplate reader. Three tests were measured to obtain the mean value. Also the inhibition rate was calculated.MAIN OUTCOME MEASURES: Growth inhibition effect of chitosan and its derivatives on the hepatoma cell line SMMC7721.RESULTS: Among the chitosan and its derivates at four kinds of concentrations, water-soluble chitosan and sulfonated chitosan could significantly inhibit the growth of SMMC7721 cells (P<0.001), and the effect was the most significant in the case of sulfonated chitosan. Treatment with water-soluble chitosan and sulfonated chitosan at the concentration of 50mg/L could inhibit the growth of SMMC7721 cells in a dose-dependent manner, and reached a peak at the concentration of 400mg/L and 800mg/L, respectively. Carboxymethyl chitosan and chitooligosaccharides showed no growth inhibition effect (P>0.05).CONCLUSION: Water-soluble chitosan and sulfonated chitosan have significant antigrowth effects on hepatoma carcinoma cells, while carboxymethyl chitosan and chitooligosaccharides are ineffective.
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Objective To study the anti-Helicobacter pylori(H. pylori) mechanisms of chitosan. Methods H. pylori was suspended in the Chitosan solutions, after 24 h and 48 h, the asparate transaminase (AST) activity and the glucose content in the supernatant s were determined, and morphological alterations of H. pylori were observed by scanning electron microscopy and transmission electron microscopy. Results The AST activity and glucose content in chitosan solutions were significant high er than those in control (P