Inhibitory action of Lipopolysaccharide-induced signal transductions by Valproic acid / Эрүүл мэндийн лаборатори

Introduction@#Valproic acid (VPA) has been used in the treatment of seizures and bipolar disorders. In the present study, we examined how VPA affected PI3K-Akt pathway in response to LPS by using mouse RAW 264.7 macrophage cells.@*Material and methods@#Mouse RAW 264.7 macrophage-like cells cultured and the cell viability checked by MTT and TUNEL assay. In addition, protein expression and protein interaction were detected by immune blotting and immune precipitation, respectively. TLR4 expression on cell surface studied by FACS analysis.@*Results@#The MTT and TUNEL assays demonstrated no significant difference between VPA at 2 mM treated and untreated control cells. VPA attenuated LPS-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen activated protein kinases (MAPKs). There was no significant difference in the TLR4 expression on the cell surface between cells treated with or without VPA. VPA inhibited LPS-induced PI3K/Akt signal transduction in a dose dependent manner.@*Conclusion@#VPA at 2mM exhibits nontoxic effect in the RAW 264.7 cells. VPA down regulates LPS-induced phosphorylation of Akt via inhibition of PI3K activation.

The key role of transcription factors on the innate immunity reaction / Эрүүл мэндийн лаборатори

Background@#The effect of lipopolysaccharide (LPS) on valproic acid (VPA)-induced cell death was examined by using mouse RAW 264.7 macrophage cells. @*Materials and methods, results@#LPS inhibited the activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) and prevented VPA-induced apoptosis. LPS inhibited VPA-induced p53 activation and pifithrin-α as a p53 inhibitor as well as LPS prevented VPA-induced apoptosis. LPS abolished the increase of Bax/Bcl-2 ratio, which is a critical indicator of p53-mediated mitochondrial damage, in response to VPA. The nuclear factor (NF)-κB inhibitors, Bay 11-7082 and parthenolide, abolished the preventive action of LPS on VPA-induced apoptosis. A series of toll-like receptor (TLR) ligands, Pam3CSK4, poly I:C, and CpG DNA as well as LPS prevented VPA-induced apoptosis. @*Conclusion@#Taken together, LPS was suggested to prevent VPA-induced apoptosis via activation of anti-apoptotic NF-κB and inhibition of pro-apoptotic p53 activation.

Study on influence of the CpG DNA on activation of IFN-γ signaling transduction regulatory proteins / Монголын Анагаах Ухаан

Introduction@#When human body encounters external pathogens primary/innate immunity cells are activated by recognizing them and secondary/adaptive immunity is activated consecutively. In our previous study, we revealed that there is a synergistic action between TLR9 and IFN-γ signaling in the endothelial cells. @*Purpose@#To determine the role of negative and positive regulator proteins on the IFN-γ/TLR9 signaling pathway. @*Methods@#In this study, murine endothelial cell (END-D) culture was used. END-D cells pre-treated with TLR9 ligand CpG DNA and then stimulated with IFN-γ. The negative (SHP-2, SOCS1, PIAS1) and positive (p38) regulator protein expression was detected by Western blotting. @*Results and Conclusion@#Treatment by TLR9 ligand CpG DNA and IFN-γ increased positive regulator p38 phosphorylation in 0.5 hour. CpG DNA inhibited IFN-γ negative regulator PIAS1 protein expression in 6 hour and SOCS1 and SHP-2 expression could not affect in 4 hour.

Role of negative regulators on the TLR7 ligand/IFN-γ signaling in the endothelial cells / Эрүүл мэндийн лаборатори

Introduction@#Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. The SOCS1 and SHP2 proteins are negative-feed loop inhibitors of signaling of JAK/STAT and TLRs pathways.@*Purpose@#To determine negative regulator protein activation which is activated through TLR7 ligand/IFN-γ signal transduction in endothelial cells. @*Methods@#We used mouse aortic linear endothelial cell (END-D); protein expressio was detected by western blotting @*Results@#We analyzed a time dependent stimulation effects of negative regulator proteins stimulated by TLR7 ligand/IFN-γ in endothelial cell cultures. Imiquimod of 10 μg/ml treatment of 1 hr was followed by 100 ng/ml IFN-γ stimulation for 1-8hr to analysis of negative regulator SOCS1 and SHP2 protein expression. </br> In untreated cells, there was low activations of negative regulator SOCS1 and SHP2 proteins. IFN-γ stimulation alone had increased SOCS1 and SHP2 protein expressions, also imiquimod treatment highly <i>elevated</i> SOCS1 and SHP2 expressions. However imiquimod and IFN-γ doubled treatment have decreased activation of negative regulator SOCS1 and SHP2 proteins. These findings suggest SOCS1 and SHP2 proteins are inhibitors in the TLR7 ligand/IFN-γ signaling. @*Conclusion@#Negative regulators, SOCS1 and SHP2 strongly suppressed activations of TLR7 ligand/IFN-γ signaling

The effect of TLR9 ligand on IFN-ү signaling / Эрүүл мэндийн лаборатори

Introduction@#The aim of this research project is to elucidate the crosstalk of innate and adaptive immune reactions against the DNA containing bacteria.@*@#This study held in the Core laboratory, Science Technology Center, Mongolian National University of Medical Sciences (MNUMS). Murine aortal endothelial cells, END-D cultured and the cell viability checked by MTT assay. In addition, the NO production, protein and gene expression studied by Griess Reagent assay, R.T-PCR and immunoblotting, respectively. @*Results@#0.1µM, 1µM and 10µM of TLR9 ligand exhibited no cytotoxic action against the cells by MTT assay. IFN-ү alone induced NO production in END-D cells. In the other hand, TLR9 ligand at 0.1µM, 1µM and 10µM up-regulated IFN-ү induced NO production in dose dependent manner. RTPCR results exhibit that TLR9 ligand up regulates iNOS mRNA. Immunoblotting analysis showed the enhanced iNOS protein expression and phosphorylation of STAT1 in cells pre-treated with TLR9 ligand.@*Discussion@#We have demonstrated CpG DNA, TLR9 ligand, up-regulates IFN-ү induced NO via enhanced IFN-ү signaling. The result of Western Blotting and RT-PCR support the up-regulation of NO. CpG DNA can be used as agent against virus and bacteria. Further research need to be conducted. @*Conclusion@#TLR9 ligand, CpG DNA up-regulates IFN-ү induced NO production in time and dose dependent manner. TLR9 ligand augments the expression of iNOS mRNA and STAT1 phosphorylation in response to IFN-ү.

EFFECT OF TLR7 LIGAND ON SIGNAL TRANSDUCTION OF INTERFERON GAMMA / Шинэ санаа Шинэ нээлт

@#BACKGROUND: Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. TLR7 is expressed on monocytes, macrophages and dendritic cells, T cell, B cell and eosinophiles. TLR7, originally identified as recognizing imidaquinoline, loxibrine, broprimine and ssRNA, ssRNA viruses such as vesicular stomatitis virus, influenza A virus and human immunodefiency virus. It is known that virus ssRNA affects signaling molecule of IFN-y. Objective: To determine gene and protein activation of IFN-y signal transduction by TLR7 ligand in the endothelial cells. MATERIAL: In study we used mouse aortic linear endothelial cell which is cultured (END-D) in 5% heat- inactivated fetal calf serum (FCS), medium (DMEM) containing antibiotic mix(penicillin G, streptomycin, amphotericin B) at 37°C (5% CO2). Endothelial cells treated with synthetic IFN-γ and imiquimodligands, then the NO (nitric oxide) concentration in the supernatant is determined by Griess reagent. Endothelial cells are cultured in 6 well cell culture plate and in each well 2*104cells are expected to be grown for 24 hours of culture. Then, the cells are treated with synthetic IFN-γ and имиквимод ligand for 6 hours and the NO signaling gene activation iNOS mRNA expression which is induced by IFN-γ is determined by RT-qPCR. Endothelial cells are cultured in 12 well cell culture plate and in each well 2*104 cells are expected to be grown for 18 hours of culture. Then, the cells are treated with synthetic IFN-γ and imiquimodligands for 24 hours and the NO signaling protein iNOS expression which is induced by IFN-γ is determined by western blotting. The experiment was conducted as representation mean of at least three test results. The difference between statistical probabilities is determined by the “Students” t test. The p<0.01 value is assumed to be statistically different. RESULTS: TLR7 ligand imiquimodaugmented interferon gamma induced nitric oxide production TLR7 ligand imiquimodincreased interferon gamma induced iNOS mRNA gene expression. TLR7 ilgand imiquimodup-regulated interferon gamma induced iNOS protein expression. CONCLUSIONS: TLR7 ligand imiquimod augments IFN-γ signaling in the endothelial cells. This synergistic effect has revealed in the levels of gene and protein expression.

Evaluation of risk factors and predictive biomarkers in the acute coronary disease / Монголын Анагаах Ухаан

Introduction. The acute coronary disease (ACD), broadly encompass the clinical states unstable angina (UA) and acute myocardial infarction (AMI), especially affects adults due to cause the impairment of work ability, associates reducement of life quality and high expenses of medical treatment, and induces leading cause of sever complication and death.Materials and Methods. In this study, 44 ACD patients and 33 healthy subjects enrolled into case and control group, respectively. Relationships of primary and intermediate risk factors between cases and healthy subjects were determined by questionnaire research and clinical examinations. Measurements such as C reactive protein (CRP), cholesterol, triglycerides, low-density lipoprotein (LDH), high-density lipoprotein (HDL), trooping I, and mean platelet volume (MPV) were analyzed by clinical laboratory assays. The SPSS12 statistical software was used for all statistical calculations.Results. Statistical significant differences of hypertension and smoking were observed in ACD patients (UA and AMI) (P<0.01) compared with healthy subjects by independent samples T test. Body mass (BM), waist-to-hip ratio (WHR), body mass index (BMI) were significantly different in patients with UA, but WHR, hip were significantly different in patients with AMI. The levels of biochemical measurements such as cholesterol, triglycerides, and glucose were significantly higher in patients with AMI (р<0.01), whereas glucose concentration was significantly higher in patients with UA (р<0.05). However, a kind of inflammatory markers, CRP was a risk factor in the patients with ACD (UA and AMI), whereas MPV was a risk factor for AMI only. In the ANOVA test, which was confirming analysis on the results of independent samples Ttest, overweight (BM), abdominal obesity (WHR, hip) measurements, parameter of glucose metabolism(glucose) and some inflammatory markers (CRP, MPV) were significantly different between study groups. Relationships by determined Pierson`s correlation, were observed between overweight parameters (BM, BMI) and biomarkers of fatty acid metabolism (cholesterol, LDL, HDL, triglycerides). The BM of overweight parameters and the WHR of abdominal obesity measurements were strongly associated with increased level of glucose.Conclusion. Primary risk factors including hypertension and smoking; parameters of the overweight or abdominal obesity such as BM, WHR, BMI and hip; biochemical measurements as cholesterol, triglycerides and glucose; and some inflammatory biomarkers as well as CRP and MPV were risk factors in the ACD.