EDN1 is a vasoconstrictor peptide originally isolated from porcine endothelial cells

ound that isorhamnetin, a 30 -methylated flavonol, produced a strong antiviral effect. The results of this study serve to clarify the molecular mechanism of the anti-influenza GFT-505 effect of isorhamnetin. Materials and Methods Ethics All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee,Konkuk University. 2 / 21 Antiviral Effect of Isorhamnetin against Influenza Six-week-old female C57BL/6 mice were purchased from KOATECH and housed in filter-top cages and in specific pathogen-free animal facility at the Korea Research Institute of Bioscience and Biotechnology. The virus challenge in mice was employed using anesthesia to minimize the animal suffering. Anesthesia of mice was conducted by intramuscular injection of 40mg/kg of Zoletil50, and 5 mg/kg of Rompun. The monitoring of the mice conditions was performed twice a day. We carried out the humane endpoint during the experiment of the mice survival rate. For this purpose, we euthanized using CO2 gas when the body weight starting to decrease to 70% of the original body weight. Cells, Virus, and Compounds Madin Darby Canine Kidney cells were obtained from the American Type Culture Collection. MDCK cells PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763407 were routinely maintained using minimum essential media supplemented with 10% fetal bovine serum,and 100U/mL penicillin/streptomycinat 37C humid incubator with a 5% CO2 atmosphere. The influenza A virus Puerto Rico/8/34 was kindly provided by the Immunogenetics Laboratory of the Department of Animal Biotechnology of Konkuk University. Before viral infection, MDCK cells were washed twice with phosphate buffered saline and cultured in virus growth medium containing MEM without FBS, 100U/mL penicillin/streptomycin, 2 g/mL trypsin TPCK, and 0.3% bovine serum albumin. Virus was serially diluted and inoculated into the eggs, and then the virus titers were calculated as log10EID50/ml. At the cellular level, virus titration was determined via the hemagglutination assay and by the cytopathic effect of MDCK cells induced by viral infection, and expressed as 50% tissue culture infectious doses, which were calculated based on the method of Reed and Muench.In mice, 50% mouse lethal dose was defined as 50% egg infective dose PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761601 that led to 50% mice mortality that calculated according to Reed and Muench. The flavonoids quercetin, kaempferol, isorhamnetin, diosmetin, and eriodictyol were purchased from the Indofine Chemical Company, Hillsborough, NJ, USA. The flavonoids and PD-98059 were dissolved in dimethylsulfoxide.The final concentration of DMSO in the culture media is 0.2%. Tamiflu was purchased from Roche-Korea Co. Ltd. Cytotoxicity and antiviral assays To determine the 50% cytotoxic concentration for each of the tested flavonoid compounds, MDCK cells were seeded onto 96-well plates at a density of 1 104 cells per well and incubated overnight for 80% confluence. MDCK cells were washed twice with PBS and the culture medium was replaced with medium containing serially diluted flavonoids for 48 hr at 37C in a 5% CO2atmosphere. After the incubation period, the flavonoid-containing media was replaced with new media containing 10% EZ-Cytox, and the cells were incubated in the dark for 3 hr at 37C in a 5% CO2 atmosphere. The optical density was measured at 480 nm using an x-Mark spectrophotometer. Cytotoxicity was estimated by comparing the cell survival rate of the flavonoids-treated cells and t