گیاه ارموستاکیس بینالودنسیس: انتخاب مناسب برای بیماریهای التهابی ژینژیوال

In-vitro wound and LPS induction In-vitro wound was induced by scrapping the 2-3 mm of surface layer of hGFs creating a scratch to the cells followed by mentioned incubation method.16 In order to induce inflammation, 1 µg/ml bacterial endotoxin (Lipopolysaccharides (LPS)) was added 1 hour after E. binalodensis extract application on cells. Experimental groups First, the effective concentration of E. binalodensis on hGFs’ proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test. 1, 10, 100 and 1000 µg/ml of the methanolic E. binalodensis extracts in 1% Dimethyl sulfoxide (DMSO) were selected for MTT test. Regarding the results of MTT test, the work was performed in five groups: 1. Control, 2. Scratch, 3. Scratch + Extract, 4. Scratch + LPS, 5. Scratch + LPS + Extract. Groups 1, 2, 4 were pretreated with the same amount of DMSO. Extract was performed 1 hour prior to LPS and scratch and cells were incubated for 48 hours. MTT assay The effect of E. binalodensis on hGFs proliferation was assessed by MTT test. MTT assay is based on the reduction of MTT to formazan crystals by mitochondria of viable cells.17 First, cells which were 48 hours incubated with 1, 10, 100 and 1000 µg/ml of the methanolic extracts of E. binalodensis, were seeded at a density of 1 × 104 cells/well in a 96-well plate. Second, the medium was removed and 10 µL MTT was added to the wells and incubated for 4 hours. After observing formazan crystals, 200 µL DMSO was added to dissolve the crystals. Finally, the absorbance of the plate was read at 450 nm by spectrophotometer (Specord 250, Analytik Jena), cell viability was expressed relative to the control group which was regarded as 100%. The test was repeated three times. ELISA assay of inflammatory markers In order to evaluate the main inflammatory markers involved in periodontal diseases, the protein levels of IL-1β, IL-6 and TNF-a in culture supernatants were measured by enzymelinked immunosorbent assay (ELISA). Human IL-1β ELISA Kit (ab100562), Human TNF alpha ELISA Kit (ab181421), Human IL-6 ELISA Kit (ab46027) were purchased. For this purpose, 50 µL of cells were added to a 96-well plate. 50 µL of the antibody cocktail, regarding the type of measured marker, was added to each well and incubated for 2 hours on a plate shaker with 400 rpm. After washing the plate regarding the kit instruction, 100 µL 3,3',5,5'-Tetramethylbenzidine (TMB) development solution was added and incubated. Finally, after washing and adding the stop solution, the plate absorbance was read at 450 nm by spectrophotometer. The levels of IL-1b, L-6 and TNF-a in cell culture supernatants, expressed as pg/mL, were quantified based on each corresponding standard curve. Each experiment was repeated three times.