Compositions for effective management of fibroblast-like synoviocytes mediated rheumatoid arthritis
a technology of fibroblasts and synoviocytes, applied in the direction of drug compositions, anti-noxious agents, plant/algae/fungi/lichens ingredients, etc., can solve the problems of hypersensitivity, especially immune related, and achieve the effect of inhibiting proliferation and migration
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and Culture of Fibroblast-Like Synoviocytes (FLS)
[0050]Synovial tissues were obtained from collagen induced arthritis rats under sterile conditions, according to the method followed by (Jinjun Zhao, Qingqing Onyang, Ziyou Hu, Qin Huang, Jing Wu, Ran Wang and Min Yang. A protocol for the culture and isolation of murine synovial fibroblasts. Biomedical Reports 5: 171-175, 2016).
[0051]FLS were isolated from synovial tissues by enzymatic digestion. In short, the tissues were separated into 1-mm3 blocks with microsurgery scissors. After which the tissues were incubated with 0.1% collagenase type II enzyme in DMEM supplemented with 10% FBS for enzymatic digestion at 37° C. for 45 min in an orbital shaker incubator (200 rpm).
[0052]After the incubation time, the tubes were vortexed vigorously for 1-2 min to release the cells and filtered using a 100 μm mesh strainer. The filtrate was centrifuged for 5 min at 1200 rpm, the cells were resuspended in DMEM supplemented with 10% FBS and 100 μg / m...
example 2
liferation Assay
[0053]WST-1 assay was used to measure the effect of different samples on FLS proliferation. FLS were seeded in 96-well plates at a density of 5×103 cells / well and incubated overnight at 37° C. under 5% CO2. Then, the cells were treated with different concentrations of the sample (AC3, C3, Cur, BDMC, DMC, BPS) as shown in the FIGS. 3-6 for 72 hours. After the treatment period, 100 μl WST-1 reagent (5015944001, Sigma, USA) diluted 1:10 in serum-free DMEM culture media was added to each well, and incubated at 37° C. under 5% CO2 for 2 h. The absorbance was measured at 450 nm with a reference wavelength of 610 nm. AC3 showed better inhibition of FLS proliferation at 20 μg / mL and 40 μg / mL (FIGS. 3 and 4) in a dose dependent manner. A comparative study of curcuminoid analogues in the inhibition of FLS proliferation showed a trend of BDMC>curcumin>DMC (FIG. 5) at 20 μg / mL, whereas BPS showed only 10% inhibition and the combination was much effective than the Boswellin PS (B...
example 3
e Analysis
[0054]FLS cells (8-104 cells / well) were seeded in 24-well plates and incubated overnight at 37° C. under 5% CO2. After synchronization in serum-free medium for 24 h, cells were treated with or without the presence of samples in DMEM 10% FBS for 24 h. After the treatment period, the cells were collected, suspended in ice cold PBS. After centrifugation, the cells were fixed by adding 70% ice cold ethanol dropwise to the pellet while vortexing and stored at 4° C. for 30 min. The cells were stained with propidium iodide (PI) solution (50 μg / ml) for 30 min at 37° C. in the dark. DNA content was analyzed by flow cytometry (BD FACS Celesta flow cytometer). AC3, BDMC among curcuminoids showed maximum inhibition of FLS replication (FIGS. 7, 8, 12, and 14), and FLS was arrested at G0 / G1 phase. Boswellin PS (BPS) showed inhibition only at 80 μg / mL (FIG. 8, FIG. 16). The effect of combination (AC3+BPS) on cell-cycle inhibition was better than the individual samples used (AC3 and BPS),...
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