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H. He, W. Li, S. Tseng; Amniotic Membrane Extract Suppresses Activation and Induces Apoptosis in RAW264.7 (Macrophage) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2395.
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Activated macrophages play a pivotal role in initiating, maintaining and resolving host inflammatory responses, but they also exert deleterious effects on the host by producing excessive free radicals, lytic enzymes and inflammatory cytokines, which could cause tissue damages and chronic inflammation. Previously we reported that cryopreserved amniotic membrane facilitates macrophage apoptosis. By using amniotic membrane extract (AME), we would like to explore deeper how amniotic membrane may exert an anti-inflammatory activity through suppression of resting and activated macrophages.
RAW264.7 cells, a mouse macrophage cell line, were cultured on plastic in DMEM with 10% FBS and stimulated with or without 200U/ml IFN-γ or/and 1µg/ml LPS. AME was prepared from cryopreserved amniotic membrane and added at different concentrations. Cell spreading was determined under microscopic observation. G- or F-actin and such cell surface markers as CD80, CD86 and MHC II were examined by immunostaining or Western blotting analysis. Cell viability/apoptosis were assayed by MTT, Live/Dead and Cell Death Detection ELISA. Secreted cytokine levels of TNF-α, IL-6 and IL-10 in conditioned media were quantitated with ELISA assays.
In both resting and activated (by IFN-γ, LPS or IFN-γ/LPS) RAW264.7 cells, AME suppressed cell spreading and reduced actin filaments based on phalloidin staining and Western blotting of Triton X-100 extracted cell lysates. Western blot and immunostaining showed that AME down-regulated the expression of CD80, CD86 and MHC class II antigen. Cell growth/viability was inhibited while cell apoptosis was enhanced by AME. Accordingly, secreted pro-inflammatory cytokines such as TNF-α and IL-6 was reduced but anti-inflammatory cytokine IL-10 was up-regulated.
Collectively, these results suggest that similar to amniotic membrane, AME retains anti-inflammatory activities and does so by down-regulating activation and inducing apoptosis in macrophages, especially when they are activated by LPS and IFN-γ.
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