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Pentoxifylline Can Reduce the Inflammation Caused by LPS after Inhibiting Autophagy in RAW264.7 Macrophage Cells.

ABSTRACT: Pentoxifylline (PTX), as a methylxanthine derivative and nonspecific phosphodiesterase inhibitor, has the characteristics of anti-inflammatory and partial inflammatory process inhibition. However, the regulatory effect of PTX on inflammatory cytokines is unclear. Autophagy can regulate the activation of inflammasomes and then inhibit inflammation as previously described. Our study attempts to explore the relationship between autophagy and PTX-mediated regulation of inflammasome suppression. Macrophage-like RAW264.7 cells were studied as the in vitro macrophage model. We investigated the anti-inflammatory effect caused by PTX with time and dose response against the LPS-induced inflammatory factors (TNF-α, IL-1β). Western blot detected the levels of autophagy-related proteins Beclin-1 and LC3, as well as the signal pathways of AMPK and p-AMPK. Fluorescence microscope and transmission electron microscope were used to observe the autophagy bodies in cells influenced by PTX. The autophagy in cells inhibited by PTX exhibited dose- and time-dependent effects, and PTX alleviated LPS-induced inflammation caused by retarded autophagy. Furthermore, in RAW264.7 macrophage cells, our data indicated that AMPK signaling perhaps functioned importantly in repressed autophagy. In addition, in RAW264.7 macrophages, our data suggested that AMPK signaling might play an important role in inhibiting autophagy during the process of PTX ameliorating LPS-mediated inflammation.

SUBMITTER: Ruan D

PROVIDER: S-EPMC7987419 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectivesBiosurfactants with anti-inflammatory activity may alleviate skin irritation caused by synthetic surfactants in cleaning products. Sophorolipid (SL) is a promising alternative to synthetic surfactants. However, there are few reports on the anti-inflammatory activity of SL and the underlying mechanism. The purpose of this work is to verify that lipopolysaccharide (LPS)-induced inflammation could be inhibited through targeting the pathway of nuclear factor-κB (NF-κB) in RAW264.7 cells.MethodsThe influence of SL on cytokine release was investigated by LPS-induced RAW264.7 cells using ELISA. The quantification of the protein expression of corresponding molecular markers was realized by Western blot analysis. Flow cytometry was employed to determine the levels of Ca2+ and reactive oxygen species (ROS). The relative expression of inducible nitric oxide synthase (INOS) and cyclooxygenase-2 (COX-2) was determined by RT-PCR. An immunofluorescence assay and confocal microscope were used to observe the NF-κB/p65 translocation from the cytoplasm into the nucleus. The likely targets of SL were predicted by molecular docking analysis.ResultsSL showed anti-inflammatory activity and reduced the release of inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide (NO). The experimental results show that SL suppressed the Ca2+ and ROS levels influx in the LPS-induced RAW264.7 cells and alleviated the LPS-induced expression of iNOS and COX-2, the LPS-induced translocation of NF-κB (p65) from the cytoplasm into the nucleus, and the expression of phosphorylated proteins such as p65 and IκBα. Furthermore, molecular docking analysis showed that SL may inhibit inflammatory signaling by competing with LPS to bind TLR4/MD-2 through hydrophobic interactions and by inhibiting IKKβ activation through the hydrogen bonding and hydrophobic interactions.ConclusionThis study demonstrated that SL exerted anti-inflammatory activity via the pathway of NF-κB in RAW264.7 cells.

Project description:Obesity has become a major threat to public health and is accompanied by chronic low-grade inflammation, which leads to various pathological developments. Lunasin, a natural seed peptide, exhibits several biological activities, such as anti-carcinogenesis, anti-inflammatory, and antioxidant activities. However, the mechanism of action of lunasin in obesity-related inflammation has not been investigated. The aim of this study was to explore whether lunasin could reduce the inflammation induced by obesity-related mediators in RAW264.7 cells and 3T3-L1 adipocytes and whether it could attenuate the crosstalk between the two cell lines. RAW264.7 cells were cultured in leptin-containing medium, adipocyte-conditioned medium (Ad-CM), or co-cultured with 3T3-L1 cells to mimic the physiology of obesity. The data showed that the secretion of pro-inflammatory cytokine interleukin-1β (IL-1β) was inhibited by lunasin after leptin activation of RAW264.7 cells. In addition, lunasin decreased monocyte chemoattractant protein-1 (MCP-1) and IL-1β secretions in the Ad-CM model. Cytokine MCP-1, IL-6, tumor necrosis factor (TNF)-α, and IL-1β secretions were significantly decreased by leptin or Ad-CM plus lipopolysaccharide stimulation. Subsequently, the co-culture of the two cells refined the direct relation between them, resulting in apparently increased MCP-1, and decreased IL-6 levels after lunasin treatment. In 3T3-L1 adipocytes, lunasin also exhibited anti-inflammatory property by inhibiting MCP-1, plasminogen activator inhibitor-1, and leptin productions stimulated by (TNF)-α, lipopolysaccharide, or RAW264.7 cell-conditioned medium. This result revealed that lunasin acts as a potential anti-inflammatory agent not only in macrophages but also in adipocytes, disrupting the crosstalk between these two cells. Therefore, this study suggests the intake of lunasin from diet or as a supplement, for auxiliary prevention or therapy in obesity-related inflammatory applications.

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Pentoxifylline Can Reduce the Inflammation Caused by LPS after Inhibiting Autophagy in RAW264.7 Macrophage Cells.

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