Exploring the mechanism of luteolin by regulating microglia polarization based on network pharmacology and in vitro experiments

Abstract

Neuroinflammation manifests following injury to the central nervous system (CNS) and M1/M2 polarization of microglia is closely associated with the development of this neuroinflammation. In this study, multiple databases were used to collect targets regarding luteolin and microglia polarization. After obtaining a common target, a protein-protein interaction (PPI) network was created and further analysis was performed to obtain the core network. Molecular docking of the core network with luteolin after gene enrichment analysis. In vitro experiments were used to examine the polarization of microglia and the expression of related target proteins. A total of 77 common targets were obtained, and the core network obtained by further analysis contained 38 proteins. GO and KEGG analyses revealed that luteolin affects microglia polarization in regulation of inflammatory response as well as the interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways. Through in vitro experiments, we confirmed that the use of luteolin reduced the expression of inducible nitric oxide synthase (iNOS), IL-6, TNF-α, p-NFκBIA (p-IκB-α), p-NFκB p65, and MMP9, while upregulating the expression of Arg-1 and IL-10. This study reveals various potential mechanisms by which luteolin induces M2 polarization in microglia to inhibit the neuroinflammatory response.

Figure 1

Flow chart of the research work.

Figure 2

Venn diagram. The figure shows that luteolin and microglia polarization have a total of 77 intersecting targets that can be used as potential targets for drug action.

Figure 3

PPI graph and core network. (a) PPI relationship network, the edge corresponding to each node represents its degree of correlation with other nodes in the network. (b) Core network, the darker the color and larger the shape of the node, the more important the node is in the core network. (c) Key nodes in the core network. The nodes are labeled with the corresponding target names and MCC algorithm score ranking. The darker the color, the more critical the node is in the core network.

Figure 4

Histogram of the top 20 GO and KEGG enrichment terms. Terms of BP (a), MF (b), CC (c) and KEGG signaling pathway (d) are shown. In the bar graphs, the smaller the p-value and the longer the bars, the higher the enrichment.

Figure 5

Signaling pathway and biological process-target relationship diagram. The red V-shaped nodes represent the corresponding signaling pathways and biological processes, the yellow circular nodes and blue diamond nodes represent the targets associated with each pathway, where the blue prismatic nodes represent NFKBIA and MMP9, which are present in all three pathways.

Figure 6

Molecular docking models of luteolin with NFKBIA (a), TNF (b), MMP9 (c), IL-6 (d) and IL-10 (e).

Figure 7

Luteolin suppressed pro-inflammatory mediators production and promoted anti-inflammatory M2 markers expression in LPS-stimulated primary microglia cells. (a) Identification of primary microglia cells. Immunostaining using an antibody targeting Iba1 (green), which is a marker for microglia cells and DAPI (blue) stain for cell nucleus. Scale bars: 100 µm. (b) Primary microglia cells were incubated with various concentrations of luteolin (1, 2.5, 5, 10, 20 μM) for 24 h to investigate the cytotoxicity. (c–g) Primary microglia cells were pretreated with the indicated concentrations of luteolin for 2 h followed by stimulation with 500 ng/mL LPS for another 24 h or (h–k) 12 h. (c) CCK8 assays. (d–g) The media were collected and the concentrations of NO, TNF-α, IL-6 and IL-10 were determined using the Griess reagent or ELISA kits. (h,i) Luteolin suppressed the iNOS and IL-6 mRNA expression, (j,k) promoted the mRNA expression of M2 microglial markers (Arg1 and IL-10) in LPS-stimulated primary microglia cells as determined by qRT-PCR. (l,m) The protein expression of Arg1 in primary microglia cells by western blot assay. *p < 0.05, **p < 0.01 compared with the control group, ^#p < 0.05, ^##p < 0.01 compared with the LPS group (a,h–m, n = 3, b–g, n = 5).

Figure 8

(A) The MMP-9 mRNA expression determined by qRT-PCR. (B) Representative images from Western blots for the proteins. (C) MMP-9/GAPDH ratio. (D) p-p65/p65 ratio. (E) p-IκB-α/IκB-α ratio. *p < 0.05, **p < 0.01 compared with the control group, ^#p < 0.05, ^##p < 0.01 compared with the LPS group (n = 3).

Figure 9

Mechanisms related to microglia polarization. Microglia can differentiate from resting state to M1 or M2 type upon stimulation. Luteolin induces microglia differentiation to M2 to promote the release of anti-inflammatory factors, reduce the inflammatory response and protect the CNS.