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. 2024 Aug 13;25(16):8789.
doi: 10.3390/ijms25168789.

Anti-Neuroinflammatory Effect of Ombuin from Rhamnus erythroxylon Pall. Leaves in LPS-Induced BV-2 Microglia by Targeting Src and Suppressing the PI3K-AKT/NF-κB Signaling Pathway

Yanjie Bian  1 , Nan Qiao  1 , Suyun Han  1 , Jixiang Gao  1 , Xiaofang Lv  1 , Lihuan Yuan  1 , Linjing Zhang  1 , Zuofu Wei  1
Affiliations

Anti-Neuroinflammatory Effect of Ombuin from Rhamnus erythroxylon Pall. Leaves in LPS-Induced BV-2 Microglia by Targeting Src and Suppressing the PI3K-AKT/NF-κB Signaling Pathway

Yanjie Bian et al. Int J Mol Sci. .

Abstract

The leaves of Rhamnus erythroxylon Pall. are widely used as tea substitutes in northwest China for their fragrant aroma, anti-irritability, and digestion-enhancing properties. Ombuin, a main flavonoid compound found in the leaves, exhibited notable anti-inflammatory and antioxidant effects. However, its potential role in treating neuroinflammatory-related diseases remains unexplored. Thus, this study aims to evaluate the anti-neuroinflammatory effects of ombuin and to explore the underlying molecular mechanisms. According to our findings, ombuin dramatically reduced the release of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1β, nitric oxide (NO), and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further analysis, including transcriptomics, network pharmacology, molecular docking, and cellular heat transfer assays, revealed that Src was a direct target of ombuin. Western blot analysis showed that ombuin effectively suppressed Src phosphorylation and inhibited the downstream expressions of p-PI3K p85, p-AKT1, p-IKKα/β, p-IκBα, and nuclear factor κB (NF-κB). Meanwhile, the repression of Src significantly reversed the anti-neuroinflammatory activity of ombuin. Our results identified Src as a direct target of ombuin and implied that ombuin exerted an anti-neuroinflammatory effect by inhibiting Src phosphorylation and suppressing the activation of the PI3K-AKT and NF-κB pathways, which might provide an alternative therapeutic strategy for neurodegenerative diseases.

Keywords: BV-2; LPS; Src; neuroinflammation; ombuin.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Ombuin inhibited LPS-induced neuroinflammation in BV-2 cells. (A) Chemical structure of ombuin. (B,C) Effects of ombuin on cell viability in the presence or absence of LPS. (D–I) The production of NO (D), IL-6 (E), IL-1β (F), TNF-α (G), and ROS (H,I) in LPS-induced BV-2 cells. After pretreatment with a series of ombuin concentrations for 1 h, BV-2 cells were stimulated with 1 μg/mL LPS for 24 h and then assayed by MTT, Griess, ELISA, and ROS detection assays. The scale bar represents 20 μm in (H). All values are expressed as the mean ± SEM for three independent experiments. ## p < 0.01 and ### p < 0.001 indicate significance compared with the control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significance compared with the LPS group.
Figure 2
Figure 2
Network pharmacology was utilized to predict the targets of ombuin in neuroinflammation. (A) Neuroinflammation potential therapeutic targets vs. ombuin targets. (B) The protein–protein interaction (PPI) network of the potential therapeutic targets for neuroinflammation affected by ombuin. (C) KEGG Enrichment Scatter Plot displaying the top 30 KEGG pathways, with pathways listed in a bubble chart format.
Figure 3
Figure 3
Analysis of Differentially Expressed Genes (DEGs) by Transcriptomics. (A,B) Volcano plot analysis of all DEGs among the control group, the LPS group, and the LPS + ombuin (50 μM) group. n = 3. (C) The heat map displays the 33 overlapping genes. In this diagram, red indicates higher expression levels, and green indicates lower expression levels. (D) Gene ontology (GO) enrichment analysis.
Figure 4
Figure 4
Network of ombuin and 199 targets.
Figure 5
Figure 5
(A) PPI network of core targets. (B) KEGG analysis, the size and color of a node are indicative of its degree in the network; nodes with a larger size and a deeper orange color represent targets with higher degrees. (C) The ombuin-targets-pathways network illustrates ombuin’s role in the treatment of neuroinflammation.
Figure 6
Figure 6
Src is a direct target of ombuin. (A) Molecular docking patterns of key targets with ombuin: (a) PIK3R1, (b) EGFR, (c) Src, (d) PTK2, (e) KDR. (B) BV-2 cells were pre-treated with 50 μM ombuin for 4 h, after which CETSA was performed using a temperature gradient ranging from 37 °C to 62 °C. The lysates were analyzed by Western blot with an Src antibody. (C) Relative band intensity. The expression levels of the Src protein in each group at different temperatures were normalized by dividing by the gray scan value of the Src protein at 37 °C to calculate the relative band intensity.
Figure 7
Figure 7
Src knockdown reversed ombuin-mediated neuroinflammatory inhibition. (A) The levels of Src and phosphorylated Src (p-Src) proteins were assessed using Western blot analysis following a 24 h treatment with ombuin and/or LPS. (B) The efficiency of Src siRNA transfection in BV-2 cells. (C–E) BV-2 cells were transfected with either non-targeting control (NC) siRNA or Src siRNA. After 24 h, the cells were further treated with ombuin/LPS for an additional 24 h. Then, the impact of Src knockdown on the production of TNF-α (C), IL-1β (D), and NO (E) in BV-2 cells was measured by ELISA. (F,G) Western blot analysis was used to evaluate the protein levels of the PI3K-AKT pathway, the NF-κB signaling pathway, and inducible nitric oxide synthase (iNOS) in BV-2 cells post-Src knockdown. (H–M) Quantitative analysis of the influence of Src silencing on downstream PI3K-AKT and NF-κB signaling pathways, as well as the expression of the inflammatory mediator iNOS protein. Data are presented as the mean ± SEM of three independent experiments. ## p < 0.01 indicates significance compared with the control group; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicate significance; ‘ns’ indicates not significant.
Figure 8
Figure 8
The proposed signaling mechanism explains the effects of ombuin on LPS-induced neuroinflammation in BV-2 cells.
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References

    1. Woodburn S.C., Bollinger J.L., Wohleb E.S. The semantics of microglia activation: Neuroinflammation, homeostasis, and stress. J. Neuroinflamm. 2021;18:258. doi: 10.1186/s12974-021-02309-6. - DOI - PMC - PubMed
    1. Maher P. The Potential of Flavonoids for the Treatment of Neurodegenerative Diseases. Int. J. Mol. Sci. 2019;20:3056. doi: 10.3390/ijms20123056. - DOI - PMC - PubMed
    1. Li S.Y., Zhou Y.L., He D.H., Liu W., Fan X.Z., Wang Q., Pan H.F., Cheng Y.X., Liu Y.Q. Centipeda minima extract exerts antineuroinflammatory effects via the inhibition of NF-κB signaling pathway. Phytomedicine. 2020;67:153164. doi: 10.1016/j.phymed.2019.153164. - DOI - PubMed
    1. Singh D. Astrocytic and microglial cells as the modulators of neuroinflammation in Alzheimer’s disease. J. Neuroinflamm. 2022;19:206. doi: 10.1186/s12974-022-02565-0. - DOI - PMC - PubMed
    1. Decandia D., Gelfo F., Landolfo E., Balsamo F., Petrosini L., Cutuli D. Dietary Protection against Cognitive Impairment, Neuroinflammation and Oxidative Stress in Alzheimer’s Disease Animal Models of Lipopolysaccharide-Induced Inflammation. Int. J. Mol. Sci. 2023;24:5921. doi: 10.3390/ijms24065921. - DOI - PMC - PubMed

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