To investigate the mechanism of Citri Grandis Exocarpium in ameliorating obesity⁃related inflammation through network pharmacology and in vitro cell experiment. Methods (1) The main active components of Citri Grandis Exocarpium were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Effect targets of the main active components were retrieved from TCMSP, SwissTargetPrediction, and HIT databases. Obesity⁃ and inflammation⁃related targets were obtained from GeneCards® and OMIM® databases. The intersection targets of effect targets of the main active components of Citri Grandis Exocarpium with inflammation⁃ and obesity⁃related targets of Citri Grandis Exocarpium were acquired by using the Venny online tool. A protein⁃protein interaction (PPI) network was constructed by employing the STRING database, and core targets were identified by using the Cytoscape 3.9.1 software. The Gene Ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed by employing the DAVID database. Molecular docking and visual processing of the main active components with corresponding proteins of core targets of Citri Grandis Exocarpium were conducted by using the CB⁃Dock2 tool. (2) 3T3⁃L1 pre⁃adipocytes were induced to differentiate for 14 days, with oil red O staining kit performed on the 7th, 10 th, and 14th day, respectively, to observe lipid droplet formation. Mature 3T3⁃L1 adipocytes were treated with different concentrations (0 μmol/L, 1 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmol/L, 40 μmol/L, 80 μmol/L, and 120 μmol/L) of naringin, and cell viability was detected via the CCK⁃8 assay to screen the effect concentration of naringin. Mature 3T3⁃L1 adipocytes were obtained and divided into control group, lipopolysaccharide group, lipopolysaccharide+20 μmol/L naringin group, or lipopolysaccharide+40 μmol/L naringin group. Except for the control group, 3T3⁃L1 adipocytes of the remaining groups were stimulated with lipopolysaccharide to construct an adipocyte inflammation model. The lipopolysaccharide+20 μmol/L naringin and lipopolysaccharide+40 μmol/L naringin groups received corresponding concentrations of naringin for treatment. The real⁃time fluorescent quantitative PCR was adopted to measure mRNA expressions of Toll⁃like receptor 4 (TLR4), nuclear factor kappa B (NF⁃κB), tumor necrosis factor (TNF)⁃α, interleukin (IL)⁃6, and IL⁃1β, while the Western blot was adopted to measure protein expressions of TLR4, NF⁃κB, and TNF⁃α in cells of various groups. Results (1) Ten main active components of Citri Grandis Exocarpium were screened, including naringin, naringenin, nobiletin, and apigenin, etc. A total of 410 effect targets of the main active components of Citri Grandis Exocarpium, 1556 obesity⁃related targets, and 11 268 inflammation⁃related targets were screened, with 167 intersection targets. The core targets of Citri Grandis Exocarpium on obesity⁃related inflammation included IL⁃6, TNF, IL⁃1β, C⁃X⁃C motif chemokine ligand 8 (CXCL8), interferon γ, NF⁃κB, and TLR4, etc. The intersection targets were mainly involved in biological processes such as response to exogenous stimuli, positive regulation of gene expression, and cellular response to lipopolysaccharide, in cellular components such as receptor complexes, extracellular regions, endoplasmic reticulum lumen, plasma membrane, and cell surface, and in molecular functions such as enzyme binding, identical protein binding, nuclear receptor activity, and protein binding, as well as in signaling pathways such as hypoxia⁃inducible factor 1 signaling pathway, IL⁃17 signaling pathway, phosphatidylinositol 3⁃kinase⁃protein kinase B signaling pathway, TNF signaling pathway, Toll⁃like receptor signaling pathway, NF⁃κB signaling pathway, mitogen⁃activated protein kinase signaling pathway, Th17 differentiation, Th1 and Th2 differentiation, Janus kinase⁃signal transducer and activator of transcription signaling pathway. The results of molecular docking confirmed that favorable binding ability between the main active components and corresponding proteins of core targets in Citri Grandis Exocarpium was indicated. (2) According to the CCK⁃8 assay results, 20 μmol/L and 40 μmol/L naringin were selected for effect concentrations in this study. Lipid droplet size and number in 3T3⁃L1 pre⁃adipocytes increased progressively with the extension of induction time, on the 14th day, they differentiated into mature 3T3⁃L1 adipocytes. Compared to the control group, the lipopolysaccharide group exhibited elevated mRNA expressions of TLR4, NF⁃κB, TNF⁃α, IL⁃6, and IL⁃1β, and elevated protein expressions of TLR4, NF⁃κB, TNF⁃α, which were decreased after naringin intervention. Conclusion Citri Grandis Exocarpium can ameliorate obesity⁃related inflammation, and its mechanism may be related to the main component naringin, which exerts anti⁃inflammatory effect through down⁃regulating TLR4/NF⁃κB signaling pathway.

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