Proteomic identification of NF-κB-related targets associated with the anti-inflammatory activity of Gomisin R in LPS-stimulated RAW264.7 cells

BACKGROUND: Kadsura longipedunculata Finet et Gagnep. belongs to the Kadsura Kaempf.exJuss. Vaccinium of the family Magnoliaceae. It mainly distributes in regions such as Guangxi, Sichuan, Fujian and Yunnan of China. Commonly used as traditional Chinese medicine for anti-inflammation and analgesia, it has a long history in folk medicine. In recent years, research on the active components and pharmacological activities of K. longipedunculata Finet et Gagnep. has received extensive attention. Many studies have reported that lignans are the characteristic active components, among which the gomisin group of compounds has been frequently reported, including Gomisin A, G, N, and M2. Our previous research found that another such compound, Gomisin R, could significantly inhibit the secretion of inflammatory factors in LPS-induced RAW264.7 mononuclear macrophages and reduce the expression of NF-κB p65 protein. However, the anti-inflammatory signaling pathways and target sites of Gomisin R remain unclear. PURPOSE: To explore the anti-inflammatory signaling pathways and target sites of Gomisin R, clarify its anti-inflammatory molecular mechanism, provide experimental references for pharmacological research and clinical application, and help discover new drug treatment targets and develop new drugs. METHODS: The experimental subjects were classified into three groups: control group, LPS model group, and Gomisin R group. An in vitro inflammation model was established by inducing RAW264.7 cells with LPS. The anti-inflammatory related signaling pathways and target proteins of Gomisin R were screened by data independent acquisition (DIA) proteomics sequencing. The target proteins were verified by Western blot, and the binding activity of Gomisin R with the target proteins was predicted by molecular docking technology. RESULTS: Compared with the LPS model group, 163 differentially expressed proteins (DEPs) were identified from the cellular components in the Gomisin R group. 90 proteins among which were upregulated and 73 proteins were downregulated. Four enrichment pathways were screened: NF-κB signaling pathway, AMPK signaling pathway, mTOR signaling pathway, and PI3K-Akt signaling pathway. The classical inflammatory pathway was the NF-κB signaling pathway. Verification showed that Gomisin R mainly downregulated 4 proteins in the NF-κB signaling pathway: CCL4, PARP1, TNFRSF11A, and ERC1, thereby exerting anti-inflammatory effects and showing good binding activities with these 4 proteins. CONCLUSION: This study identified CCL4, PARP1, TNFRSF11A, and ERC1-related proteins, which may be potential molecular targets for the anti-inflammatory effect of Gomisin R. Since these four proteins are all enriched in the NF-κB signaling pathway, it is speculated that the anti-inflammatory mechanism of Gomisin R might involve regulation of the NF-κB signaling pathway to down-regulate the protein levels of CCL4, PARP1, TNFRSF11A, and ERC1, thereby to inhibit the inflammatory response. This study provides a reference for further developing Gomisin R into a small molecule natural anti-inflammatory drug.