Kaempferol exerts therapeutic effect on rheumatoid arthritis by regulating cell senescence: a study based on network pharmacology and in vitro experiments

To investigate the mechanism of kaempferol in the treatment of rheumatoid arthritis（RA）based on network pharmacology and in vitro experiments.

Kaempferol targets were queried by TCMSP and SwissTarget databases, and RA related functional targets were searched in the GeneCards and DisGeNET databases. After the cross between kaempferol and RA targets through Venny2.1.0, STRING database was used for protein-protein interaction（PPI）analysis, and molecular docking between kaempferol and the targets was conducted. Mouse mononuclear leukemia RAW264.7 cells were treated by lipopolysaccharide（LPS）and kaempferol, and divided into control group, LPS group, LPS+Kaempferol group. MTT assay was used to determine the cytotoxicity of RAW264.7 cells after kaempferol treatment. LPS-induced RAW264.7 cells were used to construct a cellular inflammatory model. The change in aging and aging-related inflammatory factors were detected in each treatment group. The content of β-galactosidase was detected by SA-β-Gal, and the expression of IL-6 was detected by cellular immunofluorescence assay.

A total of 138 targets of kaempferol and 76 potential targets of kaempferol against RA were obtained. PPI analysis showed that TNF, EGFR, PTGS2 and AKT1 may be the core targets of kaempferol against RA. The results of molecular docking indicated that kaempferol could form stable complexes with the core targets of TNF, EGFR, PTGS2 and AKT1. The results of MTT assay displayed that the treatment of 0.125 to 16 μmol/L kaempferol for 24 h promoted the proliferation of RAW264.7 cells（P<0.05）, the doses of 0.125 to 8 μmol/L for 48 h exerted similar effect on the proliferation（P<0.05）, but the dose of 0.125 to 16 μmol/L for 72 h had no such significant effect. The results of qPCR and Western blotting showed that kaempferol decreased the expression of cell senescence markers p16 and p21 and promoted the expression of cell cycle markers CCND1 and CCNE1（P<0.05）. Cellular immunofluorescence assay indicated that kaempferol reduced the expression of IL-6（P<0.05）. SA-β-Gal staining displayed that LPS treatment promoted β-galactosidase level in RAW264.7 cells（P<0.05）, and this effect was reversed by kaempferol treatment（P<0.05）.

Kaempferol may regulate the expression of IL-1β, IL-6 and TNF-α and p16/p21 senescence related genes, prevent premature aging of immune cells, and thus play a therapeutic role for RA.