To explore the regulative effect of autophagy-related protein ATG5 on the expression of type Ⅱ collagen in human chondrocytes through affecting autophagy function.

Transcriptome data of 13 human cartilage tissue samples were downloaded from GEO database to analyze the correlation between ATG5 and COL2A1 expression. Western blotting and immunofluorescence assay were used to detect the expression of type Ⅱ collagen（Collagen Ⅱ, COL2）in human chondrocytes C28/I2 after rapamycin（Rapa）and bafilomycin A1（BafA1）treatment. Human COL2A1 promoter plasmid was constructed, and dual-luciferase reporter assay was applied to detect the effect of overexpression of ATG5 on COL2A1 promoter activity. mRFP-GFP-LC3 autophagy dual fluorescent virus was used to dynamically monitor the changes in autophagic flux of C28/I2 cells with overexpression and knockdown of ATG5. Western blotting was used to detect the effects of Rapa and BafA1 on COL2, LC3Ⅱ/LC3Ⅰ expression levels after overexpression or knockdown of ATG5.

The results of correlation analysis showed a positive correlation between the mRNA expression of ATG5 and COL2A1 in human chondrocytes samples. Rapa and BafA1 significantly promoted and inhibited the expression of COL2 in C28/I2 cells（P<0.05）, respectively. The human COL2A1 promoter plasmid was successfully constructed, and the transcriptional activity of the COL2A1 promoter was up-regulated by the overexpression of ATG5（P<0.01）. Treatment with autophagy dual-fluorescence virus resulted in LC3-GFP fluorescence quenching after overexpression of ATG5（P<0.01）, suggesting autophagy activation; and a greater fluorescence intensity of LC3-GFP was observed after knockdown of ATG5（P<0.05）, demonstrating autophagy inhibition. Rapa obviously promoted the expression of COL2 and LC3Ⅱ/LCⅠ, which were up-regulated by ATG5（P<0.05）, but BafA1 showed the opposite effect of Rapa（P<0.05）.

Overexpression of ATG5 up-regulates the expression of type Ⅱ collagen, and the regulation of ATG5 on the expression of type Ⅱ collagen depends on autophagy in chondrocytes.

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.