Effects of Senescent Hepatocytes Exosomes on Inflammatory Chondrocytes Induced by LPS and Intervention Effect of Paeonia Lactiflora

OBJECTIVE  To explore the effects of senescence hepatocellular exosomes on LPS-induced chondrocytes and the intervention effect of Paeonia lactis.METHODS  The primary hepatocytes of mice were extracted and the optimal concentration of the decoction of Paeonia lactis was selected by CCK-8. Subsequently, liver cells were randomly divided into the normal group, the senescence group and the Paeonia lactide group. The senescence of liver cells was induced by 0.3 mm hydrogen peroxide, and the gene and protein expressions of p16, p21 and p53 were detected to observe the senescence of liver cells and the intervention effect of Paeonia lactis. Exosomes were extracted from the supernatant of liver cells in each group, and identified by electron microscopy, particle size analysis, and tetrapod CD9, CD63, CD81. Chondrocytes were cultivated in mice, the content of TNF-α and IL-1β in cell supernatant were determined with or without LPS. The intake of chondrocytes were observed by PHK67 dye marker, and LPS-induced chondrocytes were treated with exosomes collected from the supernatant of each liver cell groups. Both gene and protein expression of chondrocyte matrix degradation-synthetic markers MMP3, MMP13, SOX9, ADAMTS5 were detected.RESULTS  100 μg·mL^-1 was the optimal concentration of the decoction of Paeonia lactis. After hydrogen peroxide intervention, the gene and protein expression of cell senescence markers p16, p21 and p53 in the senescence group were all higher than those in the normal group (P < 0.05), but were lower in the Paeonia lactis group than those in the senescence group (P < 0.05). Exosomes collected by liver cells from each group presented a bistratal membranaceous structure, with no difference in morphology and size. The enrichment of the exosomes in particle sizes all met the range of 40-120 nm, the representative particle sizes were enriched at 116.8 nm, and the abundance was 98%. The exosome markers CD9, CD63, and CD81 were all positively expressed. The levels of pro-inflammatory cytokines TNF-α and IL-1β in chondrocytes supernatant increased after LPS stimulation compared with before (P < 0.01), and PHK67 staining reagent was used to confirm the uptake of chondrocyte exosomes in each group. The expression levels of genes and proteins of chondrocyte matrix degradation-synthetic markers MMP3, MMP13, SOX9 and ADAMTS5 in the senescence group were all higher than that in the normal group (P < 0.05), while the exosomes in the Paeonia lactis group were lower than that in the senescence group (P < 0.05).CONCLUSION  Ageing phenotype hepatocyte exosomes can aggravate the LPS-induced degeneration of inflammatory chondrocytes, and the decoction of Paeonia lactiflora has a benign intervention in this process.