Resveratrol protection against IL-1β-induced chondrocyte damage via the SIRT1/FOXO1 signaling pathway

Abstract

Purpose: Osteoarthritis (OA) is a common joint disease characterized by cartilage degeneration, synovial inflammation, osteophytes, and subchondral osteosclerosis. This study investigated the effects of resveratrol (RES) on extracellular matrix (ECM), autophagy, and apoptosis in OA pathogenesis via the SIRT1/FOXO1 pathway.

Methods: The microenvironment of OA chondrocytes was stimulated in vitro by adding 10 ng/mL of IL-1β to primary Wistar rat chondrocyte. Western blotting, immunofluorescence, quantitative real-time PCR, and transmission electron microscopy (TEM) were used for analysis.

Results: In the presence of IL-1β, RES increased the expression of silent information regulator (SIR) 1 protein and the phosphorylation level of forkhead transcription factor (FOXO) 1. It also promoted chondrocyte autophagy, increased the expression of SOX9 and aggrecan, inhibited chondrocyte apoptosis and matrix breakdown, and protected chondrocytes from IL-1β damage. After a SIRT1 inhibitor or FOXO1 inhibitor was added, the protective effect of RES on chondrocytes was significantly weakened. Our results suggest that RES regulates the ECM metabolism, autophagy, and apoptosis of OA chondrocytes through the SIRT1/FOXO1 pathway to ameliorate IL-1β-induced chondrocyte injury.

Conclusion: RES protects chondrocytes from IL-1β-induced damage by activating SIRT1/FOXO1 signaling and holds potential in OA treatment.

Keywords: Apoptosis; Autophagy; Chondrocytes; Extracellular matrix; Osteoarthritis; Resveratrol.

Fig. 1

The SIRT1/FOXO1 pathway was downregulated in OA cartilage cells from clinical specimens. A, D Western blot analysis shows the expression of target proteins SIRT1, FOXO1, and p-FOXO1. B, E The relative expression of SIRT1, FOXO1, and p-FOXO1 was quantified by normalization with GAPDH. C, F The mRNA expression levels of SIRT1 and FOXO1 in chondrocytes were quantified by quantitative real-time PCR. G, H The concentration of P-Foxo1 protein in cartilage tissues was detected by immunofluorescence, and quantitative immunofluorescence analysis was performed using ImageJ software. All data are presented as the mean ± SEM (n = 3), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

Effect of RES on chondrocyte proliferation. A Chemical structure of RES. B The effect of RES on chondrocyte proliferation was assayed using CCK-8 to determine the optimal drug concentration for intervening cells, and the RES concentration was selected at 50 µM for all subsequent experiments

Fig. 3

RES attenuates IL-1β-induced chondrocyte damage via the SIRT1/FOXO1 pathway. A Western blot shows the expression of target proteins SIRT1, FOXO1, p-FOXO1, aggrecan, SOX9, MMP13, and ADAMTS5, which are associated with the SIRT1/FOXO1 pathway and apoptosis. B The relative expression of SIRT1, FOXO1, p-FOXO1, aggrecan, SOX9, MMP13, and ADAMTS5 was quantified by normalization with GAPDH. C–H The mRNA expression levels of SIRT1, FOXO1, p-FOXO1, aggrecan, SOX9, MMP13, ADAMTS5 in chondrocytes were quantified by quantitative real-time PCR. a–e Corresponds to control, IL-1β, IL-1β + RES, IL-1β + RES + AS, and IL-1β + RES + EX-527, respectively. AS = FOXO1 inhibitor, EX-527 = SIRT1 inhibitor. All data are presented as the mean ± SEM (n = 3), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

RES improves IL-1β-induced autophagy in chondrocytes via the SIRT1/FOXO1 pathway. A–C Expression of LC3, ATG5, and Beclin 1 genes associated with chondrocyte autophagy was quantified using quantitative real-time PCR. D Groups of intracellular autophagic vesicles were observed by transmission electron microscopy. a–e Correspond to control, IL-1β, IL-1β + RES, IL-1β + RES + AS, and IL-1β + RES + EX-527, respectively. All data are presented as the mean ± SEM (n = 3), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

Effect of RES on IL-1β-induced apoptosis levels in chondrocytes. A Western blot analysis shows the expression of BCL-2, BAX, caspase-3, genes associated with chondrocyte apoptosis. B The relative expression of BCL-2, BAX, and caspase-3 was quantified via normalization to GAPDH. a–e Correspond to control, IL-1β, IL-1β + RES, IL-1β + RES + AS, and IL-1β + RES + EX-527, respectively. C–E Levels of caspase-3, BAX, and BCL-2 in chondrocytes were quantified using quantitative real-time PCR. H Detection of chondrocyte proliferation in each group using CCK-8. F, G Detection of chondrocyte proliferation in each group using TUNEL. All data are presented as the mean ± SEM (n = 3), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001