FABP4 knockdown suppresses inflammation, apoptosis and extracellular matrix degradation in IL-1β-induced chondrocytes by activating PPARγ to regulate the NF-κB signaling pathway

Abstract

Osteoarthritis (OA) is a common degenerative disease that can lead to severe joint pain and loss of function, seriously threatening the health and normal life of patients. At present, the pathogenesis of OA remains to be clarified. Recent studies have shown that fatty acid‑binding protein 4 (FABP4) is increased in the plasma and synovial fluid of patients with OA. However, the effect of FABP4 on OA is unclear. The present study established IL‑1β‑induced ATDC5 cells with FABP4 knockdown. Next, cell viability was detected with Cell Counting Kit‑8 assay. The content of inflammatory factors, prostaglandin E2 and glycosaminoglycan (GAG) was detected via ELISA. The levels of reactive oxygen species (ROS) and superoxide dismutase (SOD) in cells were detected by using ROS and SOD kits, respectively. TUNEL staining was used to detect the apoptosis level. Western blotting was used to detect the expression levels of proteins. The results revealed that FABP4 was upregulated in IL‑1β‑induced ATDC5 cells. Knockdown of FABP4 increased cell viability, reduced inflammatory damage, oxidative stress and apoptosis in IL‑1β‑induced ATDC5 cells. Following FABP4 knockdown, the expression of matrix metalloproteinases (MMP3, MMP9 and MMP13) of IL‑1β‑induced ATDC5 cells was reduced, and the expression of GAG was promoted. FABP4 knockdown also inhibited the expression of NF‑κB p65 and enhanced peroxisome proliferator‑activated receptor (PPAR)γ expression. However, the presence of PPARγ inhibitor blocked the aforementioned effects of FABP4 on IL‑1β‑induced ATDC5 cells. In conclusion, FABP4 knockdown suppressed the inflammation, oxidative stress, apoptosis and extracellular matrix degradation of IL‑1β‑induced chondrocytes by activating PPARγ to inhibit the NF‑κB signaling pathway.

Keywords: NF-κB signaling pathway; fatty acid-binding protein 4; inflammation; osteoarthritis; peroxisome proliferator- activated receptor γ.

Figure 1.

Knockdown of FABP4 suppresses the activity and inflammatory damage of IL-1β-induced ATDC5 cells. (A) Western blotting and (B) RT-qPCR revealed that FABP4 was significantly increased in IL-1β-induced ATDC5 cells compared with that in control ATDC5 cells (n=3). Transfection efficiency of siRNA-FABP4-1 and siRNA-FABP4-2 was verified by using (C) western blotting and (D) RT-qPCR (n=4). (E) Cell Counting Kit-8 assay was performed to detect changes in the viability of IL-1β-induced ATDC5 cells (n=3). The levels of (F) TNF-α, (G) IL-6 and the (H) cell growth and regulatory factor PGE₂ were determined by using ELISAs (n=5). **P<0.01, ***P<0.001. RT-qPCR, reverse transcription-quantitative PCR; FABP4, fatty acid-binding protein 4; siRNA, small interfering RNA; PGE₂, prostaglandin E2; NC, negative control.

Figure 2.

Knockdown of FABP4 suppresses the oxidative stress and apoptosis of IL-1β-induced ATDC5 cells. (A) ROS levels in cells were detected by using a ROS ELISA kit (n=6). (B) Expression of SOD was detected by using a SOD ELISA kit (n=6). (C) Apoptosis level was detected using TUNEL staining (n=4). (D) Western blotting was used to detect the expression of Bcl-2, Bax and cleaved caspase 3 (n=3). **P<0.01, ***P<0.001. FABP4, fatty acid-binding protein 4; siRNA, small interfering RNA; ROS, reactive oxygen species; SOD, superoxide dismutase; NC, negative control.

Figure 3.

Knockdown of FABP4 suppresses matrix-degrading proteases and promotes the expression of GAG. (A) Western blotting and (B) reverse transcription-quantitative PCR assay were used to detect the expression of MMP3, MMP9, MMP13 and ADAMTS-4 (n=4). (C) Expression of GAG was detected by using a GAG ELISA kit (n=3). ***P<0.001. FABP4, fatty acid-binding protein 4; siRNA, small interfering RNA; GAG, glycosaminoglycan; ADAMTS-4, ADAM metalloproteinase with thrombospondin type 1 motif 4; NC, negative control.

Figure 4.

Knockdown of FABP4 suppresses the cell viability and inflammatory factors of IL-1β-induced ATDC5 cells by activating PPARγ. (A) Data from the Search Tool for the Retrieval of Interacting Genes/Proteins database showed that FABP4 had the potential to regulate the expression of PPARγ. (B) Expression levels of PPARγ and NF-κB p65 signaling proteins were detected using western blotting (n=3). (C) Cell Counting Kit-8 assay was performed to detect the viability of IL-1β-induced ATDC5 cells with or without PPARγ inhibitor treatment (n=6). The levels of (D) TNF-α, (E) IL-6 and (F) the cell growth and regulatory factor PGE₂ were measured by using ELISAs (n=4). *P<0.05, **P<0.01, ***P<0.001. FABP4, fatty acid-binding protein 4; PPARγ, peroxisome proliferator-activated receptor γ; PGE₂, prostaglandin E2; siRNA, small interfering RNA; NC, negative control; p-, phosphorylated; t-, total.

Figure 5.

Knockdown of FABP4 suppresses the oxidative stress and apoptosis of IL-1β-induced ATDC5 cells by activating PPARγ. (A) ROS expression in cells was detected by using a ROS ELISA kit (n=4). (B) Expression of SOD was detected by using a SOD ELISA kit (n=4). (C) Apoptosis level was detected by using TUNEL staining (n=3). (D) Western blotting was used to detect the expression of Bcl-2, Bax and cleaved caspase 3 (n=4). *P<0.05, **P<0.01, ***P<0.001. FABP4, fatty acid-binding protein 4; PPARγ, peroxisome proliferator-activated receptor γ; ROS, reactive oxygen species; SOD, superoxide dismutase; siRNA, small interfering RNA.

Figure 6.

Knockdown of FABP4 suppresses matrix-degrading enzymes and promotes the expression of GAG by activating PPARγ. (A) Western blotting and (B) reverse transcription-quantitative PCR were used to detect the expression of MMP3, MMP9, MMP13 and ADAMTS-4 (n=4). (C) Expression of GAG was detected by using a GAG ELISA kit (n=5). *P<0.05, ***P<0.001. FABP4, fatty acid-binding protein 4; PPARγ, peroxisome proliferator-activated receptor γ; ADAMTS-4, ADAM metalloproteinase with thrombospondin type 1 motif 4; GAG, glycosaminoglycan; siRNA, small interfering RNA.