Effect of cobalt ions on TNF-α and IL-6 secretion by fibroblasts surrounding hip periprosthetic membrane

Abstract

Aims: The periprosthetic fibroblast-like cells (PPFs) play an important role in aseptic loosening after total hip arthroplasty (THA). However, little is known about fibroblast metabolism in aseptic loosening. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and il-6 interleukin-6 (IL-6) are involved in periprosthetic osteolysis. Cobalt (Co) ions are capable of inducing cytokines from macrophage. In this study, we investigated the effects of Co²⁺ on glycolysis and secretion of TNF-α and IL-6 in PPFs.

Materials and methods: Fibroblasts were isolated from synovial tissues of osteoarthritis (OA) and rheumatoid arthritis (RA) patients, as well as from the periprosthetic pseudomembrane of patients undergoing revision surgery for aseptic loosening. Cells were cultured with or without Co²⁺. Following treatment, fibroblast viability was assessed using the MTT assay. To evaluate glycolysis, glucose uptake and lactate secretion were measured using specific assay kits. Furthermore, gene expression of key glycolysis enzymes (glucose transporter -1(GLUT1), hexokinase-2(HK2)) was analyzed by quantitative real-time PCR (qPCR), while protein expression of protein kinase B (AKT) and phosphorylated AKT (pAKT) was detected via Western blotting. Finally, TNF-α and IL-6 secretion into the culture supernatant was quantified using enzyme-linked immunosorbent assay (ELISA) kits.

Results: Increased glucose uptake and lactic acid secretion occurred in PPFs. Exposure to Co²⁺ significantly increased glucose uptake, lactate secretion, GLUT1/HK2 mRNA expression, and TNF-α/IL-6 levels in PPFs. This Co²⁺-induced enhancement of glycolysis and cytokine secretion was dependent on glycolytic activity, as inhibition with 2-deoxy-D-glucose (2-DG) reduced all measured parameters. Furthermore, Co²⁺ stimulation increased pAKT protein expression in PPFs, indicating activation of the PI3K/AKT pathway. Consistent with this, treatment with the phosphatidylinositol three kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 attenuated the Co²⁺-induced increases in glucose uptake, lactate secretion, GLUT1/HK2 mRNA, and TNF-α/IL-6 levels.

Conclusion: Our findings suggest that Co²⁺ enhances TNF-α and IL-6 secretion in PPFs by upregulating glycolysis. This glycolytic regulation of cytokine production appears to be mediated by the PI3K/AKT signaling pathway, identifying it as a potential novel therapeutic target for preventing aseptic loosening.

Keywords: Co2+; aseptic loosening; fibroblast; glycolysis; proinflammatory cytokines.

FIGURE 1

Glucose and lactate levels. After passaging the cells for 4 times, we used glucose assay kit and lactate assay kit to measure glucose (a) and lactate (b) levels. Results in a–b are pooled from three different cell lines. Values are the mean ± SEM. ** = P < 0.05; *** = P < 0.01; **** = P < 0.001.

FIGURE 2

Glycolysis and secretory function of PPFs after Co²⁺ stimulation. (a,b), the periprosthetic fibroblast-like cells were stimulated with Co²⁺ or phosphate-buffered saline (PBS) as vehicle control, for 24 h, followed by measurement of the Glucose (a) and Lactate (b). (c,d), the periprosthetic fibroblast-like cells were stimulated with Co²⁺ or phosphate-buffered saline (PBS) as vehicle control, for 24 h, the expression of messenger RNA (mRNA) for HK2 (c) and GLUT1 (d) were determined by quantitative polymerase chain reaction (qPCR). (e,f), Supernatants from PPFs cultures were prepared after 24 h of Co²⁺ stimulation and were analyzed for secretion of IL-6 (e) and TNF-α (f). Results in a–g are pooled from three different cell lines. Values are the mean ± SEM. *** = P < 0.01; **** = P < 0.001.

FIGURE 3

effect of inhibition of glycolysis on the periprosthetic fibroblast-like cells function. (a,b) (a) Lactate and (b) glucose in the supernatant 24 h after Co²⁺ or PBS stimulation in the presence or absence of glycolysis inhibitors (2-DG: 50 mM in PBS). (c) The periprosthetic fibroblast-like cells were cultured in the presence of Co²⁺ or PBS, with or without 2-DG (50 mM in PBS) or no glucose (NG) medium. Cellular proliferation was determined by MTT assay on day 4. (d,e) In the presence of Co²⁺ or PBS as carrier control, the PPFs were cultured with or without 2DG (50 mM) pretreatment. Supernatant from cell cultures was prepared after 24 h of Co²⁺ stimulation and were analyzed for secretion of IL-6 (d) and TNF-α (e). (f,g) the periprosthetic fibroblast-like cells were stimulated with Co²⁺ or PBS), with or without 2-DG (50 mM in PBS), the mRNA for GLUT1 (f) and HK2 (g) were determined by qPCR. Results in a–g are pooled from three different cell lines. Values are the mean ± SEM. ** = P < 0.05; **** = P < 0.001.

FIGURE 4

Role of PI3K/AKT pathway in increased glycolysis and functional changes of Co²⁺ stimulated periprosthetic fibroblast-like cells. (a) The periprosthetic fibroblast-like cells (n = 3 cell lines) was first incubated with an PI3K inhibitor (LY294002) at different concentrations for 1 hour and then stimulated with Co²⁺ or PBS for 15 min. P-AKT, AKT and actin expression was determined my WB. (b,c) In the presence or absence of inhibitors, Supernatant from cell cultures was prepared and the secretion of IL-6 (b) and TNF-α (c) was analyzed. (d,e) In the presence or absence of inhibitors, glucose (d) and lactate (e) in supernatant after Co²⁺ or PBS stimulation for 24 h. (f,g) The periprosthetic fibroblast-like cells were cultured with or without LY294002. The mRNA expression of GLUT1 (f) and HK2 (g) was determined by qPCR. Results in b–g are pooled from four different cell lines. Values are the mean ± SEM. *** = P < 0.01; **** = P < 0.001.