Interleukin 26 attenuates osteoblast differentiation in osteoarthritis patients by activating COX2 and NF-κB pathways

Abstract

Aims: Osteoarthritis (OA) represents the prevailing form of degenerative joint pathology. Recent investigations have revealed a heightened expression of interleukin 26 (IL-26) in various inflammatory arthritic conditions, including OA. However, the specific impacts and functions of IL-26 on osteoblasts (OBs) within the context of OA remain inadequately elucidated. This study aims to clarify the effects and underlying mechanisms of IL-26 by examining its influence on osteoblasts isolated from OA patients and a murine osteoblast cell line. Methods: Human primary osteoblasts and mouse pre-osteoblast cells were subjected to treatment with β-glycerophosphate or concurrent treatment with IL-26 to observe the effects on osteoblast differentiation. The differentiation of osteoblasts was assessed through the expression of relevant genes using reverse transcription-polymerase chain reaction (RT-PCR). Key molecular mechanisms of downstream signaling pathways were examined through immunoblotting assays. Results: Our results reveal that IL-26 mitigates osteoblast differentiation and reduces the expression of the marker alkaline phosphatase. Furthermore, the NF-κB downstream OB proliferated marker iNOS and inhibition OB differentiated marker LCN2 messenger RNA are up-regulated in IL-26 treated group. Also, phosphorylation and nuclear translocation of NF-κB p65 occur following IL-26 stimulation. Additionally, IL-26 enhances the downstream transcription factor cyclooxygenase-2 (COX2), a major player associated with iNOS. STAT1, the canonical receptor signaling pathway of IL-26 is activated. Conclusion: In summary, our findings substantiate the role of IL-26 in osteoarthritis and identify it as a potential therapeutic target for intervention in osteoarthritic pathology.

Keywords: interleukin 26; osteoarthritis; osteoblast differentiation.

Figure 1

IL-26 levels in osteoarthritis patients and impact on osteoblast cell viability. (A) Bone sample specimens were obtained from patients with osteoarthritis (OA). A Histological examination was performed by HE stains. IHC staining was used to detect the expression of IL-26 (Green) in bone tissue. DAPI (Blue) was used for nuclear staining. (B) The immunofluorescent intensity on the bone surface per unit area was quantified using ImageJ software. (C) Mouse 7F2 pre-osteoblasts, (D) HOB was planted in 96-well plates and cultured with the specified concentrations of IL-26 or IgG control group for 24 hours. Add MTT (500 μg/ml) solution and incubate for 1 hour and measure the absorbance at 570 nm. All experiments were repeated at least three times, and the data represent the mean ± standard deviation. HOB: human osteoblast cells. *P<0.05 compared to the dose 0 ng/ml group.

Figure 2

Effect of IL-26 on osteoblast differentiation. (A, C) 7F2 pre-osteoblasts and (B, D) HOB were differentiated using osteoblast differentiation medium (50 μg/ml ascorbic acid and 5 mM bGP) in the presence or absence of IL-26 60 ng/ml. (A, B) Alkaline phosphatase ALP staining and (C, D) alizarin red staining ARS were performed at 7, 14 or 21 days after differentiation medium stimulation. (bGP: β-glycerophosphate; bGP+IL-26: β-glycerophosphate+IL-26) ALP: Alkaline phosphatase assay.

Figure 3

Effect of IL-26 on mRNA expression during 7F2 and HOB osteoblast differentiation. (A-D) 7F2 pre-osteoblasts and (E-H) HOB were differentiated using osteoblast differentiation medium (50 μg/ml ascorbic acid and 5 mM bGP) in the presence or absence of IL-26 60 ng/ml. Collect RNA on the 1st, 3rd or 5th day after differentiation stimulation, extract all the RNA and take 1 μg to transcribe into cDNA, and use the cDNA as a template to add mouse and human specific primers for real-time quantitative polymerase chain reaction, detection osteoblast differentiation gene markers BGLAP, RUNX2, Spp1, and ALP. The experiment was repeated at least three times, and the data represent the mean ± standard deviation. (*P<0.05, **P<0.01, ***P<0.001).

Figure 4

Effects of IL-26 on the expressions of LCN2, OPG, iNOS and RANKL in 7F2 osteoblasts and HOB cells. (A-D) 7F2 pre-osteoblasts and (E-H) HOB were differentiated using osteoblast differentiation medium in the presence or absence of IL-26 60 ng/ml. RNA extraction was performed at 3 and 6 hours after differentiation stimulation, following the previously described method. Real-time quantitative polymerase chain reaction (qPCR) was carried out using mouse and human-specific primers for LCN2, OPG, iNOS, and RANKL genes. The experiment was repeated at least three times, and the data represent the mean ± standard deviation. (*P<0.05, **P<0.01, ***P<0.001).

Figure 5

IL-6 expression in HOB osteoblast differentiation during IL-26 stimulation. HOB was differentiated using osteoblast differentiation medium in the presence or absence of IL-26 for 1 and 3 days. The condition medium was collected after treated IL-26 to HOB. (*P<0.05 compared to the N group; #P<0.05, ##P<0.01 compared to the bGP group).

Figure 6

Effects of IL-26 on signaling pathways during HOB osteoblast differentiation. After 16 hours of serum-free culture, HOB was treated with osteoblast differentiation medium in the presence or absence of IL-26 for different hours. (A) Phosphorylated or non-phosphorylated STAT1, and STAT3, (B) COX-2 were analyzed by western blot. (C) Nuclear (Nu) and cytoplasmic (Cyto) extracts were analyzed by Western blot, and phosphorylated NF-κB proteins were detected using specific antibodies and compared with non-phosphorylated proteins. Electrophoresis was performed with the same amount of protein, TBP was used for the nucleus, and GAPDH was used as the control group for the cytoplasm.