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NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells
Published on: January 12, 2020
Fxyd5 activates the NF‑κB pathway and is involved in chondrocytes inflammation and extracellular matrix degradation
Lulu Song1, Xingxing Li1, Qingwan Sun2
1Capital University of Physical Education and Sports, Haidian, Beijing 100191, P.R. China.
Abstract:
It is known that increased inflammation and extracellular matrix (ECM) degradation in chondrocytes can promote the development of osteoarthritis (OA). The FXYD domain containing ion transport regulator 5 (Fxyd5) has been found to promote chronic inflammatory responses. The present study aimed to investigate the role of Fxyd5 in OA. Murine ATDC5 chondrocytes were transfected with short hairpin RNAs specifically targeting Fxyd5 to silence its expression. Subsequently, cells were induced with lipopolysaccharide (LPS). The protein expression levels of Fxyd5, MMPs and proteins related to ECM, apoptosis and NF‑κB signaling were detected using western blot analysis. In addition, cell viability was assessed using a Cell Counting Kit‑8 assay, while the secretion of the proinflammatory factors and those of the oxidative stress‑related markers were measured using the corresponding kits. Finally, cells were treated with the NF‑κB activator, betulinic acid (BA) and the above experiments were repeated. The results demonstrated that Fxyd5 was significantly upregulated in ATDC5 cells treated with LPS. Additionally, Fxyd5 knockdown increased cell viability, enhanced the protein expression of Bcl‑2, Aggrecan and collagen II, while reduced the expression of Bax, cleaved caspase‑3/caspase‑3, MMP3 and MMP13 in LPS‑induced ATDC5 cells. The production of IL‑1β, IL‑6 and IL‑18 as well as reactive oxygen species and malondialdehyde, and the reduction of superoxide dismutase caused by LPS in ATDC5 cells, were also reversed by Fxyd5 silencing. Fxyd5 silencing inhibited the phosphorylation of p65 and IκBα induced by LPS. Finally, BA reversed the protective effect of Fxyd5 silencing on LPS induced chondrocytes injury. In conclusion, Fxyd5 could enhance chondrocyte inflammation and ECM degradation via activating the NF‑κB signaling.
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