Fibroblast IRF7-mediated chondrocyte apoptosis affects the progression of collapse in steroid-induced osteonecrosis of the femoral head.

Abstract

Purpose: The objective of this study was to identify potential genes implicated in the "peri-collapse" synovium of osteonecrosis of the femoral head through coding gene sequencing and to further clarify their specific mechanisms via in vitro experiments.

Methods: Steroid-induced osteonecrosis of the femoral head (SIONFH) (n = 3), femoral neck fracture (FNF) (n = 3), and hip osteoarthritis (HOA) (n = 3) Synovial tissue of the hip joint was collected in total hip arthroplasty. A cellular model of SIONFH constructed from rat synovial fibroblasts by lipopolysaccharide intervention. Lentiviral technology was used to construct a model for fibroblast knockout of the Irf7 gene. HE was used to compare the characteristics of synovial tissue damage, and immunofluorescence and immunohistochemistry were used to compare the expression levels of VIM, IRF7, and IFNα. PCR, WB, and IF were used to examine Irf7 knockdown efficiency, chondrocyte proliferation (Col2a1, Aggrecan, Sox9), cartilage matrix degradation (Mmp13), and apoptosis (Bcl2, Bax, and Caspase3) expression under co-culture conditions. Crystalline violet staining was used to observe the migration rate of fibroblasts, and flow cytometry was used to detect the apoptosis level of chondrocytes under co-culture conditions.

Results: Transcriptome sequencing of synovial tissue and fibroblasts ultimately screened for six differential genes, HOOK1, RNPC3, KCNA3, CD48, IRF7, SAMD9. Compared to FNF and HOA, synovial inflammatory cell recruitment and synovial hyperplasia were more pronounced in SIONFH. IF and IHC confirmed high expression of IRF7 and IFNα in the synovium of SIONFH. PCR and WB results suggested that fibroblasts highly expressed Irf7, Hook1, Rnpc3, Kcna3, Cd48, Samd9, Il-6, and Tnfα after lipopolysaccharide intervention, and the expression levels of Il-6 and Tnfα were significantly reduced after knockdown of Irf7 (P < 0.001). In the co-culture system, fibroblasts intervened with lipopolysaccharide significantly promoted chondrocyte apoptosis, the rate of cartilage matrix degradation, while inhibiting the level of chondrocyte proliferation, and this result was significantly reversed in Irf7 knockout fibroblasts. This was supported by flow cytometry results.

Conclusions: IRF7, HOOK1, RNPC3, KCNA3, CD48, and SAMD9 as potential genes affecting the progression of SIONFH collapse. Irf7 mediates the fibroblast inflammatory response and affects the collapse process of SIONFH by influencing chondrocyte apoptosis. Thus, intervention in IRF7 holds promise as one of the key targets for reversing the collapse process of SIONFH.