ZNF667 alleviates the inflammatory damage in intervertebral disc degeneration via inhibiting NF-κB signaling pathway

Abstract

Objectives: With the aging population, the incidence of intervertebral disc degeneration (IDD) is increasing every year. The pathogenesis of IDD is complex, and there are currently no effective treatment options. This study aims to investigate the specific function and underlying mechanism of zinc finger protein 667 (ZNF667) in the inflammatory damage of nucleus pulposus cells in IDD.

Methods: Differential expression genes (DEG) associated with IDD were screened from IDD-related datasets in the Gene Expression Omnibus (GEO) (GSE124272 and GSE150408), and ZNF667, which is closely related to gene transcriptional regulation, was selected and analyzed in several IDD-related datasets (GSE124272, GSE150408, GSE56081, GSE147383, GSE23130). Nucleus pulposus tissues were collected from 3 IDD patients and 3 trauma-induced vertebral fracture patients (serving as controls). Hematoxylin and eosin (HE) staining was performed for pathological examination, and immunohistochemistry (IHC) was used to assess ZNF667 expression in the nucleus pulposus tissues. Gene set enrichment analysis (GSEA) was then employed to elucidate the potential mechanisms of ZNF667. For in vitro validation, human primary nucleus pulposus cells were treated with 10 ng/mL of interleukin-1β (IL-1β) to establish an IDD cell model, and subsequently transfected with a ZNF667 overexpression plasmid. Flow cytometry was used to evaluate cell apoptosis, enzyme-linked immunosorbent assay (ELISA) measured the levels of inflammatory factors-cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the cell culture supernatant, real-time polymerase chain reaction (RT-PCR) quantified ZNF667 mRNA expression, and Western blotting assessed protein expression levels of ZNF667, myeloid differentiation factor 88 (MyD88), P65, and phosphorylated P65 (p-P65).

Results: Analysis of both the GEO datasets and clinical tissue samples revealed that ZNF667 expression is reduced in IDD. In IDD patients, the extracellular matrix and nucleus pulposus cells are significantly diminished, and the arrangement of fibrochondrocytes is disordered. GSEA results showed that ZNF667 may be involved in biological processes such as angiogenesis, epithelial-mesenchymal transition (EMT), oxidative phosphorylation, peroxisome function, steroid biosynthesis, and the NF-κB-mediated TNF-α signaling pathway. In vitro, ZNF667 was expressed at low levels in the IL-1β-induced IDD cell model, and overexpression of ZNF667 reversed the IL-1β-induced increase in cell apoptosis, the upregulation of inflammation factors (COX-2, IL-6, TNF-α), and the increased expression of NF-κB pathway-related proteins (MyD88 and the p-P65/P65 ratio) (all P<0.05).

Conclusions: ZNF667 can alleviate nucleus pulposus cell apoptosis and inflammatory responses by inhibiting the NF-κB signaling pathway, thereby exerting a protective effect on intervertebral discs. This finding not only provides new insights into the pathogenesis of IDD but also suggests a potential therapeutic target for its treatment.

Keywords: intervertebral disc degeneration; nuclear factor-κB signaling pathway; nucleus pulposus cells; zinc finger protein 667.

图1

IDD中DEG的筛选 Figure 1 Screening for DEG in IDD A-B: Volcano plots of DEG analyzed in the GSE124272 dataset (A) and GSE150408 dataset (B); C-D: Cross-screening microarrays for genes jointly up-regulated (C) and down-regulated (D) in GSE124272 and GSE150408. DEG: Differential expression genes; IDD: Intervertebral disc degeneration; FC: Fold change; NS: Not significance.

图2

ZNF667在IDD相关数据集中的表达 Figure 2 ZNF667 expression in IDD-related datasets ***P<0.001. ZNF667: Zinc finger protein 667; IDD: Intervertebral disc degeneration.

图3

Control组和IDD组髓核组织的病理改变和ZNF667在髓核组织中的表达情况 Figure 3 Pathological changes and expression of ZNF667 in the nucleus pulposus tissues of the Control and IDD groups A: HE staining to observe the histopathological changes; B: IHC to observe the expression of ZNF667. IDD: Intervertebral disc degeneration; ZNF667: Zinc finger protein 667; HE: Hematoxylin and eosin; IHC: Immunohistochemistry technique.

图4

GSE56081数据集中ZNF667功能富集分析 Figure 4 Functional enrichment analysis of ZNF667 in GSE56081 dataset ZNF667: Zinc finger protein 667; TNF-α: Tumor necrosis factor-α; NF-κB: Nuclear factor-κB.

图5

GSE147383数据集中ZNF667功能富集分析 Figure 5 Functional enrichment analysis of ZNF667 in GSE147383 dataset ZNF667: Zinc finger protein 667; KEGG: Kyoto Encyclopedia of Genes and Genomes.

图6

在IL-1β诱导的IDD细胞模型中，细胞凋亡率增加，炎症因子表达上调，ZNF667表达下调，NF-κB信号通路被激活 Figure 6 In the IDD cell model induced by IL-1β, the apoptosis rate increases, the expression of inflammatory factors is upregulated, the expression of ZNF667 is downregulated, and the NF-κB signaling pathway is activated A: Apoptosis levels detected by flow cytometry; B: Levels of inflammatory factors COX-2, IL-6, and TNF-α detected by ELISA; C and D: mRNA expression (C) and protein expression (D) of ZNF667 detected by real-time PCR and Western blotting, respectively; E: Protein expression of MyD88, P65, and p-P65 detected by Western blotting. Data are expressed as mean±standard deviation, n=3. *P<0.05, **P<0.01 vs the Control group. IL-1β: Interleukin-1β; IDD: Intervertebral disc degeneration; ZNF667: Zinc finger protein 667; NF-κB: Nuclear factor-κB; PE: Phycoerythrin; FITC: Fluorescein isothiocyanate; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ELISA: Enzyme-linked immunosorbent assay; COX-2: Cyclooxygenase-2; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; MyD88: Myeloid differentiation factor 88; p-P65: Phosphorylation-P65.

图7

ZNF667 过表达对IL-1β诱导的IDD细胞模型炎性损伤和NF-κB信号通路的影响 Figure 7 Effects of ZNF667 overexpression on IL-1β induced inflammatory injury and NF-κB signaling pathway in IDD cell model A and B: mRNA expression (A) and protein expression (B) of ZNF667 detected by real-time PCR and Western blotting, respectively; C: Apoptosis levels detected by flow cytometry; D: Levels of inflammatory factors COX-2, IL-6, and TNF-α detected by ELISA; E: Protein expression of MyD88, P65, and p-P65 detected by Western blotting. Data are expressed as mean±standard deviation, n=3. *P<0.05, **P<0.01 vs the IL-1β+oe-NC group. ZNF667: Zinc finger protein 667; IL-1β: Interleukin-1β; IDD: Intervertebral disc degeneration; NF-κB: Nuclear factor-κB; PE: Phycoerythrin; FITC: Fluorescein isothiocyanate; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ELISA: Enzyme-linked immunosorbent assay; COX-2: Cyclooxygenase-2; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-α; MyD88: Myeloid differentiation factor 88; p-P65: Phosphorylation-P65.