Effects of etanercept, a tumor necrosis factor receptor fusion protein, on primary cell cultures prepared from intact human intervertebral disc tissue

Abstract

The aim of the present study was to investigate the effects of etanercept (ETA), a tumor necrosis factor (TNF) inhibitor, on human cell cultures prepared from intact intervertebral disc tissue. ETA is used as a treatment for cases of rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis and ankylosing spondylitis accompanied by moderate or severe joint pain. ETA was applied to primary cell cultures [annulus fibrosus and nucleus pulposus (NP) from intact intervertebral disc tissue]. Cell cultures without ETA treatment served as the control group. Morphological and quantitative molecular analyses of the two groups were performed. The number of viable cells and cell proliferation decreased in the ETA-treated cultures as compared with those in the control group. Furthermore, in the treatment group, the chondroadherin gene, an NP-specific marker, was not expressed after 24 h. By contrast, the cartilage oligo matrix protein was expressed 24, 48 and 72 h post-ETA treatment, while its expression was significantly lower than that in the control group. In addition, the expression of interleukin-1β, as well as matrix metallopeptidase-7 and −19, was markedly decreased. Overall, the cell proliferation and gene expression in the ETA-treated cells were significantly different from those in the control group (P<0.05). These results suggest that the treatment duration and dosage of TNF inhibitors, which are used to suppress active inflammation, should be considered in the clinical setting. These biological agents may delay the healing of intervertebral disc tissue damage by slowing cell proliferation and altering gene expression via anabolic and catabolic pathways.

Preclinical ex-vivo Testing of Anti-inflammatory Drugs in a Bovine Intervertebral Degenerative Disc Model

“…A clinical study showed that single intradiscal administration of 10 mg Etanercept on patients with LBP could alleviate pain levels for 8 weeks after follow-up (Sainoh et al, 2016). Furthermore, Caliskan and coworkers investigated the effect of Etanercept on primary cell cultures (first-passage cultures of NP and AF cells) from intact human IVD tissue at doses of 0.1, 0.25, 0.5, 1, and 2 mg/mL (Caliskan et al, 2019). The Etanercept doses of >0.5 mg/mL were found to have cytotoxic effects.…”

Does Nimodipine, a Selective Calcium Channel Blocker, Impair Chondrocyte Proliferation or Damage Extracellular Matrix Structures?

“…CHAD proteins bind to integrins, cell surface proteoglycans, and COL2A1; they play a role in regulating chondrocyte signaling and cartilage homeostasis. COL2A1 is an important cartilage-specific matrix protein, and the existence of COL2A1 in normal cartilage is a marker for anabolism [15, 16]. COL2A1 gene expression is regulated by transcription or growth factors, and an increased expression of this gene leads to the induction of ECM production [15, 16].…”

“…COL2A1 is an important cartilage-specific matrix protein, and the existence of COL2A1 in normal cartilage is a marker for anabolism [15, 16]. COL2A1 gene expression is regulated by transcription or growth factors, and an increased expression of this gene leads to the induction of ECM production [15, 16]. HIF1 is a transcription factor that regulates the transcription of a wide range of genes involved in cell survival [9, 12].…”

“…The viability of the control group was assumed to be 100%. After proliferation and inhibition of proliferation were calculated using the “ Test OD/Control ODX100 ” and “ 1- Test OD/Control OD ” formulas, respectively, data were recorded for statistical analysis [9-16].…”

“…To obtain complementary DNA (cDNA), 50 ng RNA was reverse-transcribed via a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat#4368814) using a thermal cycler (ProFlex, Thermo Fisher Scientific) in accordance with the manufacturer’s instructions. For qRT-PCR analysis, all the genes were amplified using TaqMan ® Gene Expression Assays for CHAD (Cat#4331182, Hs00154382_m1), HIF1α (Cat#4331182, Hs00153153_m1), COL2A1 (Cat#4331182, Hs00264051_m1), and internal control genes (housekeeping genes: actin beta (ACTB, Cat#4331182, Hs01060665_g1) in accordance with the manufacturer’s protocol [11, 15, 16]. Each gene was amplified using an RT-PCR reaction mix prepared with 1 μl TaqMan Gene Expression Assay, 10 ml of TaqMan Gene Expression Master Mix (Cat.…”