Effect of a nonprotein bioactive agent on the reduction of cyclooxygenase-2 and tumor necrosis factor-alpha in human intervertebral disc cells in vitro.

OBJECT: Neurotropin is a nonprotein extract from the inflamed skin of rabbits inoculated with vaccinia virus. In the present study the authors sought to clarify the focal antiinflammatory effects of Neurotropin in intervertebral disc cells, and these effects were compared with those induced by the selective cyclooxygenase (COX)-2 inhibitor 6-methoxy-2-naphthylacetic acid (nabumetone). METHODS: Six human intervertebral disc specimens were harvested during spinal surgery for lumbar disc herniation. Cells were stimulated with 500 pg/ml of interleukin (IL)-1beta in the presence of various concentrations of Neurotropin (0, 10(-5), 10(-4), and 10(-3) Neurotropin Units/ml) or 50 microg/ml of nabumetone for 3 hours. The mRNA was extracted for polymerase chain reaction (PCR), and real-time PCR was used to quantify the mRNA levels of COX- 2, tumor necrosis factor (TNF)-alpha, and phospholipase A2. Cyclooxygenase-2, TNFalpha, and prostaglandin E2 (PGE2) protein concentrations were each determined by enzyme-linked immunosorbent assay. RESULTS: Neurotropin was found to significantly suppress the expression of COX-2 and TNFalpha at mRNA levels as well as the concentration of COX-2 at protein levels in a dose-dependent manner. Nabumetone was found to significantly increase COX-2 at mRNA levels but directly suppress the concentration of PGE2 in culture medium. CONCLUSIONS: Results in this study suggest that Neurotropin has an analgesic effect through the suppression of COX-2 and TNFalpha in a focal area, and nabumetone shows this same effect through the suppression of PGE2 production. Thus, Neurotropin could decrease pain by blocking the central pain pathway or increasing focal antiinflammatory effects.

Immunohistochemical demonstration of advanced glycation end products and the effects of advanced glycation end products in ossified ligament tissues in vitro.

STUDY DESIGN: This study correlates advanced glycation end products with ossified ligament tissues of the cervical spine in vitro. OBJECTIVE: To investigate the effect of advanced glycation end products on ossification of the spinal ligaments in vitro. SUMMARY OF BACKGROUND DATA: We have hypothesized that an accumulation of advanced glycation end products in the spinal ligament might result in some observable change in specific growth factors responsible for ossification in the spinal ligaments. METHODS: Samples of the posterior longitudinal and yellow ligaments were harvested from patients (n = 5) with ossification of the posterior longitudinal ligament, and analyzed for the presence of advanced glycation end products and their receptor advanced glycation end product receptor by immunohistochemistry. Real-time polymerase chain reaction (PCR) was used to quantify the messenger ribonucleic acid (mRNA) levels of bone morphogenetic protein (BMP)-2, BMP-7, alkaline phosphatase, an osteoblast-specific transcription factor 1 (Cbfa1), and osteocalcin from yellow ligament cells treated with advanced glycation end products. RESULTS: Immunohistochemical analysis revealed that advanced glycation end products and advanced glycation end product receptor were localized to within the posterior longitudinal and yellow ligaments. Advanced glycation end products were found to increase significantly the expression of BMP-2, BMP-7, Cbfa1, and osteocalcin at the mRNA levels after treatment with advanced glycation end products (1 microg/mL). CONCLUSIONS: This is the first report to investigate the correlation, if any, between the ossified spinal ligament and advanced glycation end products. These results suggested that accumulation in advanced glycation end products and their interaction with advanced glycation end product receptor were 1 of the important risk factors in the process of ossification in the spinal ligaments.

Positive feedback loop of interleukin-1beta upregulating production of inflammatory mediators in human intervertebral disc cells in vitro.

OBJECT: Interleukin-1beta (IL-1beta) induces neurological symptoms in intervertebral disc herniation (IDH). Recently, the existence of a positive feedback loop of IL-1beta, which encourages an inflammatory reaction or degeneration in the cells of tendon, has been reported. The authors hypothesized that there is a positive feedback loop of IL-1beta in the cells of IDH. METHODS: Eight human intervertebral disc specimens were harvested during spinal surgery for lumbar disc herniation. The cells were stimulated in serum-free medium with or without exogenous IL-1beta. The messenger RNA (mRNA) was extracted for reverse-transcription polymerase chain reaction (PCR) and real-time PCR to quantify the mRNA of endogenous IL-1beta, IL-6, cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs). The cells were then stimulated in serum-free medium with or without exogenous IL-1beta, and then exogenous IL-1beta was removed. After 2, 4, and 6 days, the medium was collected, and enzyme-linked immunosorbent assay was used to measure the protein concentration of endogenous IL-1beta. The mRNA expressions of endogenous IL-1beta, IL-6, COX-2, and MMPs were increased significantly depending on the concentration of exogenous IL-1beta. The protein concentration of endogenous IL-1beta was increased over time. CONCLUSIONS: There was a positive feedback loop of IL-1beta in the cells of IDH. Furthermore, the productions of IL-6, COX-2, MMP-1, and MMP-3 were upregulated as a result of the increasing concentration of IL-1beta in a positive feedback loop of IL-1beta. The authors concluded that this positive feedback loop of IL-1beta upregulated the production of mediators and thus can cause cessation of symptoms in IDH.