Accelerated premature stress-induced senescence of young annulus fibrosus cells of rats by high glucose-induced oxidative stress

Abstract

Purposes Diabetes mellitus (DM) is thought to be an important aetiological factor in intervertebral disc degeneration. A glucose-mediated increase of oxidative stress is a major causative factor in development of diseases associated with DM. The aim of this study was to investigate the effect of high glucose on mitochondrial damage, oxidative stress and senescence of young annulus fibrosus (AF) cells. Methods AF cells were isolated from four-week-old young rats, cultured, and placed in either 10 % FBS (normal control) or 10 % FBS plus two different high glucose concentrations (0.1 M and 0.2 M) (experimental conditions) for one and three days. We identified and quantified the mitochondrial damage and reactive oxygen species (ROS) (oxidative stress). We also identified and quantified the occurrence of senescence and telomerase activity. Finally, the expressions of proteins were determined related to replicative senescence (p53-p21-pRB) and stress-induced senescence (p16-pRB). Results Two high glucoses enhanced the mitochondrial damage in young rat AF cells, which resulted in an excessive generation of ROS in a dose-and time-dependent manner for one and three days compared to normal control. Two high glucose concentrations increased the occurrence of senescence of young AF cells in a dose-and time-dependent manner. Telomerase activity declined in a dose-and timedependent manner. Both high glucose treatments increased the expressions of p16 and pRB proteins in young rat AF cells for one and three days. However, compared to normal control, the expressions of p53 and p21 proteins were decreased in young rat AF cells treated with both high glucoses for one and three days. Conclusions The present study demonstrated that high glucose-induced oxidative stress accelerates premature stress-induced senescence in young rat AF cells in a doseand time-dependent manner rather than replicative senescence. These results suggest that prevention of excessive generation of oxidative stress by strict blood glucose control could be important to prevent or to delay premature intervertebral disc degeneration in young patients with DM.

N-Cadherin Attenuates High Glucose-Induced Nucleus Pulposus Cell Senescence Through Regulation of the ROS/NF-κB Pathway

“…Previous epidemiological studies reported that DM patients exhibit a higher incidence of disc degenerative diseases than patients without DM, and DM patients with disc degenerative diseases are much younger than non-DM patients [4, 7, 8]. Notably, several basic researchers demonstrated that high glucose accelerated disc cell senescence in vitro [9, 10]. These findings suggest that a high glucose environment reduces the healthy status of disc NP cell biology.…”

“…NP cells (NP group), NP-N-CDH-NC cells (NP-N-CDH-NC group) and NP-N-CDH cells (NP-N-CDH group) were cultured in 10% FBS culture medium with 0.2 M glucose for 3 days under standard conditions (37°C, 20% O 2 and 5% CO 2 ) to investigate the effects of N-CDH overexpression on NP cell senescence under the high glucose condition. The high glucose concentration used was based on previous studies [9, 10]. …”

Molecular Mechanisms of Intervertebral Disc Degeneration

“…159 NF-kB signaling is known to play a critical role in a cell's response to inflammation and damage, and an increase in its activity has been connected to IDD with accumulated oxidative stress. 129,139,159,160 Pharmacologic and genetic systemic inhibition of NF-kB activity has been shown to reduce associated IDD in a mouse model. 141 Symptomatic discs have been shown to have higher levels of pro-inflammatory cytokines, TNF-α, IL1β, IL-6, and IL-8, which are considered be associated with the NF-kB pathway.…”

“…133,134 With mitochondrion dysfunction as a known source of excessive ROS production, the role of mitochondrion-dependent ROS has been described in various disc cells including human and rat NP and AF cells. [135][136][137][138][139] And more specifically, excessive ROS production has been reported in degenerative discs of rats. 140 Several studies have shown hydrogen peroxide to down regulate the expression of collagen type II and aggrecan in both human and rat disc cells.…”

Intervertebral Disk Degeneration and Repair

“…Increasing levels of ROS have been associated with increasing levels of DDD, inhibition of proliferation of NP cells, and activation of the senescent signal pathways to induce cell cycle arrest of NP cells. [33][34][35] Reducing nutrient supply and growth factors (Insulinlike Growth Factor (IGF-1), Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF)) at the level of the IVD has been shown to increase rates of disk cell senescence. Specifically, IGF-1 has been shown to prevent disk cell senescence induced by oxidative damage.…”