High glucose‐induced excessive reactive oxygen species promote apoptosis through mitochondrial damage in rat cartilage endplate cells

Abstract

Diabetes mellitus (DM) is an important factor in intervertebral disc degeneration (IDD). Apoptosis of cartilage endplate (CEP) cells is one of the initiators of IDD. However, the effects of high glucose on CEP cells are still unknown. Therefore, we conducted the present study to evaluate the effects of high glucose on CEP cells and to identify the mechanisms of those effects. Rat CEP cells were isolated and cultured in 10% foetal bovine serum (FBS, normal control) or high-glucose medium (10% FBS + 0.1 M glucose or 10% FBS + 0.2 M glucose, experimental conditions) for 1 or 3 days. In addition, CEP cells were treated with 0.2 M glucose for 3 days in the presence or absence of alpha-lipoic acid (ALA, 0.15 M). Flow cytometry was performed to identify and quantify the degree of apoptosis. The expression of reactive oxygen species (ROS) was assessed by flow cytometry, and mitochondrial damage (mitochondrial membrane potential) was assessed by fluorescence microscopy. Furthermore, the expression levels of cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, and cytochrome c were evaluated by Western blotting. High glucose significantly increased apoptosis and ROS accumulation in CEP cells in a dose- and time-dependent manner. Meanwhile, a disrupted mitochondrial membrane potential was detected in rat CEP cells cultured in the two high glucose concentrations. Incubating in high glucose enhanced the expression levels of cleaved caspase-3, cleaved caspase-9, Bax, and cytochrome c but decreased the level of the anti-apoptotic protein Bcl-2. ALA inhibited the expression of cleaved caspase-3, cleaved caspase-9, Bax, and cytochrome c but enhanced the expression of Bcl-2. ALA also prevented disruption of the mitochondrial membrane potential in CEP cells. This study demonstrates that high glucose-induced excessive reactive oxygen species promote mitochondrial damage, thus causing apoptosis in rat CEP cells in a dose- and time-dependent manner. ALA could prevent mitochondrial damage and apoptosis caused by high glucose in CEP cells. The results suggest that appropriate blood glucose control may be the key to preventing IDD in diabetic patients. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2476-2483, 2018.

High glucose promotes annulus fibrosus cell apoptosis through activating the JNK and p38 MAPK pathways

“…In addition, our results also showed a positive dose-effect relationship between glucose concentration and AF cell apoptosis. In line with us, several studies also showed that high glucose promotes disc cell apoptosis, such as CEP cells and notochordal cells [20,21]. Hence, inhibiting high glucose-induced disc cell apoptosis may be an effective strategy to retard disc degeneration in DM patients.…”

“…Recently, some epidemiological studies have demonstrated that diabetes facilitates disc degeneration, and DM patients have a higher incidence of disc degeneration than non-DM patients [3,[27][28][29]. Several basic researches have investigated the responses of disc cells to high glucose treatment and reported that high glucose significantly promotes senescence of disc NP cells, notochordal cells and AF cells [17][18][19], and induces apoptosis of disc CEP cells and notochordal cells [20,21]. However, the effects of high glucose on AF cell apoptosis are unclear.…”

“…Previously, some studies have reported that a high glucose environment significantly promotes senescence of disc NP cells, notochordal cells and AF cells [17][18][19]. Moreover, several studies have showed that a high glucose environment induces apoptosis of disc CEP cells and notochordal cells [20,21]. However, few studies have observed the effects of a high glucose environment on disc AF cell apoptosis.…”

Long non‑coding RNA MALAT1 promotes high glucose‑induced rat cartilage endplate cell apoptosis via the p38/MAPK signalling pathway

“…Since the endplate capillaries are responsible for supplying nutrients to the intervertebral discs, degeneration of the endplates is thought to be a risk factor for IDD, as it can hinder the transport of nutrients and eventually lead to IDD (6)(7)(8). Therefore, CEPs may be particularly susceptible to the vasculopathy associated with dM and increasing evidence has revealed that dM plays an important role in ceP degeneration (2,(9)(10)(11)(12). Fields et al revealed that endplate sclerosis, increased endplate thickness and decreased endplate porosity were observed in rats with type 2 diabetes (9).…”

“…Fields et al revealed that endplate sclerosis, increased endplate thickness and decreased endplate porosity were observed in rats with type 2 diabetes (9). Furthermore, high-glucose was revealed to induce the apoptosis of rat cartilage endplate cells in a dose-and time-dependent manner (12).…”