Role Of Mitochondrial Pathway In Oxidative Stress-induced Apoptosis In Rat Nucleus Pulposus Cells

BackgroundDegenerative disc disease (DDD) is one of the major causes of low back pain which is a common clinical problem in orthopedics and causes a significant socioeconomic problem. Current treatment strategies for disc degeneration include fusion, disc replacement and discectomy. However, these strategies are generally treated in later stage of disc degeneration without treating the underlying cause or restoring biomechanical function. Some surgerys only improve the symptoms caused by disc degeneration, and some will even aggravate decompensated lumbar instability and adjacent segments. There is, therefore, an urgent need to develop biological therapies for intervertebral disc degeneration that can both relieve painful symptoms and recover the disc function to normal status. Typically, the intervertebral disc degeneration begin about20years old, and intervertebral disc degeneration is more and more serious with age. Although the mechanism of DDD is still not fully understood, excessive intervertebral disc cell apoptosis has been suggested to play a key role in promoting the DDD and there is growing evidence indicating that the mechanism may be related to oxidative stress. In recent years, many studies have found that apoptosis is closely related to degeneration of intervertebral disc tissue.Intervertebral disc cells is only1percent of the intervertebral disc, but they can produce extracellular matrix synthesis factors, such as type Ⅰ and type Ⅱ collagen, metal protease, prostaglandins, nitric oxide, etc.These factors play an important role in maintaining the health of the intervertebral disc. In a healthy disc, the dynamic balance of extracellular matrix synthesis and catabolism need to rely on the regulation of these factors.However, under pathological conditions, the phenotypic change of nucleus pulposus cells as well as the number of reduced,nucleus pulposus cells cannot keep the balance between anabolism and catabolism so as to induce the degeneration of intervertebral disc.Recent study found that there are two major apoptosis signaling pathways:the intrinsic mitochondrial pathway and extrinsic death receptor pathway. In the mitochondrial pathway, apoptosis stimulating factors, such as hypoxia, ischemia and active oxygen, can increase the permeability of the mitochondrial outer membrane, causing the release of cytochrome c from mitochondria into the cytoplasm. Cytochrome c associates with APAF1and caspase-9to form the apoptosome, which activates effector caspases and subsequently lead to cell death by apoptosis.The Bcl-2family proteins, such as anti-apoptotic protein Bcl-2and pro-apoptotic protein Bax, are biomarkers for the intrinsic pathway. Over expression of Bcl-2prevent the efflux of cytochrome c from the mitochondria and thus prevent cells from undergoing apoptosis. Conversely, over-expression of Bax and its translocation to mitochondria has been shown to promote the release of cytochrome c into cytosol.Death signals can spread through death receptor from extracellular to intracellular, Death receptor is a group of death domains of membrane receptor, belongs to the tumor necrosis factor receptor (TNFR) gene superfamily. Death receptors can accept the death signals and activate the cell apoptosis mechanism. A domain is a cysteine rich extracellular, membrane area for a period of67～70amino acid sequence, and is closely related to cell apoptosis signal transduction, protein hydrolysis function, called the Deathdomain. At present, there are five kinds of death receptor, including Fas, TNFR-1, DR3, DR4and DR55. Fas is an important death receptor on cell surface, and apoptosis signals received by Fas can through the two signaling pathways triggered programmed cell death. In a Type Ⅰ pathway, binding of Fas ligand (FasL) on the Fas receptor (Fas) leads to strong caspase-8activation at the DISC which bypasses mitochondria directly leading to activation of procaspase-3and subsequently to apoptosis. In a Type Ⅱ pathway, however, only a little DISC is formed, so the caspase cascade cannot be propagated directly and has to be amplified via mitochondria. In this pathway, a little amount of active caspase-8cleaves Bid, a proapoptotic member of the Bcl-2family. The truncated Bid (t-Bid) targets the outer mitochondrial membrane and induces the release of cytochrome c, subsequently leading to apoptosis.Reactive oxygen species (ROS) is one of the important causes of oxidative stress.Reactive oxygen species including enclosed O2-, OH-and H2O2and other harmful intermediate. Under normal conditions, the body has a series of antioxidant defense system:the antioxidant enzymes, such as superoxide dismutase (SOD), glutathione, glycosides peptide peroxidase (gsh-px) and catalase; Other molecule, such as the TRX, gansu valley oboro skin and vitamin A, C, E, etc. Normally, free radical production and antioxidant defence system are in a dynamic balance. When the balance is broken, oxidative stress occurs. Free radical formation can promote lipid peroxidation, DNA strand breaks, and modification or degradation of biomolecules, eventually leading to cell death. ROS can make the extracellular matrix degradation, characterized by inhibition of proteoglycan synthesis and destroy the structure of extracellular matrix protein and protease activity. Excess intracellular ROS could serve as a second messenger to trigger apoptosis signal. At present, many studies have found that high level of ROS can induce nucleus pulposus cells apoptosis and accelerate the degeneration of intervertebral disc.Mitochondria are the main place producing the ROS, and are the most sensitive position about the ROS’ effects. Excess ROS leads to the oxidative stress towards mitochondria, induces a decreased mitochondrial membrane potential, and further induces the cell apoptosis. The mitochondrion is considered a key regulatory center of apoptosis. The loss of mitochondrial membrane potential (ATm) was one of the early critical events in the induction of cellular apoptosis. The loss of mitochondrial membrane potential will cause cytochrome c release into the cytosol.Cytochrome c associates with APAF1and caspase-9to form the apoptosome, which activates effector caspases and subsequently lead to cell death by apoptosis.Howerer, it is unknown whether mitochondrial pathway play a critical role in oxidative stress-induced apoptosis in nucleus pulposus cells. ROS may serve as potential targets in treatment of disc degeneration. To investigate the role of mitochondrial pathway in oxidative stress-induced apoptosis in rat nucleus pulposus cells is of great significance.ObjectiveThe aim of the present study was to investigate the apoptotic effect of mitochondrial pathway in oxidative stress-induced apoptosis in the rat nucleus pulposus cells. This study will establish the principle and experimental foundations for further prevention and treatment of degenerative disc disease.Methods1, Male Sprague-Dawley rats obtained from the Experiment Animal Center of Southern Medical University (SCXK2011-0015, Guangzhou) were enrolled in the study. Rats were euthanized by an overdose of pentobarbital (100mg/kg body weight), and the lumbar intervertebral discs were collected from the spinal column. The gel-like nucleus pulposus (NP) was separated from the annulus fibrosus, using a dissecting microscope under aseptic condition. Nucleus pulposus tissue was treated by Trypsin and collagenase, and then the cells were cultured in DMEM/F12medium. The rat NP cell was identified by collagen Ⅱ histoimmunostaining and immunofluorescence stain.2, NP cells were harvested and seeded into tissue culture plates. Experiments were performed after subconfluent cells were serum-starved overnight,and then cells in fresh medium were stimulated by H2O2(50μM,100μM,200μM,400μM) for6h. Cell proliferation and viability analysis were studied using Cell Counting Kit-8.The apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit and Hoechest33258staining.3Levels of intracellular ROS was detected using2,7-dic hloro dihy drofluorescein diacetate, DCFH-DA. Mitochondrial membrane potential was determined by the JC-1staining.4, Apoptosis-related proteins (Bcl-2, Bax, cytochrome c, and caspase-3) were investigated by western blotting.5, All of the experiments were repeated at least three times and the results were expressed as mean±standard deviation. Statistical analysis was performed using SPSS13.0software. All data involving multiple groups were analyzed with one-way ANOVA, pairwise comparisons using LSD method test. A difference with P<0.05was defined as significance.Results1,Nucleus pulposus cells exhibit spherically shaped morphology and chon drocyte-like morphology under phase-contrast microscope. By immuno histoc hemical staining and immunofluorescence staining, cells were demonstrated to exhi bit collagen Ⅱ protein expression. Therefore, cells can be identified as nucleus pulposus cells.2, CCK8assay showed that cells exposed to H2O2significantly decerease the survival of NP cells compared to the control group (P<0.01). Flow cytometry show the rate of cell apoptosis was (5.31.87)%in the control group,While the rate of cell apoptosis were risen to (22.43±2.66)%with the treatment of H2O2(100μM) for6h and (32.5±2.22)%with the treatment of H2O2(200μM) for6h, the differences were statistically significant (P<0.01)3, Flow cytometric analysis showed that H2O2application increased the intracellular ROS generation compared to the control group (P<0.01).Furthermore, cells treat with H2O2alone exhibited an obvious reduction of ΔΨmcompared to the control, as indicated by a decrease in red (JC-1aggregates)/green (JC-1monomers) ratio (p<0.05)4, Western blot analysis showed that H2O2treatment resulted in decreased Bcl-2expression and increased Bax, cytochrome-c, cleaved caspase-3expression compared to the control groups (P<0.01)Conclusion1, Hydrogen peroxide can induce rat nucleus pulposus cells apoptosis in vitro;2,Oxidative stress could induce apoptosis of nucleus pulposus cells through activating mitochondrial pathway with changes in increased the intracellular ROS generation, reduction of mitochondrial membrane potential(ΔΨm), decreased Bcl-2and increased Bax expression, release of cytochrome c, and activation of caspase-3.3,Mitochondrial pathway play a critical role in oxidative stress-induced nucleus pulposus cells apoptosis. ROS and mitochondrial pathway may serve as potential targets in treatment of disc degeneration.