Hypoxia (HYpX) induced-overload ca 2+ entry results in increase of mitochondrial oxidative stress, inflammation and apoptosis in several neurons. Ca 2+ permeable TRPM2 channel was gated by ADPribose (ADPR) and reactive oxygen species (ROS), although its activity was modulated in HYPX-exposed neurons by resveratrol (RSV). The aim of this study was to evaluate if a therapy of RSV can modulate the effect of HYPX in the TRPM2 expressing SH-SY5Y neuronal and HEK293 (no expression of TRPM2) cell lines. The SH-SY5Y and HEK293 cells were divided into four groups as control, RSV (50 μM and 24 hours), and HYPX and RSV + HYPX. For induction of HYPX in the cells, CoCl 2 (200 μM and 24 hours) incubation was used. HYPX-induced intracellular Ca 2+ responses to TRPM2 activation were increased in the SH-SY5Y cells but not in the HEK293 cells from coming H 2 o 2 and ADPR. RSV treatment improved intracellular ca 2+ responses, mitochondrial function, suppressed the generation of cytokine (IL-1β and tnf-α), cytosolic and mitochondrial ROS in the SH-SY5Y cells. Intracellular free Zn 2+ , apoptosis, cell death, PARP-1, TRPM2 expression, caspase −3 and −9 levels are increased through activating TRPM2 in the SH-SY5Y cells exposed to the HYPX. However, the values were decreased in the cells by RSV and TRPM2 blockers (ACA and 2-APB). In SH-SY5Y neuronal cells exposed to HYPX conditions, the neuroprotective effects of RSV were shown to be exerted via modulation of oxidative stress, inflammation, apoptosis and death through modulation of TRPM2 channel. RSV could be used as an effective agent in the treatment of neurodegeneration exposure to HYPX.Extensive death in neurons was induced by acute hypoxia, because disability and mortality of the neurons were increased by acute hypoxia 1 . Low blood flow to the tissue and low oxygen content of blood result in hypoxia and ischemic condition 2 . Cell survival decreased in the absence of oxygen, because ATP generation requires oxygen consumption in mitochondria 3 . Mitochondria is a main source of reactive oxygen species (ROS) generation 4 . Accumulating evidence indicates that the hypoxia and ischemic conditions result in excessive ROS generation, inflammation and apoptosis through the increase of membrane depolarization in mitochondria of neurons 5,6 . The increase of mitochondrial membrane depolarization was induced by the increase of intracellular free Ca 2+ ([Ca 2+ ] i ) concentration. Recently, hypoxia-induced mitochondria ROS generation was inhibited through modulation of voltage gated calcium channel (VGCC) in the heart cells by resveratrol (RSV) treatment 7,8 . Hence, RSV can be useful for treatment of hypoxia in neuronal cells by modulation of mitochondrial ROS generation and the subject should be clarified in the hypoxia-induced SH-SY5Y neuronal cells.Several neuronal physiological functions such as mitochondria and cell development are triggered by the changes of the [Ca 2+ ] i concentration 4 . In addition, several neurotoxicity functions such as apoptosis and inflammation in hypoxia are...

The aim of the present study was to determine whether pharmaceutical preparations with pregabalin (PGB) as an active ingredient, which are widely prescribed by clinicians, exert toxic effects on human primary nucleus pulposus (NP) and annulus fibrosis (AF). Primary human cell cultures were obtained from intact (n=6) and degenerated (n=6) tissues resected from the two groups of patients. Different doses of PGB were applied to these cultures and cells were subjected to molecular analyses at 0, 24 and 48 h. Cell vitality, toxicity and proliferation were assessed using a spectrophotometer. The expression of chondroadherin (CHAD), a (member of the NP-specific protein family), hypoxia-inducible factor-1α (HIF-1α) and type II collagen (COL2A1) was measured using reverse transcription-quantitative polymerase chain reaction. The results revealed that cell intensity increased in a time-dependent manner and cell vitality continued in the cultures without pharmaceuticals. Cell proliferation was suppressed in the PGB-treated cultures independent from the dose and duration of application. PGB was demonstrated to suppress the expression of CHAD and HIF-1α. In contrast, COL2A1 gene expression was not revealed in any experimental group. The present study utilized an in vitro model and the PGB active ingredient used herein may not be representative of clinical applications; however, the results demonstrated that PGB has a toxic effect on NP/AF cell cultures containing primary human intervertebral disc tissue. In summary, the use of pharmacological agents containing PGB may suppress the proliferation and differentiation of NP/AF cells and/or tissues, which should be considered when deciding on an appropriate treatment regime.

Cell culture setups are of paramount importance because they may influence the pattern of changes in the gene expressions of the cells used in these setups.

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The aim of the present study was to investigate the effects of three different formulations of hyaluronic acid (HA): Low molecular weight (MW) Sinovial One®, medium MW Viscoplus® and high MW Durolane®, on chondrocyte proliferation and collagen type II (COL2A1), hypoxia-inducible factor 1α (HIF-1α) and chondroadherin (CHAD) expression in primary chondrocyte cultures. Standard primary chondrocyte cultures were established from osteochondral tissues surgically obtained from 6 patients with gonarthrosis. Cell morphology was evaluated using an inverted light microscope; cell proliferation was determined with a MTT assay and confirmed with acridine orange/propidium iodide staining. Levels of CHAD, COL2A1 and HIF-1α expression were assessed using specific TaqMan gene expression assays. The results demonstrated the positive effect of HA treatment on cell proliferation, which was independent from the MW. COL2A1 expression increased in the medium and high MW HA treated groups. It was observed that HIF-1α expression increased in the high MW treated group alone. CHAD expression increased only in the medium MW HA treated group. Evaluation of gene expression revealed that levels of expression increased as the duration of HA application increased, in the medium and high MW HA treated groups. In terms of increased viability and proliferation, a longer duration of HA application was more effective. Taken together, it may be concluded that the administration of medium and high MW HA may be a successful way of treating diseases affecting chondrocytes in a clinical setting.