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Study on the Effect and Intervention of Schwann Cells Apoptosis Induced by Intermittent High Glucose Via Oxidative Stress

Author: SunLianQing
Tutor: LuJuMing
School: PLA Postgraduate Medical School
Course: Internal Medicine
Keywords: hyperglycemia peripheral neuropathy Schwann cells oxidative stress apoptosis antioxidant Alpha lipoic acid Salvianolic acid B
CLC: R587.2
Type: PhD thesis
Year: 2012
Downloads: 178
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Abstract


Objective:To investigate the inhibitory effects of Alpha lipoic acid (ALA) and Salvianolic acid B (Sal B) on the intermittent high glucose (IHG)-induced oxidative stress-induced mitochondrial pathway activation and Schwann cells (SCs) apoptosis in vitro.Methods:SCs were primarily cultured and were verified by immunostaining for S-100protein. The cultured SCs were treated in duplicate consistently with5.6mM of glucose as the control (con), consistently with50mM of glucose as HG, with5.6and50mM glucose altering every8hrs as IHG, with IHG in the presence of5μmol/L,50μmol/L and500μmol/L of ALA or0.1μmol/L,1μmol/L and10μmol/L of Sal B for48hrs, respectively. There are two osmotic controls, one was that treating the cells with44.4mM mannitol plus5.6mM glucose; the other was an intermittent high osmotic control, which treated the cells with5.6mM glucose or44.4mM mannitol plus5.6mM glucose (total50mM) alternatively at the interval of8hours.Apoptosis was confirmed by the Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and Annexin V/PI method.The concentration of8-hydroxy-2-deoxy Guanosine (8-OHdG) was detected by Elisa. Intracellular ROS generation and mitochondrial transmembrane potential (△ψm) were detected by flow cytometry analysis. Quantitative real-time reverse transcriptase PCR was performed to analyze the expression levels of bax and bcl-2.Western blot were performed to analyze the expression levels of some important transcription factors and proteins, such as bcl-2, bax, AIF, cyto c, caspase-9, caspase-3and PARP.Results:(1) The relative levels of intracellular hydrogen peroxides and superoxide anions showed a marked increase in SCs that were exposed to HG group compared with normal glucose exposure and further increased under IHG conditions. The percentages of depolarized cells in HG and IHG groups significantly increased and the concentrations of8-OHdG in the HG and IHG groups were significantly increased than that in the control.Treatment with HG and IHG up-regulated the release of cytochrome c, AIF nuclear translocation and Bax expression of protein and mRNA, but up-regulated the Bcl-2expression of protein and mRNA. In addition, treatment with HG and IHG increased the activation of caspase-9and caspase-3and the cleavage of PARP in SCs. The percentages of apoptotic cells were increased exposed to HG and substantially more in cells exposed to the IHG.(2) The cytotoxic effect of IHG was significantly more potent than that of HG and the cytotoxicy of IHG is not related to osmolarity, instead, it is a direct effect of glucose on the cell.(3) Treatment with ALA inhibited the IHG-induced oxidative stress by reducing ROS production and8-OHdG levels, mitochondrial depolarization and apoptosis in SCs. Furthermore, treatment with ALA down-regulated the IHG-induced release of cytochrome c, AIF nuclear translocation and Bax expression, but mitigated the IHG-mediated down-regulation of Bcl-2expression in SCs. In addition, treatment with ALA attenuated the IHG-induced activation of caspase-9and caspase-3and minimized the cleavage of PARP in SCs. Clearly, IHG induced mitochondrial dysfunction, and triggered high frequency of SCs apoptosis in both the caspase-dependent and the AIF-mediated caspase-independent pathway. More importantly, ALA inhibited the activation of molecular pathways of apoptosis in a dose-dependent manner.(4) Sal B inhibited the IHG-induced oxidative stress by reducing ROS production and8-OHdG levels, mitochondrial depolarization and apoptosis in SCs in a dose-dependent manner. Furthermore, Sal B down-regulated the IHG-induced release of cytochrome c, AIF nuclear translocation and Bax expression, but mitigated the IHG-mediated down-regulation of Bcl-2expression in SCs. In addition, treatment with Sal B attenuated the IHG-induced activation of caspase-9and caspase-3and minimized the cleavage of PARP in SCs.Conclusions:(1) IHG and HG induced SCs apoptosis via oxidative stress and mitochondrial pathway played a key role in the process.(2) IHG and HG induced SCs apoptosis in both caspase-dependent and caspase-independent pathways.(3)The cytotoxic effect of IHG was significantly more potent than that of HG and is not related to osmolarity, instead, it is a direct effect of glucose on the cell.(4) ALA and Sal B antagonized the IHG-induced oxidative stress-related activation of mitochondrial pathway and apoptosis in SCs.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetic coma and other complications
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