波动性高糖对氧化应激诱导的雪旺细胞凋亡通路的影响及干预研究
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摘要
目的：研究高糖尤其是波动性高糖对氧化应激反应诱导的雪旺细胞凋亡及其信号转导通路的影响,同时在细胞水平上观察α-硫辛酸及中药单体丹参酚酸B是否具有干预波动性高糖通过氧化应激所诱导的细胞凋亡的作用,并进一步探明其作用机制。
方法：原代培养大鼠雪旺细胞,分别加入不同条件培养48小时：(1)正常对照组(Con)：葡萄糖5.6mmol/L;(2)稳定性高糖组(HG)：葡萄糖50mmol/L;(3)波动性高糖组(IHG)：细胞交替培养在含葡萄糖5.6mmol/L及50mmol/L中,8小时换液1次；(4)渗透压对照组：细胞交替培养在含葡萄糖5.6mmol/L及含葡萄糖5.6mmol/L+甘露醇44.4mmol/L的培养基中,8小时换液1次；(5)α-硫辛酸大剂量组：波动性高糖+500μ mol/L α-硫辛酸；(6)α-硫辛酸中剂量组：波动性高糖+50μ mol/L α-硫辛酸；(7)α-硫辛酸小剂量组：波动性高糖+5μ mol/Lα-硫辛酸；(8)丹参酚酸B大剂量组：波动性高糖+10μ mol/L丹参酚酸B；(9)丹参酚酸B中剂量组：波动性高糖+1μ mol/L丹参酚酸B；(10)丹参酚酸B小剂量组：波动性高糖+0.1μ mol/丹参酚酸B。流式细胞术通过DCFH-DA及DHE荧光探针检测细胞内ROS含量,JC-1荧光探针检测细胞线粒体膜电位变化；Elisa法检测细胞内8-OHdG含量;Annexin V/PI及TUNEL法检测细胞凋亡;实时荧光定量PCR法检测bcl-2及bax mRNA的表达；Western blot检测bcl-2、bax、 AIF、cyto c、caspase-9、caspase-3及PARP的表达。
结果：(1)稳定性高糖和波动性高糖提高了雪旺细胞内ROS水平,增加了DNA氧化损伤标志物8-OHdG的生成,降低线粒体膜电位,下调雪旺细胞内bcl-2蛋白及mRNA的表达,上调雪旺细胞内bax蛋白及mRNA的表达,促进cyto C从线粒体到胞浆的释放及AIF的核转位,同时促进了caspase-9、caspase-3及PARP的活化,并增加了雪旺细胞的凋亡。(2)波动性高糖对雪旺细胞氧化应激及凋亡的影响比稳定性高糖更为明显,且这种作用与渗透压作用无关。(3)α-硫辛酸可以降低雪旺细胞内ROS水平及8-OHdG农度,从而改善了波动性高糖对雪旺细胞的氧化应激损伤。同时,α-硫辛酸提高了线粒体膜电位,上调了bcl-2蛋白及mRNA的表达,下调了bax蛋白及mRNA的表达,抑制了线粒体cyto C的释放及AIF的核转位,降低了PARP、caspase-9及caspase-3(?)勺活化,通过caspase依赖性及caspase非依赖性通路抑制了雪旺细胞凋亡。α-硫辛酸的这种作用在一定剂量范围内呈剂量依赖性的趋势。(4)丹参酚酸B降低了雪旺细胞内ROS及8-OHdG水平,提高线粒体膜电位,上调bcl-2蛋白及mRNA的表达,下调bax蛋白及mRNA的表达,抑制cyto c从线粒体到胞浆的释放及AIF从线粒体到细胞核的转位,降低了PARP、caspase-9及caspase-3的活化水平。
结论：(1)波动性高糖和稳定性高糖可以通过氧化应激反应促进雪旺细胞的凋亡,线粒体在此过程中起着重要作用。(2)雪旺细胞的凋亡是通过caspase依赖性及caspase非依赖性途径共同实现的。(3)波动性高糖的损伤作用更为明显,且与渗透压无关。(4)α-硫辛酸和丹参酚酸B可以通过抑制氧化应激反应从线粒体途径抑制了波动性高糖所导致的雪旺细胞凋亡。
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.
方法：原代培养大鼠雪旺细胞,分别加入不同条件培养48小时：(1)正常对照组(Con)：葡萄糖5.6mmol/L;(2)稳定性高糖组(HG)：葡萄糖50mmol/L;(3)波动性高糖组(IHG)：细胞交替培养在含葡萄糖5.6mmol/L及50mmol/L中,8小时换液1次；(4)渗透压对照组：细胞交替培养在含葡萄糖5.6mmol/L及含葡萄糖5.6mmol/L+甘露醇44.4mmol/L的培养基中,8小时换液1次；(5)α-硫辛酸大剂量组：波动性高糖+500μ mol/L α-硫辛酸；(6)α-硫辛酸中剂量组：波动性高糖+50μ mol/L α-硫辛酸；(7)α-硫辛酸小剂量组：波动性高糖+5μ mol/Lα-硫辛酸；(8)丹参酚酸B大剂量组：波动性高糖+10μ mol/L丹参酚酸B；(9)丹参酚酸B中剂量组：波动性高糖+1μ mol/L丹参酚酸B；(10)丹参酚酸B小剂量组：波动性高糖+0.1μ mol/丹参酚酸B。流式细胞术通过DCFH-DA及DHE荧光探针检测细胞内ROS含量,JC-1荧光探针检测细胞线粒体膜电位变化；Elisa法检测细胞内8-OHdG含量;Annexin V/PI及TUNEL法检测细胞凋亡;实时荧光定量PCR法检测bcl-2及bax mRNA的表达；Western blot检测bcl-2、bax、 AIF、cyto c、caspase-9、caspase-3及PARP的表达。
结果：(1)稳定性高糖和波动性高糖提高了雪旺细胞内ROS水平,增加了DNA氧化损伤标志物8-OHdG的生成,降低线粒体膜电位,下调雪旺细胞内bcl-2蛋白及mRNA的表达,上调雪旺细胞内bax蛋白及mRNA的表达,促进cyto C从线粒体到胞浆的释放及AIF的核转位,同时促进了caspase-9、caspase-3及PARP的活化,并增加了雪旺细胞的凋亡。(2)波动性高糖对雪旺细胞氧化应激及凋亡的影响比稳定性高糖更为明显,且这种作用与渗透压作用无关。(3)α-硫辛酸可以降低雪旺细胞内ROS水平及8-OHdG农度,从而改善了波动性高糖对雪旺细胞的氧化应激损伤。同时,α-硫辛酸提高了线粒体膜电位,上调了bcl-2蛋白及mRNA的表达,下调了bax蛋白及mRNA的表达,抑制了线粒体cyto C的释放及AIF的核转位,降低了PARP、caspase-9及caspase-3(?)勺活化,通过caspase依赖性及caspase非依赖性通路抑制了雪旺细胞凋亡。α-硫辛酸的这种作用在一定剂量范围内呈剂量依赖性的趋势。(4)丹参酚酸B降低了雪旺细胞内ROS及8-OHdG水平,提高线粒体膜电位,上调bcl-2蛋白及mRNA的表达,下调bax蛋白及mRNA的表达,抑制cyto c从线粒体到胞浆的释放及AIF从线粒体到细胞核的转位,降低了PARP、caspase-9及caspase-3的活化水平。
结论：(1)波动性高糖和稳定性高糖可以通过氧化应激反应促进雪旺细胞的凋亡,线粒体在此过程中起着重要作用。(2)雪旺细胞的凋亡是通过caspase依赖性及caspase非依赖性途径共同实现的。(3)波动性高糖的损伤作用更为明显,且与渗透压无关。(4)α-硫辛酸和丹参酚酸B可以通过抑制氧化应激反应从线粒体途径抑制了波动性高糖所导致的雪旺细胞凋亡。
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.
引文
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