中药筋脉通对糖尿病周围神经病变大鼠氧化应激及细胞凋亡的影响
|
摘要
[研究目的]
从整体、细胞及分子水平,探讨中药筋脉通对糖尿病大鼠背根神经节氧化应激及细胞凋亡的影响,以及筋脉通含药血清对高糖培养背根神经节神经元氧化应激及细胞凋亡的影响。
[研究方法]
第一部分动物实验：采用链脲佐菌素(STZ)60mg/kg一次性腹腔内注射的方法制造糖尿病大鼠模型。随机分为6组：模型(DM)组、筋脉通小剂量(J小)、中剂量(J中)和大剂量(J大)组、维生素C(VC)组及正常对照(Con)组,每组各10只。成模后予灌胃给药,筋脉通小、中、大剂量组分别按成人剂量5、10、20倍给药；维生素C组按成人剂量10倍给药；模型组和正常对照组予灌服等量蒸馏水。检测各组治疗前及治疗后4周、8周、12周及16周的体重和血糖变化；所有实验大鼠于灌胃16周后处死取背根神经节(DRG),处死前检测机械痛阈值；采用尼氏染色观察DRG形态学变化,免疫组化法和qRT-PCR法检测大鼠DRG中NADPH氧化酶p22-phox亚基、CytC、Bcl-2及Caspase-3的表达, TUNEL法检测细胞凋亡。
第二部分细胞实验：
1.雄性SD大鼠随机分为3组,分别灌胃筋脉通(5只)、维生素C(5只)或蒸馏水(10只)制备含药血清和正常对照血清。从孕15天(E15)的SD胎鼠背根神经节取材制备背根神经节神经元(DRGn),采用胰蛋白酶消化法、Neurobasal无血清神经元专用培养基+B27添加剂+NGF培养基进行培养,经密度梯度离心法+差速贴壁法进行纯化,抗NSE抗体进行细胞鉴定。
2.将DRGn随机分为4组：正常对照组、50、75、100mmol/L葡萄糖组,分别培养12、24、48h后MTT法检测不同浓度葡萄糖对DRGn细胞活性的影响,初步确定高糖浓度及指标检测适宜时间点。
3.将DRGn随机分为6组：正常对照组、高糖组、筋脉通含药血清原液(JMT1：1)组、稀释1倍筋脉通含药血清(JMT1：2)组、稀释2倍筋脉通含药血清(JMT1：4)组、维生素C组,培养24h后MTT法检测不同浓度筋脉通含药血清对高糖培养DRGn细胞活性的影响,选择最佳筋脉通含药血清浓度。
4.将实验分为4组：正常对照(Con)组、高糖(Glu)组、筋脉通(JMT)组、维生素C(VC)组,培养24h后,采用荧光探针法检测DRGn中超氧阴离子、线粒体膜电位(MMP)水平,Western Blot+qRT-PCR法检测Bcl-2和Caspase-3的mRNA及其蛋白表达,TUNEL法检测细胞凋亡。
[研究结果]
第一部分动物实验
1.血糖和体重监测：造模后各组大鼠血糖与Con组相比均明显升高(P<0.01)。药物干预后,治疗组血糖在同一时间点与DM组相比无明显差异(P>0.05),治疗组间血糖在各时间点均无明显差异(P>0.05)。造模后各组大鼠体重无明显差异(P>0.05)。药物干预后,DM组及各治疗组体重在同一时间点均显著低于Con组(P<0.01),治疗组间体重在各时间点均无明显差异(P>0.05)。
2.机械痛阈值检测：与Con组相比,J中组的机械痛阈值未见明显差异(P>0.05),DM组及其余治疗组的机械痛阈值均显著降低(P<0.01)。与DM组相比,各治疗组的机械痛阂值均明显升高(P<0.01)。
3.氧化应激相关指标：DM组大鼠DRG中NADPH氧化酶p22-phox亚基蛋白表达较Con组显著升高(P<0.01)；与DM相比,各治疗组的NADPH氧化酶p22-phox亚基蛋白表达显著降低(P<0.05或P<0.01),其中筋脉通大剂量组作用最好,明显优于维生素C组(P<0.01)。
4.细胞凋亡相关指标：与Con组相比,DM大鼠DRG中Cyt C蛋白表达、Caspase-3mRNA及其蛋白表达、细胞凋亡率均显著升高(P<0.01),而Bcl-2mRNA及其蛋白表达显著降低(P<0.01)；与DM相比,各治疗组的Cyt C蛋白表达、Caspase-3mRNA及其蛋白表达、细胞凋亡率均显著降低,而Bcl-2mRNA及其蛋白表达显著升高(P<0.05或P<0.01),其中筋脉通大剂量组作用最好,明显优于维生素C组(P<0.01)。
第二部分细胞实验
1.细胞活性检测：①与12h相比,24h时Con组及各高糖浓度组细胞活性均明显降低(P<0.01)；48h时各组细胞活性与24h时相比无明显变化(P>0.05)。②24h时与Con组相比,各高糖浓度组的细胞活性均显著降低(P<0.05或P<0.01),75mmol/LGlu与50mmol/LGlu组相比细胞活性显著升高(P<0.01),75与100mmol/LGlu组相比差异不明显(P>0.05)。③24h时与Con组相比,50mmol/LGlu组的细胞活性显著降低(P<0.01),各干预组的细胞活性在Con组和50mmol/LGlu组之间,与50mmol/LGlu组相比差异显著(P<0.01),与Con组相比无明显差异(P>0.05),其中JMT1：1组和VC组的细胞活性未见明显差异(P>0.05)。
2.氧化应激相关指标：与Con组相比,Glu组DRGn中超氧阴离子表达显著升高(P<0.01)；JMT组超氧阴离子表达较Con组显著升高(P<0.01),较Glu组显著降低(P<0.01),其表达明显低于VC组(P<0.01)。
3.细胞凋亡相关指标：与Con组相比,Glu组DRGn中MMP水平显著降低,Caspase-3mRNA及其蛋白表达、细胞凋亡率均显著升高,而Bcl-2mRNA及其蛋白表达显著降低(P<0.01)；与Glu组相比,JMT组MMP水平显著升高,Caspase-3mRNA及其蛋白表达、细胞凋亡率均明显降低,而Bcl-2mRNA及其蛋白表达则明显升高(P<0.01),其作用明显优于维生素C组(P<0.01)。
[结论]
第一部分动物实验
1.一次性腹腔注射STZ60mg/kg后16周,DM大鼠机械痛阂值降低,DPN动物模型成功建立。
2.DPN大鼠背根神经节中NADPH氧化酶p22-phox亚基和细胞色素C蛋白表达增强,表明组织发生了氧化应激；促凋亡的Caspase-3mRNA及蛋白表达升高,而抗凋亡的Bcl-2mRNA及蛋白表达下降,致细胞凋亡率增加。
3.中药筋脉通干预能够显著改善上述变化,以筋脉通大剂量组疗效最佳,其作用优于抗氧化剂维生素C。
4.筋脉通具有防治DPN大鼠周围神经组织损伤的作用,抑制氧化应激、减少细胞凋亡是其作用的重要途径之一。此作用不是通过降低血糖实现的,且与胰岛素应用无关。
第二部分细胞实验
1.取材于E15的SD胎鼠背根神经节,采用胰蛋白酶消化法、Neurobasal无血清神经元专用培养基+B27+NGF培养基原代培养,经密度梯度离心法+差速贴壁法进行纯化,经抗NSE抗体细胞鉴定,可获得纯度较高的DRGn。
2.高糖可显著抑制DRGn细胞活性,筋脉通含药血清能够改善高糖对DRGn细胞活性的抑制。
3.高糖培养DRGn中超氧阴离子含量升高,线粒体膜电位水平下降,表明细胞发生了氧化应激；促凋亡的Caspase-3mRNA及蛋白表达升高,而抗凋亡的Bcl-2mRNA及蛋白表达下降,致细胞凋亡率增加。
4.中药筋脉通干预能够显著改善上述变化,其作用优于抗氧化剂维生素C。
5.筋脉通含药血清具有防治高糖培养DRGn细胞损伤的作用,是通过抑制高糖所致氧化应激、减少细胞凋亡来实现的。
[Objective]
To investigate protective effects of Jinmaitong (JMT), a compound Chinese herbal medicine, on oxidative stress, mitochondrial dysfunction and apoptosis in experimental diabetic neuropathy at the animal, cellular and molecular level.
[Methods]
1. In vivo experiment:A single dose of streptozotocin (STZ) was used for the induction of diabetes in male Sprague-Dawley rats. These diabetic rats were randomly divided into5groups:diabetes (DM) group, low-dose JMT group (JMT at dose of.044g/kg/d), middle-dose JMT group (JMT at dose of0.88g/kg/d), high-dose JMT group (JMT at dose of1.75g/kg/d) and vitamin C group (VC at dose of0.05g/kg/d). Ten normal rats with matching weight and age were selected as control group. All rats then received intragastric administration for16weeks (the control and DM groups were treated with distilled water) and then killed. The pain threshold to mechanical stimulation were done before death. Body weight and blood glucose were detected before and at4th,8th,12th,16th week after treatment. Morphological changes in DRG was checked by Nissl staining. The expressions of NADPH oxidase p22-phox subunit, Cyt C, Bcl-2and Caspase-3were detected by immunohistochemical method and quantitative realtime polymerase chain reaction (qRT-PCR). The apoptotic cells were detected by the Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay.
2. In vitro experiment
①Male Sprague-Dawley rats were randomly divided into control group (distilled water), JMT group (JMT at dose of1.31g/kg/d) and vitamin C group (vitamin C at dose of0.08g/kg/d) to prepared drug-containing serum. DRG are harvested from embryonic day15SD rats, dissociated in0.25%trypsin, purified by density gradient centrifugation and differential attachment technique, then cultured in Neurobasal medium supplemented with B-27additives and nerve growth factor, and immunofluorescence method with anti-NSE was used to identify them.
②DRGn were randomly divided into control group and high glucose (50,75,100mmol/L) group. After12,24and48h cultures, MTT assay was used to determine the optimal concentration of high glucose and the optimal time of drugs.
③DRGn were randomly divided into control group, high glucose group (cultured with50mmol/L glucose medium), JMT1:1group, JMT1:2group, JMT1:4group (cultured with different concentration of JMT containing serum) and VC group (cultured with VC containing serum). After24cultures, MTT assay was used to determine the optimal concentration of JMT.
④DRGn were randomly divided into high glucose group (cultured with50mmol/L glucose medium), JMT group (cultured with JMT containing serum) and VC group (cultured with VC containing serum). After24cultures, The levels of superoxide anion and MMP were determined by fluorescent probe. The expressions of Bcl-2and Caspase-3were detected by Western Blot analysis and qRT-PCR. The apoptotic cells were detected by the TUNEL assay.
[Results]
1. In vivo experiment
①Blood glucose and body weight:The blood glucose levels in DM group were much higher than those in control group (P<0.01). At different time, there was no significant difference among model groups (P>0.05). The blood glucose and body weight levels had no marked changes before and after treatment (P>0.05)
②Pain threshold to mechanical stimulation:The pain thresholds in DM group were much lower than those in control group (P<0.01). Compared with DM group, in treatment groups significantly increased (P<0.01). Among treatment groups, the the pain thresholds in middle-dose JMT group increased markedly (P<0.01)
③Oxidative stress:The protein levels of NADPH oxidase p22-phox subunit in DM group were much higher than those in control group(P<0.01). Compared with DM group, the levels of NADPH oxidase p22-phox subunit in treatment groups significantly decreased (P<0.05or P<0.01). Among treatment groups, large-dose JMT group showed better effect than VC group (P<0.01)
④Apoptosis:The cytochrome protein levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in DM group were much higher than those in control group (P<0.01), and the mRNA and protein levels of Bcl-2were much lower (P<0.01). Compared with DM group, the cytochrome protein levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in treatment groups significantly decreased (P<0.05or P<0.01), the mRNA and protein levels of Bcl-2significantly increased (P<0.05or P<0.01). Among treatment groups, large-dose JMT group showed better effect than VC group (P<0.01)
2. In vitro experiment
①MTT assay:The cell viability levels in high glucose groups and control group significantly decreased at24h compared with12h (P<0.01), there was no significant difference between24h and48h (P>0.05). Compared with control group, the cell viability levels in high glucose groups markedly decreased at24h(P<0.05or P<0.01). Among high glucose groups, the cell viability levels in50mmol/LGlu group were much higher than75and100mmol/LGlu groups(P<0.01). Compared with control group, the cell viability levels in50mmol/LGlu group significantly decreased at24h (P<0.01), The cell viability levels in treatment groups were much lower than those in50mmol/LGlu group (P<0.01). Among treatment groups, there was no difference between JMT1:1group and VC group (P>0.05)
②Oxidative stress:The levels of superoxide anion in high glucose group were much higher than those in control group (P<0.01). Compared with high glucose group, the levels of superoxide anion in JMT group significantly decreased (P<0.01) and JMT was superior to VC (P<0.01)
③Apoptosis:The MMP levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in high glucose group were much higher than those in control group (P<0.01), and the mRNA and protein levels of Bcl-2were much lower (P<0.01) Compared with high glucose group, the MMP levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in JMT groups significantly decreased(P<0.01), and the mRNA and protein levels of Bcl-2significantly increased (P<0.01). JMT was superior to VC (P<0.01)
[Conclusion]
1. In vivo experiment
①A single intraperitoneal STZ (60mg/kg) injection-induced diabetic rats showed significant hyperalgesia at16w, indicating development of DPN.
②The levels of NADPH oxidase p22-phox subunit, Cyt C, Caspase-3and the apoptosis percentage in DPN rats significantly increased compared to control rats, and Bcl-2levels significantly reduced.
③JMT treatment significantly ameliorated the alteration in hyperalgesia, NADPH oxidase p22-phox subunit, Cyt C, Bcl-2, Caspase-3and the apoptosis percentage in DPN rats. The large-dose of JMT showed the best effectiveness
④These results suggest that JMT antagonized the oxidative stress, activation of the mitochondrial pathway and apoptosis in DPN rats. And its effectiveness was independent of hypoglycemia and insulin.
2. In vitro experiment
①The purity coefficient of DRGn which were isolated from E15SD rats, dissociated in trypsin digestion, purified by density gradient centrifugation and differential attachment technique, then cultured in Neurobasal medium supplemented with B-27additives and nerve growth factor, could reach more than90%through identification of immunofluorescence method with anti-NSE.
②High glucose remarkably inhibited the cell viability levels of DRGn and JMT treatment significantly ameliorated the inhibition.
③The levels of superoxide anion, Caspase-3and the apoptosis percentage of DRGn in cultured in high glucose medium significantly increased compared to control group, but MMP and Bcl-2levels significantly reduced.
④JMT treatment significantly ameliorated the alteration in superoxide anion, MMP, Bcl-2, Caspase-3and the apoptosis percentage of DRGn in cultured in high glucose medium. And JMT was superior to VC.
⑤These results suggest that JMT containing serum antagonized the oxidative stress, activation of the mitochondrial pathway and apoptosis of DRGn in cultured in high glucose medium.
从整体、细胞及分子水平,探讨中药筋脉通对糖尿病大鼠背根神经节氧化应激及细胞凋亡的影响,以及筋脉通含药血清对高糖培养背根神经节神经元氧化应激及细胞凋亡的影响。
[研究方法]
第一部分动物实验：采用链脲佐菌素(STZ)60mg/kg一次性腹腔内注射的方法制造糖尿病大鼠模型。随机分为6组：模型(DM)组、筋脉通小剂量(J小)、中剂量(J中)和大剂量(J大)组、维生素C(VC)组及正常对照(Con)组,每组各10只。成模后予灌胃给药,筋脉通小、中、大剂量组分别按成人剂量5、10、20倍给药；维生素C组按成人剂量10倍给药；模型组和正常对照组予灌服等量蒸馏水。检测各组治疗前及治疗后4周、8周、12周及16周的体重和血糖变化；所有实验大鼠于灌胃16周后处死取背根神经节(DRG),处死前检测机械痛阈值；采用尼氏染色观察DRG形态学变化,免疫组化法和qRT-PCR法检测大鼠DRG中NADPH氧化酶p22-phox亚基、CytC、Bcl-2及Caspase-3的表达, TUNEL法检测细胞凋亡。
第二部分细胞实验：
1.雄性SD大鼠随机分为3组,分别灌胃筋脉通(5只)、维生素C(5只)或蒸馏水(10只)制备含药血清和正常对照血清。从孕15天(E15)的SD胎鼠背根神经节取材制备背根神经节神经元(DRGn),采用胰蛋白酶消化法、Neurobasal无血清神经元专用培养基+B27添加剂+NGF培养基进行培养,经密度梯度离心法+差速贴壁法进行纯化,抗NSE抗体进行细胞鉴定。
2.将DRGn随机分为4组：正常对照组、50、75、100mmol/L葡萄糖组,分别培养12、24、48h后MTT法检测不同浓度葡萄糖对DRGn细胞活性的影响,初步确定高糖浓度及指标检测适宜时间点。
3.将DRGn随机分为6组：正常对照组、高糖组、筋脉通含药血清原液(JMT1：1)组、稀释1倍筋脉通含药血清(JMT1：2)组、稀释2倍筋脉通含药血清(JMT1：4)组、维生素C组,培养24h后MTT法检测不同浓度筋脉通含药血清对高糖培养DRGn细胞活性的影响,选择最佳筋脉通含药血清浓度。
4.将实验分为4组：正常对照(Con)组、高糖(Glu)组、筋脉通(JMT)组、维生素C(VC)组,培养24h后,采用荧光探针法检测DRGn中超氧阴离子、线粒体膜电位(MMP)水平,Western Blot+qRT-PCR法检测Bcl-2和Caspase-3的mRNA及其蛋白表达,TUNEL法检测细胞凋亡。
[研究结果]
第一部分动物实验
1.血糖和体重监测：造模后各组大鼠血糖与Con组相比均明显升高(P<0.01)。药物干预后,治疗组血糖在同一时间点与DM组相比无明显差异(P>0.05),治疗组间血糖在各时间点均无明显差异(P>0.05)。造模后各组大鼠体重无明显差异(P>0.05)。药物干预后,DM组及各治疗组体重在同一时间点均显著低于Con组(P<0.01),治疗组间体重在各时间点均无明显差异(P>0.05)。
2.机械痛阈值检测：与Con组相比,J中组的机械痛阈值未见明显差异(P>0.05),DM组及其余治疗组的机械痛阈值均显著降低(P<0.01)。与DM组相比,各治疗组的机械痛阂值均明显升高(P<0.01)。
3.氧化应激相关指标：DM组大鼠DRG中NADPH氧化酶p22-phox亚基蛋白表达较Con组显著升高(P<0.01)；与DM相比,各治疗组的NADPH氧化酶p22-phox亚基蛋白表达显著降低(P<0.05或P<0.01),其中筋脉通大剂量组作用最好,明显优于维生素C组(P<0.01)。
4.细胞凋亡相关指标：与Con组相比,DM大鼠DRG中Cyt C蛋白表达、Caspase-3mRNA及其蛋白表达、细胞凋亡率均显著升高(P<0.01),而Bcl-2mRNA及其蛋白表达显著降低(P<0.01)；与DM相比,各治疗组的Cyt C蛋白表达、Caspase-3mRNA及其蛋白表达、细胞凋亡率均显著降低,而Bcl-2mRNA及其蛋白表达显著升高(P<0.05或P<0.01),其中筋脉通大剂量组作用最好,明显优于维生素C组(P<0.01)。
第二部分细胞实验
1.细胞活性检测：①与12h相比,24h时Con组及各高糖浓度组细胞活性均明显降低(P<0.01)；48h时各组细胞活性与24h时相比无明显变化(P>0.05)。②24h时与Con组相比,各高糖浓度组的细胞活性均显著降低(P<0.05或P<0.01),75mmol/LGlu与50mmol/LGlu组相比细胞活性显著升高(P<0.01),75与100mmol/LGlu组相比差异不明显(P>0.05)。③24h时与Con组相比,50mmol/LGlu组的细胞活性显著降低(P<0.01),各干预组的细胞活性在Con组和50mmol/LGlu组之间,与50mmol/LGlu组相比差异显著(P<0.01),与Con组相比无明显差异(P>0.05),其中JMT1：1组和VC组的细胞活性未见明显差异(P>0.05)。
2.氧化应激相关指标：与Con组相比,Glu组DRGn中超氧阴离子表达显著升高(P<0.01)；JMT组超氧阴离子表达较Con组显著升高(P<0.01),较Glu组显著降低(P<0.01),其表达明显低于VC组(P<0.01)。
3.细胞凋亡相关指标：与Con组相比,Glu组DRGn中MMP水平显著降低,Caspase-3mRNA及其蛋白表达、细胞凋亡率均显著升高,而Bcl-2mRNA及其蛋白表达显著降低(P<0.01)；与Glu组相比,JMT组MMP水平显著升高,Caspase-3mRNA及其蛋白表达、细胞凋亡率均明显降低,而Bcl-2mRNA及其蛋白表达则明显升高(P<0.01),其作用明显优于维生素C组(P<0.01)。
[结论]
第一部分动物实验
1.一次性腹腔注射STZ60mg/kg后16周,DM大鼠机械痛阂值降低,DPN动物模型成功建立。
2.DPN大鼠背根神经节中NADPH氧化酶p22-phox亚基和细胞色素C蛋白表达增强,表明组织发生了氧化应激；促凋亡的Caspase-3mRNA及蛋白表达升高,而抗凋亡的Bcl-2mRNA及蛋白表达下降,致细胞凋亡率增加。
3.中药筋脉通干预能够显著改善上述变化,以筋脉通大剂量组疗效最佳,其作用优于抗氧化剂维生素C。
4.筋脉通具有防治DPN大鼠周围神经组织损伤的作用,抑制氧化应激、减少细胞凋亡是其作用的重要途径之一。此作用不是通过降低血糖实现的,且与胰岛素应用无关。
第二部分细胞实验
1.取材于E15的SD胎鼠背根神经节,采用胰蛋白酶消化法、Neurobasal无血清神经元专用培养基+B27+NGF培养基原代培养,经密度梯度离心法+差速贴壁法进行纯化,经抗NSE抗体细胞鉴定,可获得纯度较高的DRGn。
2.高糖可显著抑制DRGn细胞活性,筋脉通含药血清能够改善高糖对DRGn细胞活性的抑制。
3.高糖培养DRGn中超氧阴离子含量升高,线粒体膜电位水平下降,表明细胞发生了氧化应激；促凋亡的Caspase-3mRNA及蛋白表达升高,而抗凋亡的Bcl-2mRNA及蛋白表达下降,致细胞凋亡率增加。
4.中药筋脉通干预能够显著改善上述变化,其作用优于抗氧化剂维生素C。
5.筋脉通含药血清具有防治高糖培养DRGn细胞损伤的作用,是通过抑制高糖所致氧化应激、减少细胞凋亡来实现的。
[Objective]
To investigate protective effects of Jinmaitong (JMT), a compound Chinese herbal medicine, on oxidative stress, mitochondrial dysfunction and apoptosis in experimental diabetic neuropathy at the animal, cellular and molecular level.
[Methods]
1. In vivo experiment:A single dose of streptozotocin (STZ) was used for the induction of diabetes in male Sprague-Dawley rats. These diabetic rats were randomly divided into5groups:diabetes (DM) group, low-dose JMT group (JMT at dose of.044g/kg/d), middle-dose JMT group (JMT at dose of0.88g/kg/d), high-dose JMT group (JMT at dose of1.75g/kg/d) and vitamin C group (VC at dose of0.05g/kg/d). Ten normal rats with matching weight and age were selected as control group. All rats then received intragastric administration for16weeks (the control and DM groups were treated with distilled water) and then killed. The pain threshold to mechanical stimulation were done before death. Body weight and blood glucose were detected before and at4th,8th,12th,16th week after treatment. Morphological changes in DRG was checked by Nissl staining. The expressions of NADPH oxidase p22-phox subunit, Cyt C, Bcl-2and Caspase-3were detected by immunohistochemical method and quantitative realtime polymerase chain reaction (qRT-PCR). The apoptotic cells were detected by the Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay.
2. In vitro experiment
①Male Sprague-Dawley rats were randomly divided into control group (distilled water), JMT group (JMT at dose of1.31g/kg/d) and vitamin C group (vitamin C at dose of0.08g/kg/d) to prepared drug-containing serum. DRG are harvested from embryonic day15SD rats, dissociated in0.25%trypsin, purified by density gradient centrifugation and differential attachment technique, then cultured in Neurobasal medium supplemented with B-27additives and nerve growth factor, and immunofluorescence method with anti-NSE was used to identify them.
②DRGn were randomly divided into control group and high glucose (50,75,100mmol/L) group. After12,24and48h cultures, MTT assay was used to determine the optimal concentration of high glucose and the optimal time of drugs.
③DRGn were randomly divided into control group, high glucose group (cultured with50mmol/L glucose medium), JMT1:1group, JMT1:2group, JMT1:4group (cultured with different concentration of JMT containing serum) and VC group (cultured with VC containing serum). After24cultures, MTT assay was used to determine the optimal concentration of JMT.
④DRGn were randomly divided into high glucose group (cultured with50mmol/L glucose medium), JMT group (cultured with JMT containing serum) and VC group (cultured with VC containing serum). After24cultures, The levels of superoxide anion and MMP were determined by fluorescent probe. The expressions of Bcl-2and Caspase-3were detected by Western Blot analysis and qRT-PCR. The apoptotic cells were detected by the TUNEL assay.
[Results]
1. In vivo experiment
①Blood glucose and body weight:The blood glucose levels in DM group were much higher than those in control group (P<0.01). At different time, there was no significant difference among model groups (P>0.05). The blood glucose and body weight levels had no marked changes before and after treatment (P>0.05)
②Pain threshold to mechanical stimulation:The pain thresholds in DM group were much lower than those in control group (P<0.01). Compared with DM group, in treatment groups significantly increased (P<0.01). Among treatment groups, the the pain thresholds in middle-dose JMT group increased markedly (P<0.01)
③Oxidative stress:The protein levels of NADPH oxidase p22-phox subunit in DM group were much higher than those in control group(P<0.01). Compared with DM group, the levels of NADPH oxidase p22-phox subunit in treatment groups significantly decreased (P<0.05or P<0.01). Among treatment groups, large-dose JMT group showed better effect than VC group (P<0.01)
④Apoptosis:The cytochrome protein levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in DM group were much higher than those in control group (P<0.01), and the mRNA and protein levels of Bcl-2were much lower (P<0.01). Compared with DM group, the cytochrome protein levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in treatment groups significantly decreased (P<0.05or P<0.01), the mRNA and protein levels of Bcl-2significantly increased (P<0.05or P<0.01). Among treatment groups, large-dose JMT group showed better effect than VC group (P<0.01)
2. In vitro experiment
①MTT assay:The cell viability levels in high glucose groups and control group significantly decreased at24h compared with12h (P<0.01), there was no significant difference between24h and48h (P>0.05). Compared with control group, the cell viability levels in high glucose groups markedly decreased at24h(P<0.05or P<0.01). Among high glucose groups, the cell viability levels in50mmol/LGlu group were much higher than75and100mmol/LGlu groups(P<0.01). Compared with control group, the cell viability levels in50mmol/LGlu group significantly decreased at24h (P<0.01), The cell viability levels in treatment groups were much lower than those in50mmol/LGlu group (P<0.01). Among treatment groups, there was no difference between JMT1:1group and VC group (P>0.05)
②Oxidative stress:The levels of superoxide anion in high glucose group were much higher than those in control group (P<0.01). Compared with high glucose group, the levels of superoxide anion in JMT group significantly decreased (P<0.01) and JMT was superior to VC (P<0.01)
③Apoptosis:The MMP levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in high glucose group were much higher than those in control group (P<0.01), and the mRNA and protein levels of Bcl-2were much lower (P<0.01) Compared with high glucose group, the MMP levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in JMT groups significantly decreased(P<0.01), and the mRNA and protein levels of Bcl-2significantly increased (P<0.01). JMT was superior to VC (P<0.01)
[Conclusion]
1. In vivo experiment
①A single intraperitoneal STZ (60mg/kg) injection-induced diabetic rats showed significant hyperalgesia at16w, indicating development of DPN.
②The levels of NADPH oxidase p22-phox subunit, Cyt C, Caspase-3and the apoptosis percentage in DPN rats significantly increased compared to control rats, and Bcl-2levels significantly reduced.
③JMT treatment significantly ameliorated the alteration in hyperalgesia, NADPH oxidase p22-phox subunit, Cyt C, Bcl-2, Caspase-3and the apoptosis percentage in DPN rats. The large-dose of JMT showed the best effectiveness
④These results suggest that JMT antagonized the oxidative stress, activation of the mitochondrial pathway and apoptosis in DPN rats. And its effectiveness was independent of hypoglycemia and insulin.
2. In vitro experiment
①The purity coefficient of DRGn which were isolated from E15SD rats, dissociated in trypsin digestion, purified by density gradient centrifugation and differential attachment technique, then cultured in Neurobasal medium supplemented with B-27additives and nerve growth factor, could reach more than90%through identification of immunofluorescence method with anti-NSE.
②High glucose remarkably inhibited the cell viability levels of DRGn and JMT treatment significantly ameliorated the inhibition.
③The levels of superoxide anion, Caspase-3and the apoptosis percentage of DRGn in cultured in high glucose medium significantly increased compared to control group, but MMP and Bcl-2levels significantly reduced.
④JMT treatment significantly ameliorated the alteration in superoxide anion, MMP, Bcl-2, Caspase-3and the apoptosis percentage of DRGn in cultured in high glucose medium. And JMT was superior to VC.
⑤These results suggest that JMT containing serum antagonized the oxidative stress, activation of the mitochondrial pathway and apoptosis of DRGn in cultured in high glucose medium.
引文
[1]Edwards JL, Vincent AM, Cheng HT, et al. Diabetic neuropathy:mechanisms to management [J].Pharmacol Ther,2008,120(1):1-34.
[2]中华医学会糖尿病学分会慢性并发症调查组.1991-2000年全国住院糖尿病患者慢性并发症及相关大血管病变回顾性分析[J].中国医学科学院学报,2002,24(5)：447-451.
[3]梁晓春.抗氧化治疗糖尿病周围神经病变[J].中国中西医结合杂志,2007,27(8)：680-682.
[4]潘长玉主译Joslin糖尿病学(第14版)[M].北京：人民卫生出版社,2007：872-881.
[5]Brownlee M. Biochemistry and molecular cell biology of diabetic complications [J]. Nature,2001,414(5):813-820.
[6]Vincent AM, Callaghan BC, Smith AL, et al.Diabetic neuropathy:cellular mechanisms as therapeutic targets[J].Nat Rev Neurol,2011,7(10):573-583.
[7]Leinninger GM, Edwards JL, Lipshaw MJ, et al. Mechanisms of disease: mitochondria as new therapeutic targets in diabetic neuropathy [J]. Nat Clin Pract, 2006,2(11):620-628.
[8]Kuroda J, Nakagawa K, Yamasaki T, et al.The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells[J].Genes Cells,2005,10(12):1139-1151.
[9]Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage [J]. Nature, 2000,404(6779):787-790.
[10]Green DR, Reed JC. Mitochondria and apoptosis[J]. Science,1998,281(5381): 1309-1312.
[11]Takahashi A, Masuda A, Sun M, et al. Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH [J]. Brain Res Bull,2004,62(6):497-504.
[12]梁晓春,崔丽英,郭赛珊,等.筋脉通治疗糖尿病周围神经病变的临床观察[J].中国中西医结合杂志,1999,19(9)：517-519.
[13]张克俭,梁晓春,崔丽英,等.筋脉通胶囊对糖尿病周围神经病变患者Na+-K+-ATP酶活性的影响[J].中医杂志,2001,42(3)：159-161.
[14]梁晓春,张宏,郭赛珊,等.筋脉通对糖尿病大鼠坐骨神经传导速度、醛糖还原酶及山梨醇浓度的影响[J].中国糖尿病杂志,2000,8(1)：37-39.
[15]姜楠,吴群励,屈岭,等.筋脉通胶囊对糖尿病大鼠坐骨神经形态测量学及核转录因子kB蛋白表达的影响[J].环球中医药,2012,5(2)：100-104.
[16]郝伟欣,贾力,徐惠媛,等.筋脉通对大鼠坐骨神经传导速度及红细胞抗氧化作用的影响[J].中国新药杂志,2003,12(5)：343-345.
[17]屈岭,梁晓春,吴群励,等.筋脉通对糖尿病大鼠周围神经组织神经生长因子表达的影响[J].中国中药杂志,2008,33(21)：2539-2544.
[18]王普艳,梁晓春,孙连庆,等.中药筋脉通对糖尿病大鼠周围神经睫状体神经营养因子表达的影响[J].中西医结合急救杂志,2010,17(1)：3-8.
[19]黄文智,梁晓春,王普艳,等.筋脉通对实验性糖尿病大鼠坐骨神经AGEs及其受体表达的影响[J].中华中医药杂志,2011,26(4)：806-809.
[20]李伯武,梁晓春,王普艳,等.筋脉通对糖尿病大鼠坐骨神经NADPH氧化酶p22-phox亚基表达的影响[J].医学研究杂志,2011,40(5)：4346.
[21]孙青,梁晓春,王普艳,等.筋脉通对糖尿病大鼠周围神经组织诱导型一氧化氮合酶和硝基酪氨酸表达的影响[J].中国中药杂志,2012,37(3)：348-352.
[22]王普艳.筋脉通对糖尿病大鼠坐骨神经及雪旺细胞氧化应激致细胞凋亡的影响[D].北京：北京协和医学院,2009.
[23]Qu L, Liang XC, Zhang H, et al. Effect of Jinmaitong serum on the proliferation of rat Schwann cells cultured in high glucose medium[J].Chin J Integr Med,2008,14(4): 293-297.
[24]吴群励,梁晓春,张宏,等.中药筋脉通含药血清对高糖培养雪旺细胞NF-κB表达的影响[J].世界中西医结合杂志,2011,06(6)：478-481.
[25]孙连庆,梁晓春,张宏,等.中药筋脉通对高糖培养雪旺细胞增殖及NGF表达的影响[J].中华中医药杂志,2009,24(6)：55-58.
[26]王普艳,梁晓春,张宏,等.筋脉通胶囊对高糖培养雪旺细胞睫状体神经营养因子表达的影响[J].中医杂志,2010,51(8)：736-739.
[27]赵丽,梁晓春,张宏,等.筋脉通含药血清对高糖培养的Schwann细胞诱导型一氧化 氮合酶和NADPH氧化酶p22-phox亚基表达的影响[J].中西医结合学报,2011,9(3)：299-305.
[28]朴元林,梁晓春,赵丽,等.筋脉通含药血清对高糖培养施万细胞8-羟基脱氧鸟苷和活化的caspase-3表达的影响[J].医学研究杂志,2011,40(10)：35-39.
[29]沈丽霞,牛建昭,王继峰.槲皮素对AlC13致衰老模型小鼠记忆障碍的保护作用[J].北京中医药大学学报,2007,30(9)：615-617.
[30]Ou Y, Geng P, Liao GY, et al. Intracellular GSH and ROS levels may be related to galactose-mediated human lens epithelial cell apoptosis:role of recombinant hirudin variant Ⅲ[J].Chem Biol Interact,2009,179(2-3):103-109.
[31]赵春贵,张立伟,董建华,等.肉桂酸及其衍生物对活性氧H202清除作用[J].化学研究与应用,2004,(5)：685-687.
[32]Schmeichel AM, Schmelzer JD, Low PA. Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy [J]. Diabetes,2003,52(1):165-71.
[33]Cameron NE, Cotter MA. Effects of protein kinase C beta inhibition on neurovascular dysfunction in diabetic rats:interaction with oxidative stress and essential fatty acid dysmetabolism[J]. Diatetes Metab Res Rev,2002,18(4):315-323.
[34]ObrosovaIG, Huysen CV, Fathallah L, et al. Evalution of a-adrenoceptor antagonist on diabetes induced changes in peripheral nerve function, metabolism, and antioxidative defence [J]. FASEB J,2000,14(11):1548-1558.
[35]Kandhare AD, Raygude KS, Ghosh P, et al.Neuroprotective effect of naringin by modulation of endogenous biomarkers in streptozotocin induced painful diabetic neuropathy [J]. Fitoterapia,2012 Feb 9. [Epub ahead of print]
[36]Kaeidi A, Esmaeili-Mahani S, Abbasnejad M, et al.Satureja khuzestanica attenuates apoptosis in hyperglycemic PC12 cells and spinal cord of diabetic rats[J].J Nat Med,2012,Mar2. [Epub ahead of print]
[37]沈应君主编.中药药理学[M].北京：人民卫生出版社,2000：96-97.
[38]张瑞明,李延谦.中药新药期临床研究有关问题的探讨[J].中药新药与临床药理,2004,15：66-67.
[39]Stevens MJ, Obrosova I, Cao X, et al. Effects of DL-alpha-lipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy[J].Diabetes,2000,49(6):1006-1015.
[40]Hounsom L, Horrobin DF, Tritschler H, et al. Lipoic acid-gamma linolenic acid conjugate is effective against multiple indices of experimental diabetic neuropathy[J].Diabetologia,1998,41(7):839-843.
[41]Ratnam DV, Ankola DD, Bhardwaj V, et al. Role of antioxidants in prophylaxis and therapy:A pharmaceutical perspective[J]. J Control Release,2006,113(3):189-207.
[42]Takahashi N, Morimoto S, Okigaki M, et al.Decreased plasma level of vitamin C in chronic kidney disease:comparison between diabetic and non-diabetic patients[J].Nephrol Dial Transplant,2011,26(4):1252-1257.
[43]Odermarsky M, Lykkesfeldt J, Liuba P.Poor vitamin C status is associated with increased carotid intima-media thickness, decreased microvascular function, and delayed myocardial repolarization in young patients with type 1 diabetes[J].Am J Clin Nutr,2009,90(2):447-452.
[44]Sadi G, Yilmaz O, G u ray T. Effect of vitamin C and lipoic acid on streptozotocin-induced diabetes gene expression:mRNA and protein expressions of Cu-Zn SOD and catalase[J].Mol Cell Biochem,2008,309(1-2):109-116.
[45]Lee EY, Lee MY, Hong SW, et al.Blockade of oxidative stress by vitamin C ameliorates albuminuria and renal sclerosis in experimental diabetic rats[J]. Yonsei Med J,2007,48(5):847-855.
[46]Lindroos OF. Short Nissl staining for incubated cryostat sections of the brain[J].Biotech Histochem,1991,66(4):208-209.
[47]Kiernan JA, Macpherson CM, Price A, et al.A histochemical examination of the staining of kainate-induced neuronal degeneration by anionic dyes[J].Biotech Histochem,1998,73(5):244-254.
[48]顾兵,金建波,李华南,等.神经组织染色方法的研究概况[J].中国药理学通报,2011,27(10)：1472-1475.
[49]McHugh JM, McHugh WB.Diabetes and peripheral sensory neurons:what we don't know and how it can hurt us[J].AACN Clin Issues,2004,5(1):136-149.
[50]Leusen JH, Verhoeven AJ, Roos D. Interacitons between the components of the human NADPH oxidase:Intruges in the phox family[J].J Lab Clin Med, 1996,128(5):461-476.
[51]Dohi K, Ohtaki H, Nakamachi T, et al. Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury[J]. Journal of Neuroinflammation, 2010,7(1):41.
[52]Xian Cao, Demel SL, Quinn MT, et al. Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and peri vascular nerve fibers[J].Auton Neurosci,2009,151(2):90-97.
[53]El-Benna J, Dang PM, Gougerot-Pocidalo MA, et al. P47phox, the phagocyte NADPH oxidase/NOX2 organizer:structure, phosphorylation and implication in diseases[J].Exp Mol Med,2009,41(4):217-225.
[54]Nauseef WM. Assembly of the phagocyte NADPH oxidase[J].Histochem Cell Biol,2004,122(4):277-291.
[55]Kim H, Park JS, Choi YJ, et al.Bone marrow mononuclear cells have neurovascular tropism and improve diabetic neuropathy[J].Stem Cells,2009,27(7):1686-1696.
[56]Ibi M,Matsuno K,Shiba D, et al. Reactive oxygen species derived from NOX1/NADPH oxidase enhance inflammatory pain[J]. J Neurosci,2008,28(38): 9486-9494.
[57]Cotter MA, Cameron NE.Effect of the NAD(P)H oxidase inhibitor, apocynin, on peripheral nerve perfusion and function in diabetic rats[J].Life Sci,2003,73(14): 1813-1824.
[58]Vincent AM, Perrone L, Sullivan KA, et al.Receptor for advanced glycation end products activation injures primary sensory neurons via oxidative stress[J]. Endocrinology,2007,148(2):548-558.
[59]Inoguchi T, Li P, Umeda F, et al. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells[J].Diabetes,2000,49(11):1939-1945.
[60]San Martin A, Foncea R, Laurindo FR, et al. Noxl-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products[J].Free Radic Biol Med,2007,42(11):1671-1679.
[61]Zhang L, Pang S, Deng B, et al. High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol[J].Int J Biochem Cell Biol, 2012,44(4):629-638.
[62]吴其夏主编.病理生理学(第二版)[M].北京：中国协和医科大学出版社,2003：99-111.
[63]Sima AA.New insights into the metabolic and molecular basis for diabetic neuropathy [J]. Cell Mol Life Sci,2003,60(11):2445-2464.
[64]Russell JW, Golovoy D, Vincent AM, et al.High glucose-induced oxidative stress and mitochondrial dysfunction in neurons[J]. FASEBJ,2002,16(13):1738-1748.
[65]Thornberry NA, Lazebnik Y. Caspases:enemies within[J].Science,1998(281): 1312-1316.
[66]Adams JM, Cory S.The Bcl-2 protein family:arbiters of cell survival[J]. Science,1998,281(5381):1322-1326.
[67]Vincent AM, Brownlee M, Russell JW.Oxidative stress and programmed cell death in diabetic neuropathy [J]. Ann N Y Acad Sci,2002,959:368-383.
[68]Vincent AM, Russell JW, Sullivan KA, et al.SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy [J].Exp Neurol, 2007,208(2):216-227.
[69]Kamboj SS, Vasishta RK, Sandhir R.N-acetylcysteine inhibits hyperglycemia-induced oxidative stress and apoptosis markers in diabetic neuropathy[J].J Neurochem,2010,112(1):77-91.
[70]刘玉琴主编.细胞培养实验手册[M].北京：人民军医出版社,2009：93-97.
[71]梁晓春.糖尿病周围神经病变与消渴兼证“筋痹”及其中医治疗[J].中国临床医生,2006,34(5)：17-18.
[72]Russell J W, Sullivan KA, Windebank AJ, et al. Neurons undergo apoptosis in animal and cell culture models of diabetes [J]. Neurobiol Dis,1999,6(5):347-363.
[73]Hall KE, Sheng HC, Srinivasan S, et al. Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival [J]. Brain Res, 2001,888(1):128-137.
[74]Sango K, Horie H, Inoue S, et al. Age-related changes of DRG neuronal attachment to extracellular matrix proteins in vitro[J]. Neuroreport,1993,4(6):663-666.
[75]Puttfarcken PS, Manelli AM, Arneric SP, et al. Evidence for nicotinic receptors potentially modulating nociceptive transmission at the level of the primary sensory neuron:studies with F11 cells [J]. J Neurochem,1997,69(3):930-938.
[76]Chen W, Mi R, Haughey N, et al. Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line [J]. J Peripher Nerv Syst, 2007,12(2):121-130.
[77]Raymon HK, Thode S, Zhou J, et al. Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties[J].J Neurosci,1999,19(13): 5420-5428.
[78]Banker GA, Goslin K. Culturing nerve cells (2nd ed) [M]. Boston:MIT Press,1998: 545.
[79]Malin SA, Davis BM, Molliver DC. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity [J].Nat Protoc,2007,2(1):152-160.
[80]Sergery F, Arleen R. Protocols for neural cell culture(3rd ed) [M]. New Jersey:Humona Press,2001:95-97.
[81]Yong VW, Horie H, Kim SU. Comparison of six different substrata on the plating efficiency, differentiation and survival of human dorsal root ganglion neurons in culture[J]. Dev Neurosci,1988,10(4):222-230.
[82]Sango K, Horie H, Okamura A, et al. Diabetes impairs DRG neuronal attachment to extracellular matrix proteins in vitro. Brain Res Bull[J].1995,37(5):533-537.
[83]Sango K, Horie H, Inoue S. Administration of an aldose reductase inhibitor, ONO-2235, to streptozotocin-diabetic mice restores reductions of DRG neuronal attachment to extracellular matrix proteins in vitro [J].Neurosci Lett,1999, 263(2-3):157-160.
[84]Luo ZJ, King RH, Lewin J, et al. Effects of nonenzymatic glycosylation of extracellular matrix components on cell survival and sensory neurite extension in cell culture[J]. J Neurol.2002,249(4):424-431.
[85]李全波,马文庭,刘静芷,等.大鼠背根神经节神经元细胞纯化培养的模型建立[J].中国疼痛医学杂志,2011,17(8)：494-497.
[86]张军,许百男,李翀,等.胚胎大鼠背根神经节神经元的培养与纯化[J].解放军医学杂志,2006,31(12)：1152-1154.
[87]Silver J, Miller JH. Regeneration beyond the glial scar[J].Nat Rev Neurosci, 2004,5(2):146-156.
[88]Lewin GR, Ritter AM, Mendell LM.On the role of nerve growth factor in the development of myelinated nociceptors[J].J Neurosci,1992,12(5):1896-1905.
[89]Ben-Zvi A, Yagil Z, Hagalili Y, et al.Semaphorin 3 A and neurotrophins:a balance between apoptosis and survival signaling in embryonic DRG neurons[J].J Neurochem,2006,96(2):585-597.
[90]Wang H, Zhou X W, Zhang H, et al. Analgesic effect of intrathecal S03 on rats with chronic constriction injury and its regulation of intracellular calcium ion concentration of spinal dorsal root ganglion neurons[J].Chin Pharmacol Bull,2006,22(6):693-697.
[91]隋峰,杜新亮,张畅斌,等.一种原代培养大鼠DRG神经元的新方法[J].中国药理学通报,2009,25(7)：971-973.
[92]Young L, Bilsland J, Harper S.A rapid method for determination of cell survival in primary neuronal DRG cultures[J].J Neurosci Methods,1999,30,93(1):81-89.
[93]Terashima T, Kojima H, Fujimiya M, et al.The fusion of bone-marrow-derived proinsulin-expressing cells with nerve cells underlies diabetic neuropathy[J].Proc Natl Acad Sci USA,2005,102(35):12525-12530.
[94]王丽琴,宋学琴,王晓娟,等.大鼠背根神经节细胞的纯化培养[J].脑与神经疾病杂志,2003,11(5)：265-267.
[95]Andersen PL, Doucette JR, Nazarali AJ.A novel method of eliminating non-neuronal proliferating cells from cultures of mouse dorsal root ganglia[J]. Cell Mol Neurobiol,2003,23(2):205-210.
[96]Alcazar A, Cid C.High cytotoxic sensitivity of the oligodendrocyte precursor cells to HSP90 inhibitors in cell cultures[J].Exp Neurol,2009,216(2):511-514.
[97]章静波主编.组织和细胞培养技术[M].北京：人民卫生出版社,2002：105-106.
[98]Mosmann T. Rapid col orimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays[J]. Immunol Methods,1983,65(1-2):55-63.
[99]Vincent AM, Stevens MJ, Backus C, et al. Cell culture modeling to test therapies against hyperglycemia-mediated oxidative stress and injury[J]. Antioxid Redox Signal,2005,7(11-12):1494-1506.
[100]Vincent AM, McLean LL, Backus C, et al. Short-term hyperglycemia produces oxidative damage and apoptosis in neurons[J].FASEB J,2005,19(6):638-640.
[101]Iwama H, Amagaya S, Ogihara Y. Effect of shosaikoto, a Japanese and Chinese traditional herbal medicinal mixture, on the mitogenic activity of lipopolysaccharide:a new pharmacological testing method[J].J Ethnopharmacol,1987,21 (1):45-53.
[102]黄臣虎,陆茵,高骁君,等.中药血清药理学研究进展[J].中国实验方剂学杂志,2011,17(10)：266-271.
[103]杨彦芳,王玉芹.中药复方血清药理学方法规范化探讨[J].中国中西医结合杂志,2000,20(5)：280-282.
[104]李仪奎.中药血清药理学实验方法的若干问题[J].中药新药与临床药理,1999,10(2)：95.
[105]罗焕敏.血清药理学与血浆药理学[J].中国药理学通报,2003,19(9)：1075-1076.
[106]Vincent AM, Russell JW, Low P, et al. Oxidative stress in the pathogenesis of diabetic neuropathy [J]. Endocr Rev,2004,25(4):612-628.
[107]Zherebitskaya E, Akude E, Smith DR, et al. Development of selective axonopathy in adult sensory neurons isolated from diabetic rats:role of glucose-induced oxidative stress[J]. Diabetes,2009,58(6):1356-1364.
[108]Zhao H, Kalivendi S, Zhang H, et al. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium:potential implications in intracellular fluorescence detection of superoxide [J]. Free Radic Biol Med,2003,34:1359-1368.
[109]Allen DA, Yaqoob MM, Harwood SM. Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications [J]. J Nutr Biochem, 2005,16(12):705-713.
[110]Cellerino A, Bahr M, Isenmann S. Apoptosis in the developing visual system[J]. Cell Tissue Res,2000,301(1):53-69.
[111]Graham SH, Chen J, Clark RS. Bcl-2 family gene products in cerebral ischemia and traumatic brain injury[J]. J Neurotrauma,2000,17(10):831-841.
[112]Srinivasan S, Stevens M, Wiley JW. Diabetic peripheral neuropathy:evidence for apoptosis and associated mitochondrial dysfunction[J].Diabetes,2000,49 (11):1932-1938.
[113]Honma H, Podratz JL, Windebank AJ. Acute glucose deprivation leads to apoptosis in a cell model of acute diabetic neuropathy[J].J Peripher Nerv Syst, 2003,8(2):65-74.
[114]冯健.齐墩果酸对大鼠血管平滑肌细胞中血红素氧合酶1表达的影响及其作用机制的研究[D].重庆：第三军医大学,2011.
[115]顾锦华,黄华,薛华,等.齐墩果酸对糖尿病小鼠胰岛损伤的保护作用[J].中草药,2010,41(11)：1866-1870.
[116]张兰兰,曹琼,张鹏,等.槲皮素对氧化应激大鼠星形胶质细胞的保护作用[J].中华神经医学杂志,2009,8(1)：10-12.
[117]毕伟,朱丽红,王传明,等.槲皮素抑制鱼藤酮诱导的PC12细胞凋亡[J].中国病理生理杂志,2011,27(1)：82-85.
[118]张明发,沈雅琴.红景天苷的神经保护作用[J].国际脑血管病杂志,2011,19(2)：137-141.
[119]Zhang L,Yu H,Zhao X, et al. Neuroprotective effects of salidroside against (3-anyloid-induced oxidative stress in SH-SY5Y human neuroblastoma cells2010
[120]裴晶晶,吴润,赵红斌,等.Ca2+信号介导红景天苷促进小鼠骨髓间充质干细胞向神经细胞的定向分化[J].中国组织工程研究与临床康复,2010,14(10)：1808-1812.
[121]吴春,陈林林.菟丝子黄酮体外清除自由基活性的研究[J].天然产物研究与开发,2005,17(5)：553-556.
[122]王展,方积年.具有抗氧化活性的酸性菟丝子多糖H2的研究[J].植物学报,2001,43(3)：243-248.
[123]魏晓东,刘玉萍,李晶,等.D-半乳糖致衰大鼠非酶糖基化改变及菟丝子醇提液对其作用的研究[J].中国老年学杂志,2009,29(19)：2494-2496.
[124]周中和,曲方,何祥.局部应用重组水蛭素对大鼠脑出血神经细胞凋亡的干预作用[J].临床神经病学杂志,2006,19(4)：304-306.
[125]张尉华,饶明俐,吴江,等.水蛭素对大鼠实验性脑出血神经组织的保护作用[J].中风与神经疾病杂志,2006,23(1)：44-45.
[126]杨霞,黄萍,肖晓蓉,等.中药桂皮醛对牙周致病菌的体外抑菌活性研究[J].重庆医学,2009,38(1)：75-76.
[127]贾蓓蓓,邵世和,高小焕,等.桂皮醛对大鼠体内耐甲氧西林金黄色葡萄球菌生物膜的清除效应[J].江苏大学学报(医学版),2011,21(3)：199-202,207,封3.
[128]李澎,任钧国,段昌令等.4种延胡索成分对乳鼠心肌细胞缺氧和过氧化损伤的影响[J].中国中药杂志,2010,35(1)：84-87.
[129]俞静,花宝金,朴炳奎,等.延胡索乙素对放射所致血管内皮细胞损伤的保护作用[J].中华肿瘤防治杂志,2011,18(8)：584-587,601.
[130]樊景坡.苍耳子、细辛、枸杞、白术对小鼠组织自由基代谢的影响[J].中医药信息,1994,2(1)：48.
[131]栗坤,郑景禄,白晶,等.细辛、杜仲及其合剂对D-半乳糖所致衰老大鼠NO、NOS和CAT的影响[J].中国老年杂志,2001,21(2)：132.
[1]Russell JW, Sullivan KA, Windebank AJ, et al. Neurons undergo apoptosis in animal and cell culture models of diabetes[J]. Neurobiol Dis,1999,6(5):347-363.
[2]Hall KE, Sheng HC, Srinivasan S, et al. Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival[J]. Brain Res, 2001,888(1):128-137.
[3]Sango K, Horie H, Inoue S, et al. Age-related changes of DRG neuronal attachment to extracellular matrix proteins in vitro[J].Neuroreport,l 993,4(6):663-666.
[4]Puttfarcken PS, Manelli AM, Arneric SP, et al. Evidence for nicotinic receptors potentially modulating nociceptive transmission at the level of the primary sensory neuron:studies with F11 cells[J]. J Neurochem,1997,69(3):930-938.
[5]Chen W, Mi R, Haughey N, et al. Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line[J].J Peripher Nerv Syst, 2007,12(2):121-130.
[6]Chen W, Mi R, Haughey N, et al. Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line[J].J Peripher Nerv Syst, 2007,12(2):121-130.
[7]禹晓东,罗卓荆,张琳,等.大鼠脊髓背根神经节神经元的纯化培养[J].中国脊柱脊髓杂志,2005,15(10)：591-594.
[8]王丽琴,宋学琴,王晓娟,等.胚胎大鼠背根神经节细胞的分离培养、纯化及生物学特性的研究[J].细胞生物学杂志,2003,25(5)：320-323.
[9]Russell JW, Golovoy D, Vincent AM, et al. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons[J]. FASEB J,2002,16(13):1738-1748.
[10]Vincent AM, Stevens MJ, Backus C, et al. Cell culture modeling to test therapies against hyperglycemia-mediated oxidative stress and injury [J]. Antioxid Redox Signal,2005,7(11-12):1494-1506.
[11]Srinivasan S, Stevens M, Wiley JW. Diabetic peripheral neuropathy:evidence for apoptosis and associated mitochondrial dysfunction[J].Diabetes,2000,49(11): 1932-1938.
[12]Vincent AM, McLean LL, Backus C, et al. Short-term hyperglycemia produces oxidative damage and apoptosis in neurons[J]. FASEB J,2005,19(6):638-640.
[13]Honma H, Podratz JL, Windebank AJ. Acute glucose deprivation leads to apoptosis in a cell model of acute diabetic neuropathy[J].J Peripher Nerv Syst, 2003,8(2):65-74.
[14]Ozturk G, Erdogan E, Ozturk M, et al. Differential analysis of effect of high glucose level in the development of neuropathy in a tissue culture model of diabetes mellitus: role of hyperosmolality[J].Exp Clin Endocrinol Diabetes,2008,116(10):582-591.
[15]Vincent AM, Russell JW, Low P,et al. Oxidative stress in the pathogenesis of diabetic neuropathy [J]. Endocr Rev,2004,25(4):612-628.
[16]Vincent AM, Russell JW, Sullivan KA, et al. SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy [J].Exp Neurol, 2007,208(2):216-227.
[17]Zherebitskaya E, Akude E, Smith DR, et al. Development of selective axonopathy in adult sensory neurons isolated from diabetic rats:role of glucose-induced oxidative stress[J]. Diabetes,2009,58(6):1356-1364.
[18]Vincent AM, Olzmann JA, Brownlee M, et al. Uncoupling proteins prevent glucose-induced neuronal oxidative stress and programmed cell death[J]. Diabetes, 2004,53(3):726-734.
[19]Spillson AB, Russell JW. Metabotropic glutamate receptor regulation of neuronal cell death[J]. Exp Neurol,2003,184 Suppl 1:S97-105.
[20]Berent-Spillson A, Russell JW. Metabotropic glutamate receptor 3 protects neurons from glucose-induced oxidative injury by increasing intracellular glutathione concentration[J]. J Neurochem,2007,101(2):342-354.
[21]Berent-Spillson A, Robinson AM, Golovoy D, et al. Protection against glucose-induced neuronal death by NAAG and GCP Ⅱ inhibition is regulated by mGluR3[J]. J Neurochem,2004,89(1):90-99.
[22]Kim SR, Lee DY, Chung ES, et al. Transient receptor potential vanilloid subtype 1 mediates cell death of mesencephalic dopaminergic neurons in vivo and in vitro[J]. J Neurosci,2005,25(3):662-671.
[23]Hong S, Wiley JW. Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1 [J]. J Biol Chem,2005,280(1):618-627.
[24]Hong S, Agresta L, Guo C, et al. The TRPV1 receptor is associated with preferential stress in large dorsal root ganglion neurons in early diabetic sensory neuropathy [J]. J Neurochem,2008,105(4):1212-1222.
[25]Gumy LF, Bampton ET, Tolkovsky AM. Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG[J]. Mol Cell Neurosci,2008,37(2):298-311.
[26]Tosaki T, Kamiya H, Yasuda Y, et al. Reduced NGF secretion by Schwann cells under the high glucose condition decreases neurite outgrowth of DRG neurons[J]. Exp Neurol,2008,213(2):381-387.
[27]Yu C, Rouen S, Dobrowsky RT. Hyperglycemia and downregulation of caveolin-1 enhance neuregulin-induced demyelination[J].Glia,2008,56(8):877-887.
[28]Yong VW, Horie H, Kim SU. Comparison of six different substrata on the plating efficiency, differentiation and survival of human dorsal root ganglion neurons in culture[J].Dev Neurosci,1988,10(4):222-230.
[29]Sango K, Horie H, Okamura A, et al. Diabetes impairs DRG neuronal attachment to extracellular matrix proteins in vitro[J].Brain Res Bull,1995,37(5):533-537.
[30]Sango K, Horie H, Inoue S. Administration of an aldose reductase inhibitor, ONO-2235, to streptozotocin-diabetic mice restores reductions of DRG neuronal attachment to extracellular matrix proteins in vitro [J].Neurosci Lett,1999, 263(2-3):157-160.
[31]Luo ZJ, King RH, Lewin J, et al. Effects of nonenzymatic glycosylation of extracellular matrix components on cell survival and sensory neurite extension in cell culture[J]. J Neurol,2002,249(4):424-431.
[32]Gardiner NJ, Wang Z, Luke C, et al. Expression of hexokinase isoforms in the dorsal root ganglion of the adult rat and effect of experimental diabetes[J]. Brain Res,2007,1175:143-154.
[33]Kozlova EN, Jansson L. In vitro interactions between insulin-producing beta cells and embryonic dorsal root ganglia[J]. Pancreas,2005,31(4):380-384.
[34]Craner MJ, Klein JP, Black JA, et al. Preferential expression of IGF-I in small DRG neurons and down-regulation following injury[J].Neuroreport,2002,13(13): 1649-1652.
[35]Edwards JL, Quattrini A, Lentz SI, et al. Diabetes regulates mitochondrial biogenesis and fission in mouse neurons[J]. Diabetologia,2010,53(1):160-169.
[1]Edwards JL, Vincent AM, Cheng HT, et al. Diabetic neuropathy:mechanisms to management [J].Pharmacol Ther,2008,120(1):1-34.
[2]Brownlee M. Biochemistry and molecular cell biology of diabetic complications [J]. Nature,2001,414(5):813-820.
[3]Vincent AM, Callaghan BC, Smith AL, et al.Diabetic neuropathy:cellular mechanisms as therapeutic targets[J].Nat Rev Neurol,2011,7(10):573-583.
[4]Baluchnejadmojarad T, Roghani M, Khastehkhodaie Z.Chronic treatment of silymarin improves hyperalgesia and motor nerve conduction velocity in diabetic neuropathic rat[J].Phytother Res,2010,24(8):1120-1125.
[5]Kumar A, Kaundal RK, Iyer S, et al.Effects of resveratrol on nerve functions, oxidative stress and DNA fragmentation in experimental diabetic neuropathy [J]. Life Sci,2007,80(13):1236-1244.
[6]Watcho P, Stavniichuk R, Ribnicky DM, et al.High-fat diet-induced neuropathy of prediabetes and obesity:effect of PMI-5011, an ethanolic extract of Artemisia dracunculus L[J].Mediators Inflamm,2010:268547. Epub 2010 Apr 8.
[7]郝伟欣,贾力,徐惠媛,等.筋脉通对大鼠坐骨神经传导速度及红细胞抗氧化作用的影响[J].中国新药杂志,2003,12(5)：343-345.
[8]李伯武,梁晓春,王普艳,等.筋脉通对糖尿病大鼠坐骨神经NADPH氧化酶p22-phox亚基表达的影响[J].医学研究杂志,2011,40(5)：43-46.
[9]赵丽,梁晓春,张宏,等.筋脉通含药血清对高糖培养的Schwann细胞诱导型一氧化氮合酶和NADPH氧化酶p22-phox亚基表达的影响[J].中西医结合学报,2011,9(3)：299-305.
[10]张德刚,赵瑛,夏培金,等.通络方剂改善糖尿病大鼠周围神经病变作用机制的探讨[J].中西医结合学报,2006,4(6)：601-605.
[11]Russell JW, Golovoy D, Vincent AM, et al.High glucose-induced oxidative stress and mitochondrial dysfunction in neurons[J]. FASEBJ,2002,16(13):1738-1748.
[12]Sun LQ, Xue B, Li XJ, et al.Inhibitory effects of salvianolic acid B on apoptosis of Schwann cells and its mechanism induced by intermittent high glucose[J].Life Sci,2012,90(3-4):99-108.
[13]王普艳.筋脉通对糖尿病大鼠坐骨神经及雪旺细胞氧化应激致细胞凋亡的影响[D].北京：北京协和医学院,2009.
[14]Wu LL, Chiou CC, Chang PY, et al. Urinary 8-OHDG:a marker of oxidative stess to DNA and a risk factor for cancer. atherselerosis and diabetics[J]. Clin Chim Acta,2004,339(6):1-9.
[15]Obrosova IG, Li F, Abatan OI, et al. Role of poly(ADP-ribose) polymerase activation in diabetic neuropathy[J].Diabetes,2004,53(3):711-720.
[16]朴元林,梁晓春,赵丽,等.筋脉通含药血清对高糖培养施万细胞8-羟基脱氧鸟苷和活化的caspase-3表达的影响[J].医学研究杂志,2011,40(10)：35-39.
[17]Sheftel AD, Kim SF, Ponka P. Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism[J]. J Biol Chem,2007,282(14):10480-10486.
[18]Quan S, Kaminski PM, Yang L, et al. Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats[J]. Biochem Biophys Res Common,2004; 315:509-516.
[19]Negi G, Kumar A, Sharma SS. Nrf2 and NF-κB modulation by sulforaphane counteracts multiple manifestations of diabetic neuropathy in rats and high glucose-induced changes[J].Curr Neurovasc Res,2011,8(4):294-304.
[20]Romeo L, Intrieri M, D'Agata V, et al.The major green tea polyphenol, (-)-epigallocatechin-3-gallate, induces heme oxygenase in rat neurons and acts as an effective neuroprotective agent against oxidative stress[J].J Am Coll Nutr,2009,28 Suppl:492S-499S.
[21]Huang SM, Hsu CL, Chuang HC, et al.Inhibitory effect of vanillic acid on methylglyoxal-mediated glycation in apoptotic Neuro-2A cells.Neurotoxicology [J].2008,29(6):1016-1022.
[22]李玉红,陈泽奇,叶仁群,等.加味补肝汤对糖尿病周围神经病变大鼠背根节p38丝裂素活化蛋白激酶表达的干预作用[J].中国临床康复,2006,10(7)：67-69.
[23]叶仁群,陈泽奇,熊丽丽,等.加味补肝汤对糖尿病大鼠背根神经节细胞凋亡的 影响[J].湖南中医药大学学报,2008,28(5)：44-47.
[24]黄娟,陈泽奇,熊丽丽,等.加味补肝汤对糖尿病大鼠坐骨神经中JNK丝裂原活化蛋白激酶表达的影响[J].中国医师杂志,2009,11(2)：156-159,169.
[2]中华医学会糖尿病学分会慢性并发症调查组.1991-2000年全国住院糖尿病患者慢性并发症及相关大血管病变回顾性分析[J].中国医学科学院学报,2002,24(5)：447-451.
[3]梁晓春.抗氧化治疗糖尿病周围神经病变[J].中国中西医结合杂志,2007,27(8)：680-682.
[4]潘长玉主译Joslin糖尿病学(第14版)[M].北京：人民卫生出版社,2007：872-881.
[5]Brownlee M. Biochemistry and molecular cell biology of diabetic complications [J]. Nature,2001,414(5):813-820.
[6]Vincent AM, Callaghan BC, Smith AL, et al.Diabetic neuropathy:cellular mechanisms as therapeutic targets[J].Nat Rev Neurol,2011,7(10):573-583.
[7]Leinninger GM, Edwards JL, Lipshaw MJ, et al. Mechanisms of disease: mitochondria as new therapeutic targets in diabetic neuropathy [J]. Nat Clin Pract, 2006,2(11):620-628.
[8]Kuroda J, Nakagawa K, Yamasaki T, et al.The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells[J].Genes Cells,2005,10(12):1139-1151.
[9]Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage [J]. Nature, 2000,404(6779):787-790.
[10]Green DR, Reed JC. Mitochondria and apoptosis[J]. Science,1998,281(5381): 1309-1312.
[11]Takahashi A, Masuda A, Sun M, et al. Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH [J]. Brain Res Bull,2004,62(6):497-504.
[12]梁晓春,崔丽英,郭赛珊,等.筋脉通治疗糖尿病周围神经病变的临床观察[J].中国中西医结合杂志,1999,19(9)：517-519.
[13]张克俭,梁晓春,崔丽英,等.筋脉通胶囊对糖尿病周围神经病变患者Na+-K+-ATP酶活性的影响[J].中医杂志,2001,42(3)：159-161.
[14]梁晓春,张宏,郭赛珊,等.筋脉通对糖尿病大鼠坐骨神经传导速度、醛糖还原酶及山梨醇浓度的影响[J].中国糖尿病杂志,2000,8(1)：37-39.
[15]姜楠,吴群励,屈岭,等.筋脉通胶囊对糖尿病大鼠坐骨神经形态测量学及核转录因子kB蛋白表达的影响[J].环球中医药,2012,5(2)：100-104.
[16]郝伟欣,贾力,徐惠媛,等.筋脉通对大鼠坐骨神经传导速度及红细胞抗氧化作用的影响[J].中国新药杂志,2003,12(5)：343-345.
[17]屈岭,梁晓春,吴群励,等.筋脉通对糖尿病大鼠周围神经组织神经生长因子表达的影响[J].中国中药杂志,2008,33(21)：2539-2544.
[18]王普艳,梁晓春,孙连庆,等.中药筋脉通对糖尿病大鼠周围神经睫状体神经营养因子表达的影响[J].中西医结合急救杂志,2010,17(1)：3-8.
[19]黄文智,梁晓春,王普艳,等.筋脉通对实验性糖尿病大鼠坐骨神经AGEs及其受体表达的影响[J].中华中医药杂志,2011,26(4)：806-809.
[20]李伯武,梁晓春,王普艳,等.筋脉通对糖尿病大鼠坐骨神经NADPH氧化酶p22-phox亚基表达的影响[J].医学研究杂志,2011,40(5)：4346.
[21]孙青,梁晓春,王普艳,等.筋脉通对糖尿病大鼠周围神经组织诱导型一氧化氮合酶和硝基酪氨酸表达的影响[J].中国中药杂志,2012,37(3)：348-352.
[22]王普艳.筋脉通对糖尿病大鼠坐骨神经及雪旺细胞氧化应激致细胞凋亡的影响[D].北京：北京协和医学院,2009.
[23]Qu L, Liang XC, Zhang H, et al. Effect of Jinmaitong serum on the proliferation of rat Schwann cells cultured in high glucose medium[J].Chin J Integr Med,2008,14(4): 293-297.
[24]吴群励,梁晓春,张宏,等.中药筋脉通含药血清对高糖培养雪旺细胞NF-κB表达的影响[J].世界中西医结合杂志,2011,06(6)：478-481.
[25]孙连庆,梁晓春,张宏,等.中药筋脉通对高糖培养雪旺细胞增殖及NGF表达的影响[J].中华中医药杂志,2009,24(6)：55-58.
[26]王普艳,梁晓春,张宏,等.筋脉通胶囊对高糖培养雪旺细胞睫状体神经营养因子表达的影响[J].中医杂志,2010,51(8)：736-739.
[27]赵丽,梁晓春,张宏,等.筋脉通含药血清对高糖培养的Schwann细胞诱导型一氧化 氮合酶和NADPH氧化酶p22-phox亚基表达的影响[J].中西医结合学报,2011,9(3)：299-305.
[28]朴元林,梁晓春,赵丽,等.筋脉通含药血清对高糖培养施万细胞8-羟基脱氧鸟苷和活化的caspase-3表达的影响[J].医学研究杂志,2011,40(10)：35-39.
[29]沈丽霞,牛建昭,王继峰.槲皮素对AlC13致衰老模型小鼠记忆障碍的保护作用[J].北京中医药大学学报,2007,30(9)：615-617.
[30]Ou Y, Geng P, Liao GY, et al. Intracellular GSH and ROS levels may be related to galactose-mediated human lens epithelial cell apoptosis:role of recombinant hirudin variant Ⅲ[J].Chem Biol Interact,2009,179(2-3):103-109.
[31]赵春贵,张立伟,董建华,等.肉桂酸及其衍生物对活性氧H202清除作用[J].化学研究与应用,2004,(5)：685-687.
[32]Schmeichel AM, Schmelzer JD, Low PA. Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy [J]. Diabetes,2003,52(1):165-71.
[33]Cameron NE, Cotter MA. Effects of protein kinase C beta inhibition on neurovascular dysfunction in diabetic rats:interaction with oxidative stress and essential fatty acid dysmetabolism[J]. Diatetes Metab Res Rev,2002,18(4):315-323.
[34]ObrosovaIG, Huysen CV, Fathallah L, et al. Evalution of a-adrenoceptor antagonist on diabetes induced changes in peripheral nerve function, metabolism, and antioxidative defence [J]. FASEB J,2000,14(11):1548-1558.
[35]Kandhare AD, Raygude KS, Ghosh P, et al.Neuroprotective effect of naringin by modulation of endogenous biomarkers in streptozotocin induced painful diabetic neuropathy [J]. Fitoterapia,2012 Feb 9. [Epub ahead of print]
[36]Kaeidi A, Esmaeili-Mahani S, Abbasnejad M, et al.Satureja khuzestanica attenuates apoptosis in hyperglycemic PC12 cells and spinal cord of diabetic rats[J].J Nat Med,2012,Mar2. [Epub ahead of print]
[37]沈应君主编.中药药理学[M].北京：人民卫生出版社,2000：96-97.
[38]张瑞明,李延谦.中药新药期临床研究有关问题的探讨[J].中药新药与临床药理,2004,15：66-67.
[39]Stevens MJ, Obrosova I, Cao X, et al. Effects of DL-alpha-lipoic acid on peripheral nerve conduction, blood flow, energy metabolism, and oxidative stress in experimental diabetic neuropathy[J].Diabetes,2000,49(6):1006-1015.
[40]Hounsom L, Horrobin DF, Tritschler H, et al. Lipoic acid-gamma linolenic acid conjugate is effective against multiple indices of experimental diabetic neuropathy[J].Diabetologia,1998,41(7):839-843.
[41]Ratnam DV, Ankola DD, Bhardwaj V, et al. Role of antioxidants in prophylaxis and therapy:A pharmaceutical perspective[J]. J Control Release,2006,113(3):189-207.
[42]Takahashi N, Morimoto S, Okigaki M, et al.Decreased plasma level of vitamin C in chronic kidney disease:comparison between diabetic and non-diabetic patients[J].Nephrol Dial Transplant,2011,26(4):1252-1257.
[43]Odermarsky M, Lykkesfeldt J, Liuba P.Poor vitamin C status is associated with increased carotid intima-media thickness, decreased microvascular function, and delayed myocardial repolarization in young patients with type 1 diabetes[J].Am J Clin Nutr,2009,90(2):447-452.
[44]Sadi G, Yilmaz O, G u ray T. Effect of vitamin C and lipoic acid on streptozotocin-induced diabetes gene expression:mRNA and protein expressions of Cu-Zn SOD and catalase[J].Mol Cell Biochem,2008,309(1-2):109-116.
[45]Lee EY, Lee MY, Hong SW, et al.Blockade of oxidative stress by vitamin C ameliorates albuminuria and renal sclerosis in experimental diabetic rats[J]. Yonsei Med J,2007,48(5):847-855.
[46]Lindroos OF. Short Nissl staining for incubated cryostat sections of the brain[J].Biotech Histochem,1991,66(4):208-209.
[47]Kiernan JA, Macpherson CM, Price A, et al.A histochemical examination of the staining of kainate-induced neuronal degeneration by anionic dyes[J].Biotech Histochem,1998,73(5):244-254.
[48]顾兵,金建波,李华南,等.神经组织染色方法的研究概况[J].中国药理学通报,2011,27(10)：1472-1475.
[49]McHugh JM, McHugh WB.Diabetes and peripheral sensory neurons:what we don't know and how it can hurt us[J].AACN Clin Issues,2004,5(1):136-149.
[50]Leusen JH, Verhoeven AJ, Roos D. Interacitons between the components of the human NADPH oxidase:Intruges in the phox family[J].J Lab Clin Med, 1996,128(5):461-476.
[51]Dohi K, Ohtaki H, Nakamachi T, et al. Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury[J]. Journal of Neuroinflammation, 2010,7(1):41.
[52]Xian Cao, Demel SL, Quinn MT, et al. Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and peri vascular nerve fibers[J].Auton Neurosci,2009,151(2):90-97.
[53]El-Benna J, Dang PM, Gougerot-Pocidalo MA, et al. P47phox, the phagocyte NADPH oxidase/NOX2 organizer:structure, phosphorylation and implication in diseases[J].Exp Mol Med,2009,41(4):217-225.
[54]Nauseef WM. Assembly of the phagocyte NADPH oxidase[J].Histochem Cell Biol,2004,122(4):277-291.
[55]Kim H, Park JS, Choi YJ, et al.Bone marrow mononuclear cells have neurovascular tropism and improve diabetic neuropathy[J].Stem Cells,2009,27(7):1686-1696.
[56]Ibi M,Matsuno K,Shiba D, et al. Reactive oxygen species derived from NOX1/NADPH oxidase enhance inflammatory pain[J]. J Neurosci,2008,28(38): 9486-9494.
[57]Cotter MA, Cameron NE.Effect of the NAD(P)H oxidase inhibitor, apocynin, on peripheral nerve perfusion and function in diabetic rats[J].Life Sci,2003,73(14): 1813-1824.
[58]Vincent AM, Perrone L, Sullivan KA, et al.Receptor for advanced glycation end products activation injures primary sensory neurons via oxidative stress[J]. Endocrinology,2007,148(2):548-558.
[59]Inoguchi T, Li P, Umeda F, et al. High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells[J].Diabetes,2000,49(11):1939-1945.
[60]San Martin A, Foncea R, Laurindo FR, et al. Noxl-based NADPH oxidase-derived superoxide is required for VSMC activation by advanced glycation end-products[J].Free Radic Biol Med,2007,42(11):1671-1679.
[61]Zhang L, Pang S, Deng B, et al. High glucose induces renal mesangial cell proliferation and fibronectin expression through JNK/NF-κB/NADPH oxidase/ROS pathway, which is inhibited by resveratrol[J].Int J Biochem Cell Biol, 2012,44(4):629-638.
[62]吴其夏主编.病理生理学(第二版)[M].北京：中国协和医科大学出版社,2003：99-111.
[63]Sima AA.New insights into the metabolic and molecular basis for diabetic neuropathy [J]. Cell Mol Life Sci,2003,60(11):2445-2464.
[64]Russell JW, Golovoy D, Vincent AM, et al.High glucose-induced oxidative stress and mitochondrial dysfunction in neurons[J]. FASEBJ,2002,16(13):1738-1748.
[65]Thornberry NA, Lazebnik Y. Caspases:enemies within[J].Science,1998(281): 1312-1316.
[66]Adams JM, Cory S.The Bcl-2 protein family:arbiters of cell survival[J]. Science,1998,281(5381):1322-1326.
[67]Vincent AM, Brownlee M, Russell JW.Oxidative stress and programmed cell death in diabetic neuropathy [J]. Ann N Y Acad Sci,2002,959:368-383.
[68]Vincent AM, Russell JW, Sullivan KA, et al.SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy [J].Exp Neurol, 2007,208(2):216-227.
[69]Kamboj SS, Vasishta RK, Sandhir R.N-acetylcysteine inhibits hyperglycemia-induced oxidative stress and apoptosis markers in diabetic neuropathy[J].J Neurochem,2010,112(1):77-91.
[70]刘玉琴主编.细胞培养实验手册[M].北京：人民军医出版社,2009：93-97.
[71]梁晓春.糖尿病周围神经病变与消渴兼证“筋痹”及其中医治疗[J].中国临床医生,2006,34(5)：17-18.
[72]Russell J W, Sullivan KA, Windebank AJ, et al. Neurons undergo apoptosis in animal and cell culture models of diabetes [J]. Neurobiol Dis,1999,6(5):347-363.
[73]Hall KE, Sheng HC, Srinivasan S, et al. Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival [J]. Brain Res, 2001,888(1):128-137.
[74]Sango K, Horie H, Inoue S, et al. Age-related changes of DRG neuronal attachment to extracellular matrix proteins in vitro[J]. Neuroreport,1993,4(6):663-666.
[75]Puttfarcken PS, Manelli AM, Arneric SP, et al. Evidence for nicotinic receptors potentially modulating nociceptive transmission at the level of the primary sensory neuron:studies with F11 cells [J]. J Neurochem,1997,69(3):930-938.
[76]Chen W, Mi R, Haughey N, et al. Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line [J]. J Peripher Nerv Syst, 2007,12(2):121-130.
[77]Raymon HK, Thode S, Zhou J, et al. Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties[J].J Neurosci,1999,19(13): 5420-5428.
[78]Banker GA, Goslin K. Culturing nerve cells (2nd ed) [M]. Boston:MIT Press,1998: 545.
[79]Malin SA, Davis BM, Molliver DC. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity [J].Nat Protoc,2007,2(1):152-160.
[80]Sergery F, Arleen R. Protocols for neural cell culture(3rd ed) [M]. New Jersey:Humona Press,2001:95-97.
[81]Yong VW, Horie H, Kim SU. Comparison of six different substrata on the plating efficiency, differentiation and survival of human dorsal root ganglion neurons in culture[J]. Dev Neurosci,1988,10(4):222-230.
[82]Sango K, Horie H, Okamura A, et al. Diabetes impairs DRG neuronal attachment to extracellular matrix proteins in vitro. Brain Res Bull[J].1995,37(5):533-537.
[83]Sango K, Horie H, Inoue S. Administration of an aldose reductase inhibitor, ONO-2235, to streptozotocin-diabetic mice restores reductions of DRG neuronal attachment to extracellular matrix proteins in vitro [J].Neurosci Lett,1999, 263(2-3):157-160.
[84]Luo ZJ, King RH, Lewin J, et al. Effects of nonenzymatic glycosylation of extracellular matrix components on cell survival and sensory neurite extension in cell culture[J]. J Neurol.2002,249(4):424-431.
[85]李全波,马文庭,刘静芷,等.大鼠背根神经节神经元细胞纯化培养的模型建立[J].中国疼痛医学杂志,2011,17(8)：494-497.
[86]张军,许百男,李翀,等.胚胎大鼠背根神经节神经元的培养与纯化[J].解放军医学杂志,2006,31(12)：1152-1154.
[87]Silver J, Miller JH. Regeneration beyond the glial scar[J].Nat Rev Neurosci, 2004,5(2):146-156.
[88]Lewin GR, Ritter AM, Mendell LM.On the role of nerve growth factor in the development of myelinated nociceptors[J].J Neurosci,1992,12(5):1896-1905.
[89]Ben-Zvi A, Yagil Z, Hagalili Y, et al.Semaphorin 3 A and neurotrophins:a balance between apoptosis and survival signaling in embryonic DRG neurons[J].J Neurochem,2006,96(2):585-597.
[90]Wang H, Zhou X W, Zhang H, et al. Analgesic effect of intrathecal S03 on rats with chronic constriction injury and its regulation of intracellular calcium ion concentration of spinal dorsal root ganglion neurons[J].Chin Pharmacol Bull,2006,22(6):693-697.
[91]隋峰,杜新亮,张畅斌,等.一种原代培养大鼠DRG神经元的新方法[J].中国药理学通报,2009,25(7)：971-973.
[92]Young L, Bilsland J, Harper S.A rapid method for determination of cell survival in primary neuronal DRG cultures[J].J Neurosci Methods,1999,30,93(1):81-89.
[93]Terashima T, Kojima H, Fujimiya M, et al.The fusion of bone-marrow-derived proinsulin-expressing cells with nerve cells underlies diabetic neuropathy[J].Proc Natl Acad Sci USA,2005,102(35):12525-12530.
[94]王丽琴,宋学琴,王晓娟,等.大鼠背根神经节细胞的纯化培养[J].脑与神经疾病杂志,2003,11(5)：265-267.
[95]Andersen PL, Doucette JR, Nazarali AJ.A novel method of eliminating non-neuronal proliferating cells from cultures of mouse dorsal root ganglia[J]. Cell Mol Neurobiol,2003,23(2):205-210.
[96]Alcazar A, Cid C.High cytotoxic sensitivity of the oligodendrocyte precursor cells to HSP90 inhibitors in cell cultures[J].Exp Neurol,2009,216(2):511-514.
[97]章静波主编.组织和细胞培养技术[M].北京：人民卫生出版社,2002：105-106.
[98]Mosmann T. Rapid col orimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays[J]. Immunol Methods,1983,65(1-2):55-63.
[99]Vincent AM, Stevens MJ, Backus C, et al. Cell culture modeling to test therapies against hyperglycemia-mediated oxidative stress and injury[J]. Antioxid Redox Signal,2005,7(11-12):1494-1506.
[100]Vincent AM, McLean LL, Backus C, et al. Short-term hyperglycemia produces oxidative damage and apoptosis in neurons[J].FASEB J,2005,19(6):638-640.
[101]Iwama H, Amagaya S, Ogihara Y. Effect of shosaikoto, a Japanese and Chinese traditional herbal medicinal mixture, on the mitogenic activity of lipopolysaccharide:a new pharmacological testing method[J].J Ethnopharmacol,1987,21 (1):45-53.
[102]黄臣虎,陆茵,高骁君,等.中药血清药理学研究进展[J].中国实验方剂学杂志,2011,17(10)：266-271.
[103]杨彦芳,王玉芹.中药复方血清药理学方法规范化探讨[J].中国中西医结合杂志,2000,20(5)：280-282.
[104]李仪奎.中药血清药理学实验方法的若干问题[J].中药新药与临床药理,1999,10(2)：95.
[105]罗焕敏.血清药理学与血浆药理学[J].中国药理学通报,2003,19(9)：1075-1076.
[106]Vincent AM, Russell JW, Low P, et al. Oxidative stress in the pathogenesis of diabetic neuropathy [J]. Endocr Rev,2004,25(4):612-628.
[107]Zherebitskaya E, Akude E, Smith DR, et al. Development of selective axonopathy in adult sensory neurons isolated from diabetic rats:role of glucose-induced oxidative stress[J]. Diabetes,2009,58(6):1356-1364.
[108]Zhao H, Kalivendi S, Zhang H, et al. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium:potential implications in intracellular fluorescence detection of superoxide [J]. Free Radic Biol Med,2003,34:1359-1368.
[109]Allen DA, Yaqoob MM, Harwood SM. Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications [J]. J Nutr Biochem, 2005,16(12):705-713.
[110]Cellerino A, Bahr M, Isenmann S. Apoptosis in the developing visual system[J]. Cell Tissue Res,2000,301(1):53-69.
[111]Graham SH, Chen J, Clark RS. Bcl-2 family gene products in cerebral ischemia and traumatic brain injury[J]. J Neurotrauma,2000,17(10):831-841.
[112]Srinivasan S, Stevens M, Wiley JW. Diabetic peripheral neuropathy:evidence for apoptosis and associated mitochondrial dysfunction[J].Diabetes,2000,49 (11):1932-1938.
[113]Honma H, Podratz JL, Windebank AJ. Acute glucose deprivation leads to apoptosis in a cell model of acute diabetic neuropathy[J].J Peripher Nerv Syst, 2003,8(2):65-74.
[114]冯健.齐墩果酸对大鼠血管平滑肌细胞中血红素氧合酶1表达的影响及其作用机制的研究[D].重庆：第三军医大学,2011.
[115]顾锦华,黄华,薛华,等.齐墩果酸对糖尿病小鼠胰岛损伤的保护作用[J].中草药,2010,41(11)：1866-1870.
[116]张兰兰,曹琼,张鹏,等.槲皮素对氧化应激大鼠星形胶质细胞的保护作用[J].中华神经医学杂志,2009,8(1)：10-12.
[117]毕伟,朱丽红,王传明,等.槲皮素抑制鱼藤酮诱导的PC12细胞凋亡[J].中国病理生理杂志,2011,27(1)：82-85.
[118]张明发,沈雅琴.红景天苷的神经保护作用[J].国际脑血管病杂志,2011,19(2)：137-141.
[119]Zhang L,Yu H,Zhao X, et al. Neuroprotective effects of salidroside against (3-anyloid-induced oxidative stress in SH-SY5Y human neuroblastoma cells2010
[120]裴晶晶,吴润,赵红斌,等.Ca2+信号介导红景天苷促进小鼠骨髓间充质干细胞向神经细胞的定向分化[J].中国组织工程研究与临床康复,2010,14(10)：1808-1812.
[121]吴春,陈林林.菟丝子黄酮体外清除自由基活性的研究[J].天然产物研究与开发,2005,17(5)：553-556.
[122]王展,方积年.具有抗氧化活性的酸性菟丝子多糖H2的研究[J].植物学报,2001,43(3)：243-248.
[123]魏晓东,刘玉萍,李晶,等.D-半乳糖致衰大鼠非酶糖基化改变及菟丝子醇提液对其作用的研究[J].中国老年学杂志,2009,29(19)：2494-2496.
[124]周中和,曲方,何祥.局部应用重组水蛭素对大鼠脑出血神经细胞凋亡的干预作用[J].临床神经病学杂志,2006,19(4)：304-306.
[125]张尉华,饶明俐,吴江,等.水蛭素对大鼠实验性脑出血神经组织的保护作用[J].中风与神经疾病杂志,2006,23(1)：44-45.
[126]杨霞,黄萍,肖晓蓉,等.中药桂皮醛对牙周致病菌的体外抑菌活性研究[J].重庆医学,2009,38(1)：75-76.
[127]贾蓓蓓,邵世和,高小焕,等.桂皮醛对大鼠体内耐甲氧西林金黄色葡萄球菌生物膜的清除效应[J].江苏大学学报(医学版),2011,21(3)：199-202,207,封3.
[128]李澎,任钧国,段昌令等.4种延胡索成分对乳鼠心肌细胞缺氧和过氧化损伤的影响[J].中国中药杂志,2010,35(1)：84-87.
[129]俞静,花宝金,朴炳奎,等.延胡索乙素对放射所致血管内皮细胞损伤的保护作用[J].中华肿瘤防治杂志,2011,18(8)：584-587,601.
[130]樊景坡.苍耳子、细辛、枸杞、白术对小鼠组织自由基代谢的影响[J].中医药信息,1994,2(1)：48.
[131]栗坤,郑景禄,白晶,等.细辛、杜仲及其合剂对D-半乳糖所致衰老大鼠NO、NOS和CAT的影响[J].中国老年杂志,2001,21(2)：132.
[1]Russell JW, Sullivan KA, Windebank AJ, et al. Neurons undergo apoptosis in animal and cell culture models of diabetes[J]. Neurobiol Dis,1999,6(5):347-363.
[2]Hall KE, Sheng HC, Srinivasan S, et al. Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival[J]. Brain Res, 2001,888(1):128-137.
[3]Sango K, Horie H, Inoue S, et al. Age-related changes of DRG neuronal attachment to extracellular matrix proteins in vitro[J].Neuroreport,l 993,4(6):663-666.
[4]Puttfarcken PS, Manelli AM, Arneric SP, et al. Evidence for nicotinic receptors potentially modulating nociceptive transmission at the level of the primary sensory neuron:studies with F11 cells[J]. J Neurochem,1997,69(3):930-938.
[5]Chen W, Mi R, Haughey N, et al. Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line[J].J Peripher Nerv Syst, 2007,12(2):121-130.
[6]Chen W, Mi R, Haughey N, et al. Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line[J].J Peripher Nerv Syst, 2007,12(2):121-130.
[7]禹晓东,罗卓荆,张琳,等.大鼠脊髓背根神经节神经元的纯化培养[J].中国脊柱脊髓杂志,2005,15(10)：591-594.
[8]王丽琴,宋学琴,王晓娟,等.胚胎大鼠背根神经节细胞的分离培养、纯化及生物学特性的研究[J].细胞生物学杂志,2003,25(5)：320-323.
[9]Russell JW, Golovoy D, Vincent AM, et al. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons[J]. FASEB J,2002,16(13):1738-1748.
[10]Vincent AM, Stevens MJ, Backus C, et al. Cell culture modeling to test therapies against hyperglycemia-mediated oxidative stress and injury [J]. Antioxid Redox Signal,2005,7(11-12):1494-1506.
[11]Srinivasan S, Stevens M, Wiley JW. Diabetic peripheral neuropathy:evidence for apoptosis and associated mitochondrial dysfunction[J].Diabetes,2000,49(11): 1932-1938.
[12]Vincent AM, McLean LL, Backus C, et al. Short-term hyperglycemia produces oxidative damage and apoptosis in neurons[J]. FASEB J,2005,19(6):638-640.
[13]Honma H, Podratz JL, Windebank AJ. Acute glucose deprivation leads to apoptosis in a cell model of acute diabetic neuropathy[J].J Peripher Nerv Syst, 2003,8(2):65-74.
[14]Ozturk G, Erdogan E, Ozturk M, et al. Differential analysis of effect of high glucose level in the development of neuropathy in a tissue culture model of diabetes mellitus: role of hyperosmolality[J].Exp Clin Endocrinol Diabetes,2008,116(10):582-591.
[15]Vincent AM, Russell JW, Low P,et al. Oxidative stress in the pathogenesis of diabetic neuropathy [J]. Endocr Rev,2004,25(4):612-628.
[16]Vincent AM, Russell JW, Sullivan KA, et al. SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy [J].Exp Neurol, 2007,208(2):216-227.
[17]Zherebitskaya E, Akude E, Smith DR, et al. Development of selective axonopathy in adult sensory neurons isolated from diabetic rats:role of glucose-induced oxidative stress[J]. Diabetes,2009,58(6):1356-1364.
[18]Vincent AM, Olzmann JA, Brownlee M, et al. Uncoupling proteins prevent glucose-induced neuronal oxidative stress and programmed cell death[J]. Diabetes, 2004,53(3):726-734.
[19]Spillson AB, Russell JW. Metabotropic glutamate receptor regulation of neuronal cell death[J]. Exp Neurol,2003,184 Suppl 1:S97-105.
[20]Berent-Spillson A, Russell JW. Metabotropic glutamate receptor 3 protects neurons from glucose-induced oxidative injury by increasing intracellular glutathione concentration[J]. J Neurochem,2007,101(2):342-354.
[21]Berent-Spillson A, Robinson AM, Golovoy D, et al. Protection against glucose-induced neuronal death by NAAG and GCP Ⅱ inhibition is regulated by mGluR3[J]. J Neurochem,2004,89(1):90-99.
[22]Kim SR, Lee DY, Chung ES, et al. Transient receptor potential vanilloid subtype 1 mediates cell death of mesencephalic dopaminergic neurons in vivo and in vitro[J]. J Neurosci,2005,25(3):662-671.
[23]Hong S, Wiley JW. Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1 [J]. J Biol Chem,2005,280(1):618-627.
[24]Hong S, Agresta L, Guo C, et al. The TRPV1 receptor is associated with preferential stress in large dorsal root ganglion neurons in early diabetic sensory neuropathy [J]. J Neurochem,2008,105(4):1212-1222.
[25]Gumy LF, Bampton ET, Tolkovsky AM. Hyperglycaemia inhibits Schwann cell proliferation and migration and restricts regeneration of axons and Schwann cells from adult murine DRG[J]. Mol Cell Neurosci,2008,37(2):298-311.
[26]Tosaki T, Kamiya H, Yasuda Y, et al. Reduced NGF secretion by Schwann cells under the high glucose condition decreases neurite outgrowth of DRG neurons[J]. Exp Neurol,2008,213(2):381-387.
[27]Yu C, Rouen S, Dobrowsky RT. Hyperglycemia and downregulation of caveolin-1 enhance neuregulin-induced demyelination[J].Glia,2008,56(8):877-887.
[28]Yong VW, Horie H, Kim SU. Comparison of six different substrata on the plating efficiency, differentiation and survival of human dorsal root ganglion neurons in culture[J].Dev Neurosci,1988,10(4):222-230.
[29]Sango K, Horie H, Okamura A, et al. Diabetes impairs DRG neuronal attachment to extracellular matrix proteins in vitro[J].Brain Res Bull,1995,37(5):533-537.
[30]Sango K, Horie H, Inoue S. Administration of an aldose reductase inhibitor, ONO-2235, to streptozotocin-diabetic mice restores reductions of DRG neuronal attachment to extracellular matrix proteins in vitro [J].Neurosci Lett,1999, 263(2-3):157-160.
[31]Luo ZJ, King RH, Lewin J, et al. Effects of nonenzymatic glycosylation of extracellular matrix components on cell survival and sensory neurite extension in cell culture[J]. J Neurol,2002,249(4):424-431.
[32]Gardiner NJ, Wang Z, Luke C, et al. Expression of hexokinase isoforms in the dorsal root ganglion of the adult rat and effect of experimental diabetes[J]. Brain Res,2007,1175:143-154.
[33]Kozlova EN, Jansson L. In vitro interactions between insulin-producing beta cells and embryonic dorsal root ganglia[J]. Pancreas,2005,31(4):380-384.
[34]Craner MJ, Klein JP, Black JA, et al. Preferential expression of IGF-I in small DRG neurons and down-regulation following injury[J].Neuroreport,2002,13(13): 1649-1652.
[35]Edwards JL, Quattrini A, Lentz SI, et al. Diabetes regulates mitochondrial biogenesis and fission in mouse neurons[J]. Diabetologia,2010,53(1):160-169.
[1]Edwards JL, Vincent AM, Cheng HT, et al. Diabetic neuropathy:mechanisms to management [J].Pharmacol Ther,2008,120(1):1-34.
[2]Brownlee M. Biochemistry and molecular cell biology of diabetic complications [J]. Nature,2001,414(5):813-820.
[3]Vincent AM, Callaghan BC, Smith AL, et al.Diabetic neuropathy:cellular mechanisms as therapeutic targets[J].Nat Rev Neurol,2011,7(10):573-583.
[4]Baluchnejadmojarad T, Roghani M, Khastehkhodaie Z.Chronic treatment of silymarin improves hyperalgesia and motor nerve conduction velocity in diabetic neuropathic rat[J].Phytother Res,2010,24(8):1120-1125.
[5]Kumar A, Kaundal RK, Iyer S, et al.Effects of resveratrol on nerve functions, oxidative stress and DNA fragmentation in experimental diabetic neuropathy [J]. Life Sci,2007,80(13):1236-1244.
[6]Watcho P, Stavniichuk R, Ribnicky DM, et al.High-fat diet-induced neuropathy of prediabetes and obesity:effect of PMI-5011, an ethanolic extract of Artemisia dracunculus L[J].Mediators Inflamm,2010:268547. Epub 2010 Apr 8.
[7]郝伟欣,贾力,徐惠媛,等.筋脉通对大鼠坐骨神经传导速度及红细胞抗氧化作用的影响[J].中国新药杂志,2003,12(5)：343-345.
[8]李伯武,梁晓春,王普艳,等.筋脉通对糖尿病大鼠坐骨神经NADPH氧化酶p22-phox亚基表达的影响[J].医学研究杂志,2011,40(5)：43-46.
[9]赵丽,梁晓春,张宏,等.筋脉通含药血清对高糖培养的Schwann细胞诱导型一氧化氮合酶和NADPH氧化酶p22-phox亚基表达的影响[J].中西医结合学报,2011,9(3)：299-305.
[10]张德刚,赵瑛,夏培金,等.通络方剂改善糖尿病大鼠周围神经病变作用机制的探讨[J].中西医结合学报,2006,4(6)：601-605.
[11]Russell JW, Golovoy D, Vincent AM, et al.High glucose-induced oxidative stress and mitochondrial dysfunction in neurons[J]. FASEBJ,2002,16(13):1738-1748.
[12]Sun LQ, Xue B, Li XJ, et al.Inhibitory effects of salvianolic acid B on apoptosis of Schwann cells and its mechanism induced by intermittent high glucose[J].Life Sci,2012,90(3-4):99-108.
[13]王普艳.筋脉通对糖尿病大鼠坐骨神经及雪旺细胞氧化应激致细胞凋亡的影响[D].北京：北京协和医学院,2009.
[14]Wu LL, Chiou CC, Chang PY, et al. Urinary 8-OHDG:a marker of oxidative stess to DNA and a risk factor for cancer. atherselerosis and diabetics[J]. Clin Chim Acta,2004,339(6):1-9.
[15]Obrosova IG, Li F, Abatan OI, et al. Role of poly(ADP-ribose) polymerase activation in diabetic neuropathy[J].Diabetes,2004,53(3):711-720.
[16]朴元林,梁晓春,赵丽,等.筋脉通含药血清对高糖培养施万细胞8-羟基脱氧鸟苷和活化的caspase-3表达的影响[J].医学研究杂志,2011,40(10)：35-39.
[17]Sheftel AD, Kim SF, Ponka P. Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism[J]. J Biol Chem,2007,282(14):10480-10486.
[18]Quan S, Kaminski PM, Yang L, et al. Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats[J]. Biochem Biophys Res Common,2004; 315:509-516.
[19]Negi G, Kumar A, Sharma SS. Nrf2 and NF-κB modulation by sulforaphane counteracts multiple manifestations of diabetic neuropathy in rats and high glucose-induced changes[J].Curr Neurovasc Res,2011,8(4):294-304.
[20]Romeo L, Intrieri M, D'Agata V, et al.The major green tea polyphenol, (-)-epigallocatechin-3-gallate, induces heme oxygenase in rat neurons and acts as an effective neuroprotective agent against oxidative stress[J].J Am Coll Nutr,2009,28 Suppl:492S-499S.
[21]Huang SM, Hsu CL, Chuang HC, et al.Inhibitory effect of vanillic acid on methylglyoxal-mediated glycation in apoptotic Neuro-2A cells.Neurotoxicology [J].2008,29(6):1016-1022.
[22]李玉红,陈泽奇,叶仁群,等.加味补肝汤对糖尿病周围神经病变大鼠背根节p38丝裂素活化蛋白激酶表达的干预作用[J].中国临床康复,2006,10(7)：67-69.
[23]叶仁群,陈泽奇,熊丽丽,等.加味补肝汤对糖尿病大鼠背根神经节细胞凋亡的 影响[J].湖南中医药大学学报,2008,28(5)：44-47.
[24]黄娟,陈泽奇,熊丽丽,等.加味补肝汤对糖尿病大鼠坐骨神经中JNK丝裂原活化蛋白激酶表达的影响[J].中国医师杂志,2009,11(2)：156-159,169.