【作者】 孙青；

【导师】 梁晓春；

【作者基本信息】 北京协和医学院 ， 中西医结合临床， 2012， 博士

【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。更多还原

【Abstract】 [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 experiment①Male 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 experiment①Blood 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 experiment①MTT 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 experiment①A 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 effectiveness④These 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 experiment①The 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.更多还原