【作者】 王普艳；

【导师】 梁晓春； 孙华； 田国庆；

【作者基本信息】 中国协和医科大学 ， 中西医结合临床， 2010， 博士

【摘要】 [目的]从整体、细胞及分子水平,研究中药筋脉通对糖尿病大鼠坐骨神经氧化应激及细胞凋亡的作用,以及筋脉通含药血清对高糖培养雪旺细胞氧化应激及其凋亡的影响。[方法]1.整体实验将SD雄性大鼠随机分为正常组和造模组；用链脲佐菌素(STZ,60mg/kg)一次性腹腔内注射SD大鼠的方法制作糖尿病模型。糖尿病模型成功后再随机分为模型组、筋脉通小、中、大剂量组和维生素C组,每组14只。筋脉通小、中、大组分别按成人剂量5倍、10倍和20倍给药；VC组按成人剂量10倍给药。模型组和正常组予灌服蒸馏水。所有实验大鼠灌胃16w处死。检测各组治疗前及治疗后4w、8w、12w和16w的体重和血糖变化；处死前测定大鼠的机械痛阈值；应用免疫组化法和实时荧光定量PCR法检测大鼠坐骨神经NADPH氧化酶phox p22亚基、iNOS、Bcl-2及Caspase-3的表达。2.细胞实验体外培养雪旺细胞,S-100蛋白进行鉴定。取第3代雪旺细胞,加入DMEM、50mmolGlu培养液以及含50mmolGlu的筋脉通(JMT)和维生素C(VC)含药血清进行培养；采用TUNEL法测定各组雪旺细胞凋亡的情况；应用激光扫描共聚焦显微技术和实时荧光定量PCR法检测NADPH氧化酶phox p22亚基、iNOS、Bcl-2、Caspase-3表达水平的变化。[结果]1.整体实验(1)血糖和体重造模前各组体重无明显差异(P>0.05)。造模成功后,模型组及各治疗组大鼠血糖显著高于正常组(P〈0.01)；模型组及治疗组间比较,大鼠4周、8周、12周、16周血糖均无明显差异(P>0.05)。模型组及各治疗组大鼠4周、8周、12周、16周体重显著低于正常组(P<0.01)；模型组及治疗组间比较,大鼠4周、8周、12周、16周体重均无明显差异(P>0.05)。(2)机械痛阈值检测：与正常组相比,模型组、筋脉通大、小剂量组、VC组机械痛阈值显著降低(P〈0.01)；筋脉通中剂量组无明显降低(P>0.05)。与模型组相比,各治疗组的痛阈值显著升高(P〈0.01)。与维生素C组相比,筋脉通中剂量组痛阈值有显著升高(P〈0.01)。筋脉通各剂量组间比较,筋脉通中剂量组痛阈值升高最明显(P<0.01)。(3)坐骨神经NADPH氧化酶phox p22亚基表达模型组p22亚基的积分光密度值较正常组显著升高(P<0.01)。各治疗组积分光密度值较模型组均有显著下降(P<0.01)。筋脉通中剂量组积分光密度值明显低于筋脉通小剂量组和维生素C组(P〈0.05,P<0.01)。(4)坐骨神经NADPH氧化酶phox p22亚基mRNA表达模型组p22亚基mRNA值较正常组显著升高(P<0.01)。与模型组相比,筋脉通大、中、小剂量组的mRNA值均有明显下降(P〈0.01,P<0.01,P〈0.05),维生素C组下降不明显(P>0.05)。与维生素C组相比,筋脉通小、中剂量组的p22亚基mRNA值明显下调(P〈0.05,P<0.01)。(5)坐骨神经iNOS表达模型组iNOS积分光密度值较正常组显著升高(P<0.01)。各治疗组积分光密度值较模型组均有显著降低(P<0.01)。筋脉通中剂量组的积分光密度值明显低于筋脉通小剂量组和维生素C组(P<0.01)。(6)坐骨神经Bcl-2表达模型组和各治疗组的Bcl-2积分光密度值较正常组显著下降(P<0.01)。筋脉通小、中剂量组积分光密度值较模型组均有显著增高(P<0.01)。筋脉通中剂量组的积分光密度值明显高于维生素C组(P<0.01)。(7)坐骨神经Bcl-2 mRNA表达模型组的Bcl-2 mRNA值均较正常组显著降低(P<0.01)。与模型组相比,筋脉通大、中、小剂量组的Bcl-2 mRNA值均有明显上调(P<0.01),维生素C组上调不显著(P>0.05)。与维生素C组相比,筋脉通大、中、小剂量组的Bcl-2mRNA值明显增高(P<0.01)。(8)坐骨神经Caspase-3表达模型组的Caspase-3积分光密度值较正常组显著增高(P<0.01)。筋脉通中、大剂量组积分光密度值较模型组均有显著降低(P<0.01,P<0.05)。筋脉通中剂量组的积分光密度值明显低于筋脉通小剂量组和维生素C组(P<0.05,P<0.01)。(9)坐骨神经Caspase-3 mRNA表达模型组的Caspase-3 mRNA值较正常组显著升高(P<0.01)。与模型组相比,筋脉通大、中、小剂量组mRNA值均有明显下调(P<0.01,P<0.01,P<0.05)。与维生素C组相比,筋脉通中剂量组的Caspase-3 mRNA值明显下降(P<0.05)。2.细胞实验(1) NADPH氧化酶phox p22亚基mRNA表达①Glu组雪旺细胞p22亚基mRNA表达水平较正常组明显升高(P<0.01)。②与Glu组相比,JMT组雪旺细胞mRNA表达水平明显降低(P<0.01)。③JMT组雪旺细胞p22亚基mRNA表达水平显著低于VC组(P<0.01)。(2)SC中iNOS表达①Glu组及治疗组雪旺细胞iNOS的荧光强度值较正常组明显升高(P<0.01)。②与Glu组相比,JMT组与VC组雪旺细胞iNOS的荧光强度值明显降低(P<0.01)。③JMT组的荧光强度值要明显低于VC组(P<0.01)。(3)SC中Bcl-2表达①Glu组雪旺细胞Bcl-2的荧光强度值较正常组明显降低(P<0.01)。②与Glu组相比,JMT组雪旺细胞Bcl-2的荧光强度值明显升高(P<0.01),VC组升高不明显(P>0.05)。③JMT组Bcl-2mRNA值显著高于VC组(P<0.01)。(4) Bcl-2 mRNA表达①Glu组雪旺细胞Bcl-2 mRNA表达水平较正常组明显降低(P<0.01)。②与Glu组相比,JMT组与VC组雪旺细胞Bcl-2-mRNA表达水平均增高(P<0.01)。③JMT组雪旺细胞Bcl-2 mRNA表达水平高于VC组(P<0.05)。(5)SC中Caspase-3表达①Glu组雪旺细胞Caspase-3的荧光强度值较正常组明显升高(P<0.01)。②与Glu组相比,JMT组与VC组雪旺细胞Caspase-3的荧光强度值明显降低(P<0.01)。③JMT组的荧光强度值要明显低于VC组(P<0.01)。(6) Caspase-3 mRNA表达①Glu组雪旺细胞Caspase-3mRNA表达水平较正常组明显升高(P<0.01)。②与Glu组相比,JMT组与VC组雪旺细胞Caspase-3 mRNA表达水平明显降低(P<0.01)。③JMT组雪旺细胞Caspase-3 mRNA表达水平低于VC组(P<0.01)。(7)雪旺细胞凋亡百分率①正常组未见雪旺细胞凋亡。②Glu组及其各治疗组雪旺细胞凋亡明显增加(P<0.01)。③与Glu相比,JMT组雪旺细胞的凋亡百分率明显降低(P<0.01),VC组降低不明显(P>0.05)。④JMT组雪旺细胞的凋亡百分率明显低于VC组(P<0.01)。[结论]1.整体实验(1)空腹单次腹腔注射STZ 60mg/kg后16周DPN模型建立,检测DM组大鼠机械痛阈值显著降低,证实DPN模型成功；筋脉通干预后显著提高DM大鼠的机械痛阈值。(2)DM大鼠坐骨神经NADPH氧化酶phox p22亚基、iNOS表达显著升高。(3)筋脉通能显著降低DM大鼠坐骨神经NADPH氧化酶p22亚基和iNOS的表达,优于维生素C。(4)DM大鼠坐骨神经Bcl-2表达降低,Caspase-3表达显著升高。(5)筋脉通干预后,坐骨神经Bcl-2表达明显升高,Caspase-3表达显著降低,优于维生素C。2.细胞实验(1)采用组织贴块法原代培养,经差速贴壁法+低浓度胰蛋白酶消化法+G418纯化的Wistar乳鼠坐骨神经雪旺细胞,并经S-100蛋白免疫组化鉴定,纯度可达90%左右,成功建立SC原代模型。(2)高糖培养环境下雪旺细胞NADPH氧化酶p22亚基和iNOS表达增高。(3)高糖环境培养下SC凋亡百分率明显增加,Bc卜2表达降低,而Casase-3表达显著升高。(4)筋脉通含药血清显著降低NADPH氧化酶p22亚基和iNOS的表达,优于维生素C。(5)筋脉通含药血清显著降低SC的凋亡百分率,并显著提高Bcl-2的表达,降低Caspase-3的表达,优于维生素C。[创新点]从整体、细胞及分子水平,研究中药筋脉通对糖尿病大鼠坐骨神经氧化应激及细胞凋亡的作用,以及筋脉通含药血清对高糖培养雪旺细胞氧化应激及其凋亡的影响,经检索国内外文献未见报道。更多还原

【Abstract】 [Objective]To study the effects of Jinmaitong Capsule (JMT) on cell apoptosis and oxidative stress in sciatic nerves of STZ-DM rats, as well as to study the effects of medicated serum of JMT on apoptosis and oxidative stress of Schwann Cells cultured in high glucose from the aspects of integral level, cellular level and molecular level.[Methods]1. In vivo experimentAll Sprague Dawley (SD) rats were randomly divided into normal group and "diabetic" group. "Diabetic" rats were induced by STZ which were randomly divided into model group, low-dose JMT group (treated with JMT similar to the quintupling dose of adult recommended dosage), middle-dose JMT group (similar to the decuple dose of adult recommended dosage), high-dose JMT group (similar to the twenty-fold dose of adult recommended dosage) and Vitamin C group (similar to the decuple dose of adult recommended dosage). Normal group included ten rats, other group included fourteen diabetic rats. All rats were given intragastric administration for 16 weeks (the normal and model groups were treated with distilled water) and then killed. Body weight and blood glucose were detected before and at the 4th,8th,12th,16th week after treatment. The pain threshold to mechanical stimulation with Von Frey filament were carried out before death. The expression of NADPH oxidase phox p22 subunit, iNOS, Bcl-2, Caspase-3 and their mRNAs in sciatic nerve were detected by immunohistochemical method and real-time fluorogenetic quantitative PCR respectively.2. In vitro experiment Schwann cells were cultivated and identified with S-100 protein antibody. The 3rd passage schwann cells were cultured respectively in following conditions including DMEM, high glucose(50mmol) media supplemented with 20% rat serum,50mmol glucose media containing medicated serum of JMT and Vitamin C. DMEM served as negative control. Schwann cell apoptosis is detected by TUNEL kit. the expression of NADPH oxidase phox p22 subunit, iNOS, Bcl-2 and Caspase-3 in SC were detected by real-time fluorogenetic quantitative PCR and confocal laser scanning microscope respectively.[Results]1. In vivo experiment(1) Blood glucose and body weightThe blood glucose levels of STZ-DM rats were much higher than those of normal rats (P<0.01). In all the treated groups, there were no significant differences among them compared each other or compared with model group (P>0.05). And it got the same result when concerning about body weight no matter how the rats were dealt with (P>0.05).(2) Pain threshold to mechanical stimulation with Von Frey filament: Compared with normal group, the pain thresholds of model group, high-dose and low-dose JMT group and Vitamin C group decreased extremely (P<0.01) while middle-dose JMT group didn’t lower much (P>0.05). Compared with model group, the threshold values of low-dose, middle-dose, high-dose JMT group and Vitamin C group raised strikingly (P<0.01). Compared with Vitamin C group, the threshold values of middle-dose raised strikingly (P<0.01). Compared among JMT groups, the threshold values of middle-dose were much higher than other JMT groups (P<0.01).(3) NADPH oxidase phox p22 subunit expression of sciatic nerveThe integrated option density of p22 subunit expression in STZ-DM rats was much higher than the normal (P<0.01). And the levels of p22 subunit in all the treated groups decreased notably compared with model group (P <0.01). The levels of p22 subunit in middle-dose JMT group were lower than low-dose JMT group and Vitamin C group (P<0.01). (4) NADPH oxidase phox p22 subunit mRNA expression of sciatic nerveThe levels of p22 subunit mRNA expression in STZ-DM rats were much higher than those of the normal rats (P<0.01). Compared with model group, p22 subunit mRNA expression of low-dose, middle-dose and high-dose JMT groups down-regulated noticeably (P<0.01, P<0.01, P<0.05); There was no significant difference between model group and Vitamin C group (P>0.05). Compared with Vitamin C group, p22 subunit mRNA expression of low-dose and middle-dose JMT treated groups down-regulated noticeably (P<0.05, P<0.01).(5) iNOS expression of sciatic nerveThe integrated option density of iNOS expression in STZ-DM rats was much higher than that of the normal (P<0.01). And the levels of iNOS in all the treated groups decreased notably compared with model group (P <0.01). The levels of iNOS in middle-dose JMT group were higher than those of low-dose JMT group and Vitamin C group (P<0.01).(6) Bcl-2 expression of sciatic nerveThe integrated option density of Bcl-2 expression in STZ-DM rats was much lower than that of the normal (P<0.01). And the levels of Bcl-2 in low-dose and middle-dose JMT groups increased notably compared with model group (P<0.01). The levels of Bcl-2 in middle-dose JMT group were higher than those of Vitamin C group (P<0.01).(7)Bcl-2 mRNA expression of sciatic nerveThe levels of Bcl-2 mRNA expression in STZ-DM rats were much lower than those of the normal rats (P<0.01). Compared with model group, Bcl-2 mRNA expression of low-dose, middle-dose and high-dose JMT groups up-regulated noticeably (P<0.01); There was no significant difference between model group and Vitamin C group (P>0.05). Compared with Vitamin C group, the Bcl-2 mRNA expression of low-dose, middle-dose and high-dose JMT treated groups up-regulated noticeably (P<0.01).(8) Caspase-3 expression of sciatic nerve The integrated option density of Caspase-3 expression in STZ-DM rats was much higher than the normal (P<0.01). And the levels of Caspase-3 in high-dose and middle-dose JMT groups decreased notably compared with model group (P<0.05, P<0.01). The levels of Caspase-3 in middle-dose JMT group were much lower than those of lower-dose JMT group and Vitamin C group (P<0.05, P<0.01).(9) Caspase-3 mRNA expression of sciatic nerveThe levels of Caspase-3 mRNA expression in STZ-DM rats were much higher than those of the normal rats (P<0.01). Compared with model group, Caspase-3 mRNA expression of low-dose, middle-dose and high-dose JMT groups down-regulated noticeably (P<0.01, P<0.01, P<0.05); Compared with Vitamin C group, Caspase-3 mRNA expression of middle-dose JMT treated groups down-regulated noticeably (P<0.05).2. In vitro experiment(1) NADPH oxidase phox p22 subunit mRNA expression of schwann cells①The expression of p22 subunit mRNA in schwann cells cultured in high glucose condition were much higher than those of normal condition (P <0.01).②p22 subunit mRNA expression in schwann cells of JMT group down-regulated compared with high glucose group (P<0.01)③p22 subunit mRNA expression in schwann cells of JMT group was lower than that of Vitamin C group (P<0.01).(2) iNOS expression of schwann cells①The fluorescence intensities of iNOS in schwann cells cultured in high glucose condition were much higher than those of normal condition (P<0.01).②The fluorescence intensities of iNOS in schwann cells in JMT and Vitamin C groups weakened remarkably compared with high glucose group (P<0.01).③The fluorescence intensities of iNOS in schwann cells in JMT group were much lower than that of Vitamin C groups (P<0.01).(3) Bcl-2 expression of schwann cells①The fluorescence intensities of Bcl-2 in schwann cells cultured in high glucose condition were much lower than those of normal condition (P <0.01).②The fluorescence intensities of Bcl-2 in schwann cells in JMT groups reinforced remarkably compared with high glucose group (P<0.01), There were no significant differences between Vitamin C group and high glucose group (P>0.05).③The fluorescence intensities of Bcl-2 in schwann cells in JMT group were much higher than that of Vitamin C groups (P <0.01).(4)Bcl-2 mRNA expression of schwann cells①The expression of Bcl-2 mRNA in schwann cells cultured in high glucose condition were much lower than those of normal condition (P< 0.01).②Bcl-2 mRNA expression in schwann cells of JMT and Vitamin C groups up-regulated compared with high glucose group (P<0.01)③Bcl-2 mRNA expression in schwann cells of JMT group was higher than that of Vitamin C group (P<0.01).(5) Caspase-3 expression of schwann cells①The fluorescence intensities of Caspase-3 in schwann cells cultured in high glucose condition were much higher than those of normal condition (P<0.01).②The fluorescence intensities of Caspase-3 in schwann cells in JMT and Vitamin C groups weakened remarkably compared with high glucose group (P<0.01).③The fluorescence intensities of Caspase-3 in schwann cells in JMT group were much lower than that of Vitamin C groups (P<0.01).(6) Caspase-3 mRNA expression of schwann cells①The expression of Caspase-3 mRNA in schwann cells cultured in high glucose condition were much higher than those of normal condition (P <0.01).②Caspase-3 mRNA expression in schwann cells of JMT group down-regulated compared with high glucose group (P<0.01)③Caspase-3 mRNA expression in schwann cells of JMT group was lower than that of Vitamin C group (P<0.01).(7) the percent of schwann cell apoptosis①There were no cell apoptosis in schwann cells cultured in normal condition.②The percent of cell apoptosis in schwann cells cultured in high glucose condition were much higher than those of normal condition (P<0.01).③the percent of schwann cells apoptosis of JMT group down-regulated compared with high glucose group (P<0.01), but there was no significant difference between high glucose group and Vitamin C group (P>0.05).④The percent of schwann cells apoptosis of JMT group was lower than that of Vitamin C group (P<0.01).[Conclusion]1. In vivo experiment①SZT-induced diabetic rats (single intraperitoneal injection, 60mg/kg) had hyperalgia at 16w, which demonstrated the sensory nerve fibers were injured and the DPN models were established. JMT can alleviate hyperalgia strikingly.②The NADPH oxidase phox p22 subunit and iNOS expression of sciatic nerve increased significantly in DPN rats.③JMT could down-regulate the expression of NADPH oxidase phox p22 subunit and iNOS in sciatic nerve in DPN rats.④The Bcl-2 expression of sciatic nerve degressed significantly in DPN rats, while the caspase-3 expression increased.⑤JMT could up-regulate the expression of Bcl-2 and down-regulate the expression of Caspase-3 in sciatic nerve in DPN rats; the therapeutic effect of JMT was much better than Vitamin C.2. In vitro experiment①The purity coefficient of Schwann cells isolated from the sciatic nerves of newborn Wistar rats, cultivated and purified by methods of repeated explanation, differential velocity adherent technique, low density trypsin digestion and application of G418 could reach more than 90%through identification of SABC immunohistochemical method with S-100 protein antibody.②High glucose promoted the expression of NADPH oxidase phox p22 subunit and iNOS in schwann cells obviously. ③The medicated serum of JMT could decrease the level of NADPH oxidase phox p22 subunit and iNOS in schwann cells cultured in high glucose effectively. The therapeutic effect of JMT was much better than Vitamin C.④High glucose depressed the expression of Bcl-2 and promoted the expression of Caspase-3 in schwann cells obviously, and High glucose increased the percent of schwann cell apoptosis.⑤The medicated serum of JMT could increase the level of Bcl-2 and decrease the level of Caspase-3 in schwann cells cultured in high glucose effectively, it could decrease the percent of schwann cell apoptosis effectively.The therapeutic effect of JMT was much better than Vitamin C.[Innovation]To study the effects of Jinmaitong Capsule on cell apoptosis and oxidative stress in sciatic nerve of STZ-DM rats, as well as to study the effects of medicated serum of JMT on the role of schwann cell apoptosis and oxidative stress cultured in high glucose from the aspects of integral level, cellular level and molecular level, which hasn’t been reported home and abroad. This research can provide the experimental foundation for the application of JMT to clinical treatment of DPN.[Key words]Diabetic peripheral neuropathyoxidative stresscell apoptosisSchwann cellReal-time fluorogenetic quantitative PCRTraditional Chinese Medicine Jinmaitong Capsule更多还原