筋脉通对糖尿病大鼠坐骨神经及雪旺细胞睫状神经营养因子表达的影响
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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CNTF及其mRNA表达的作用,以及筋脉通含药血清对高糖培养雪旺细胞的增殖与分泌CNTF的影响。
【方法】
1.整体实验
制作糖尿病大鼠模型,随机分为模型对照组、筋脉通小剂量、中剂量、大剂量组及神经妥乐平组,每组10只。以体重、鼠龄及性别相匹配的正常大鼠10只作为正常对照组。筋脉通小、中、大组分别按成人剂量5倍、10倍和20倍给药;神经妥乐平组按成人剂量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,并检测大鼠坐骨神经CNTF及CNTF-mRNA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)和神经妥乐平(Ntp)含药血清进行培养,48h后行MTT比色试验观察雪旺细胞增殖活性的变化,采用实时荧光定量PCR法检测CNTF-mRNA的表达,以及激光扫描共聚焦显微技术检测CNTF蛋白表达水平的变化。
【结果】
1.整体实验
(1)血糖和体重
STZ-DM大鼠血糖值均显著高于正常组(P＜0.01);药物干预后,治疗组血糖在同一时间点与模型组相比无明显差异(P＞0.05);治疗组间血糖在各时间点均无明显差异(P＞0.05)。干预前后,各组大鼠的体重在同一时间点无明显差异(P＞0.05)。
(2)坐骨神经CNTF表达
模型组和各治疗组的积分光密度值较正常组显著下降(P＜0.01)。各治疗组积分光密度值较模型组均有显著增高(P＜0.01)。神经妥乐平组和筋脉通中、小剂量组的积分光密度值明显高于筋脉通大剂量组(P＜0.01)。
(3)坐骨神经CNTF-mRNA表达
模型组及各治疗组的CNTF-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、小剂量组和神经妥乐平组的CNTF-mRNA值均有明显上调(P＜0.01)。与神经妥乐平组相比,筋脉通大、中、小剂量组的CNTF-mRNA值明显增高(P＜0.01)。
2.细胞实验
(1)MTT比色实验
原代培养雪旺细胞在50mmolGlu环境培养48h后,OD值显著降低(P＜0.01)。与50mmolGlu组相比,JMT组与Ntp组的OD值显著升高(P＜0.05);JMT组及Ntp组比较OD值无统计学差异(P＞0.05)。
(2)激光扫描共聚焦显微镜检测
①50mmolGlu组及治疗组雪旺细胞CNTF的荧光强度值较正常组明显降低(P＜0.01)。②与50mmolGlu组相比,JMT组与Ntp组雪旺细胞CNTF的荧光强度值明显升高(P＜0.01)。③JMT组与Ntp组比较无显著差异(P＞0.05)。
(3)QT-PCR法检测
①50mmolGlu组及治疗组雪旺细胞CNTF-mRNA表达水平较正常组明显降低(P＜0.01)。②与50mmolGlu组相比,JMT组与Ntp组雪旺细胞CNTF-mRNA表达水平增高(P＜0.01)。③JMT组雪旺细胞CNTF-mRNA表达水平高于Ntp组(P＜0.05)。
【结论】
1.整体实验
(1)造模后16周,DPN大鼠坐骨神经CNTF及CNTF-mRNA表达水平显著下降,减弱了受损神经纤维的再生修复能力。(2)筋脉通可上调DPN大鼠坐骨神经CNTF及其mRNA的表达,提示筋脉通具有防止DPN大鼠周围神经组织进一步损伤、促进受损神经纤维再生修复的功能。(3)筋脉通减轻DPN大鼠坐骨神经损伤的作用不是通过降低血糖实现的。
2.细胞实验
(1)高糖培养环境对雪旺细胞的增殖有明显抑制作用。(2)高糖培养环境使雪旺细胞CNTF及CNTF-mRNA表达能力明显降低。(3)筋脉通含药血清可有效促进高糖培养雪旺细胞的增殖能力,并上调雪旺细胞表达CNTF及其mRNA的水平。
【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CNTF及其mRNA表达的作用,以及筋脉通含药血清对高糖培养雪旺细胞的增殖与分泌CNTF的影响,经检索国内外文献未见报道。
【Objective】
To study the effects of Jinmaitong Capsule on CNTF and its mRNA expression in sciatic nerve of STZ-DM rats,as well as to study the effects of medicated serum of JMT on the role of proliferation and elaborating CNTF of Schwann Cells cultured in high glucose from the aspects of integral level, cellular level and molecular level.
【Methods】
1.In vivo experiment
Fifty SZT-induced diabetic rats were randomly divided into 5 groups including 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 Neurotropin group(similar to the decuple dose of adult recommended dosage). Ten normal rats matching with weight,age and sex served as normal control group.All rats were given intragastric administration for 16 weeks(the normal and model groups were treated with distilledwater) and then killed. Body weight and blood glucose were detected before and at the 4th,8th,12th, 16th week after treatment.The expression of CNTF and its mRNA in sciatic nerve were detected respectively:
2.In vitro experiment
Schwann cells were cultivated and were 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 Neutrophin.DMEM served as negative control.After 48h, MTT colorimetric assay was adopted to measure schwann cells proliferation. Furthermore,CNTF and its mRNA levels were detected in schwann cells cultured in dirrerent media at 48h.
【Results】
1.In vivo experiment
(1) Blood glucose and body weight
—The 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) CNTF expression of sciatic nerve
The integrated option density of CNTF expression in STZ-DM rats was much lower than the normal(P＜0.01).And the levels of CNTF in all the treated groups increased notably compared with model group(P＜0.01).The levels of CNTF in low-dose JMT group,middle-dose JMT group and Neurotropin group were higher than high-dose JMT group(P＜0.01).
(3)CNTF-mRNA expression of sciatic nerve
The levels of CNTF-nRNA expression in STZ-DM rats were much lower than those of the normal rats(P＜0.01).Compared with model group,CNTF-mRNA expression of treated groups up-regulated noticeably(P＜0.01),middle-dose JMT group and Neurotropin group.CNTF-mRNA expression of JMT treated groups were higher than Neurotropin group(P＜0.01).
2.In vitro experiment
(1) MTT colorimetric assay
Schwann cells isolated from newborn rats primary cultured in different medium.The OD values of high glucose group diminished significantly compared with those of normal group 48h later(P＜0.01).The OD values of JMT and Neutrophin group increased greatly compared with 50mMGlu group(P＜0.05) at 48h and there were no significant differences between these two treated groups(P＞0.05).
(2)Confocal laser scanning microscope examination
①The fluorescence intensities of CNTF in schwann cells cultured in high glucose condition were much weaker than those of normal condition(P＜0.01).②The fluorescence intensities of CNTF in schwann cells in JMT and Neurotropin groups reinforced remarkably compared with high glucose group (P＜0.01).③There were no significant differences between these two treated groups(P＞0.05).
(3) QT-PCR
①The expression of CNTF-mRNA in schwann cells cultured in high glucose condition were much weaker than those of normal condition(P＜0.01).②CNTF-mRNA expression in schwann cells of JMT group and Neurotropin group up-regulated compared with high glucose group(P＜0.01)③CNTF-mRNA expression in schwann cells of JMT group was higher than that of Neurotropin group(P＜0.05).
【Conclusion】
1.In vivo experiment
①The significant degressions of CNTF and CNTF-mRNA expression of sciatic nerve in DPN rats attenuated the ability to reparative regeneration.
②JMT could up-regulate the expression of CNTF and CNTF-mRNA in sciatic nerve in DPN rats.
③JMT could not only prevent the progression of nerve dysfunction and degeneration but also to promote regeneration of degenerated nerve fibers. And its efficacy was independent of hypoglycemia.
2.In vitro experiment
①High glucose inhibited the proliferation of Schwann cells remarkably and this action was correlated with the concentration of glucose.
②High glucose depressed the expression of CNTF and its mRNA in schwann cells obviously.
③The medicated serum of JMT could promote schwann cells cultured in high glucose to proliferate and elaborate CNTF and its mRNA effectively.
【Innovation】
To study the effects of Jinmaitong Capsule on CNTF and its mRNA expression in sciatic nerve of STZ-DM rats,as well as to study the effects of medicated serum of JMT on the role of proliferation and elaborating CNTF of schwann Cells 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 basic theory and experimental foundation for the application of JMT to clinical treatment of DPN.
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CNTF及其mRNA表达的作用,以及筋脉通含药血清对高糖培养雪旺细胞的增殖与分泌CNTF的影响。
【方法】
1.整体实验
制作糖尿病大鼠模型,随机分为模型对照组、筋脉通小剂量、中剂量、大剂量组及神经妥乐平组,每组10只。以体重、鼠龄及性别相匹配的正常大鼠10只作为正常对照组。筋脉通小、中、大组分别按成人剂量5倍、10倍和20倍给药;神经妥乐平组按成人剂量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,并检测大鼠坐骨神经CNTF及CNTF-mRNA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)和神经妥乐平(Ntp)含药血清进行培养,48h后行MTT比色试验观察雪旺细胞增殖活性的变化,采用实时荧光定量PCR法检测CNTF-mRNA的表达,以及激光扫描共聚焦显微技术检测CNTF蛋白表达水平的变化。
【结果】
1.整体实验
(1)血糖和体重
STZ-DM大鼠血糖值均显著高于正常组(P＜0.01);药物干预后,治疗组血糖在同一时间点与模型组相比无明显差异(P＞0.05);治疗组间血糖在各时间点均无明显差异(P＞0.05)。干预前后,各组大鼠的体重在同一时间点无明显差异(P＞0.05)。
(2)坐骨神经CNTF表达
模型组和各治疗组的积分光密度值较正常组显著下降(P＜0.01)。各治疗组积分光密度值较模型组均有显著增高(P＜0.01)。神经妥乐平组和筋脉通中、小剂量组的积分光密度值明显高于筋脉通大剂量组(P＜0.01)。
(3)坐骨神经CNTF-mRNA表达
模型组及各治疗组的CNTF-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、小剂量组和神经妥乐平组的CNTF-mRNA值均有明显上调(P＜0.01)。与神经妥乐平组相比,筋脉通大、中、小剂量组的CNTF-mRNA值明显增高(P＜0.01)。
2.细胞实验
(1)MTT比色实验
原代培养雪旺细胞在50mmolGlu环境培养48h后,OD值显著降低(P＜0.01)。与50mmolGlu组相比,JMT组与Ntp组的OD值显著升高(P＜0.05);JMT组及Ntp组比较OD值无统计学差异(P＞0.05)。
(2)激光扫描共聚焦显微镜检测
①50mmolGlu组及治疗组雪旺细胞CNTF的荧光强度值较正常组明显降低(P＜0.01)。②与50mmolGlu组相比,JMT组与Ntp组雪旺细胞CNTF的荧光强度值明显升高(P＜0.01)。③JMT组与Ntp组比较无显著差异(P＞0.05)。
(3)QT-PCR法检测
①50mmolGlu组及治疗组雪旺细胞CNTF-mRNA表达水平较正常组明显降低(P＜0.01)。②与50mmolGlu组相比,JMT组与Ntp组雪旺细胞CNTF-mRNA表达水平增高(P＜0.01)。③JMT组雪旺细胞CNTF-mRNA表达水平高于Ntp组(P＜0.05)。
【结论】
1.整体实验
(1)造模后16周,DPN大鼠坐骨神经CNTF及CNTF-mRNA表达水平显著下降,减弱了受损神经纤维的再生修复能力。(2)筋脉通可上调DPN大鼠坐骨神经CNTF及其mRNA的表达,提示筋脉通具有防止DPN大鼠周围神经组织进一步损伤、促进受损神经纤维再生修复的功能。(3)筋脉通减轻DPN大鼠坐骨神经损伤的作用不是通过降低血糖实现的。
2.细胞实验
(1)高糖培养环境对雪旺细胞的增殖有明显抑制作用。(2)高糖培养环境使雪旺细胞CNTF及CNTF-mRNA表达能力明显降低。(3)筋脉通含药血清可有效促进高糖培养雪旺细胞的增殖能力,并上调雪旺细胞表达CNTF及其mRNA的水平。
【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CNTF及其mRNA表达的作用,以及筋脉通含药血清对高糖培养雪旺细胞的增殖与分泌CNTF的影响,经检索国内外文献未见报道。
【Objective】
To study the effects of Jinmaitong Capsule on CNTF and its mRNA expression in sciatic nerve of STZ-DM rats,as well as to study the effects of medicated serum of JMT on the role of proliferation and elaborating CNTF of Schwann Cells cultured in high glucose from the aspects of integral level, cellular level and molecular level.
【Methods】
1.In vivo experiment
Fifty SZT-induced diabetic rats were randomly divided into 5 groups including 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 Neurotropin group(similar to the decuple dose of adult recommended dosage). Ten normal rats matching with weight,age and sex served as normal control group.All rats were given intragastric administration for 16 weeks(the normal and model groups were treated with distilledwater) and then killed. Body weight and blood glucose were detected before and at the 4th,8th,12th, 16th week after treatment.The expression of CNTF and its mRNA in sciatic nerve were detected respectively:
2.In vitro experiment
Schwann cells were cultivated and were 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 Neutrophin.DMEM served as negative control.After 48h, MTT colorimetric assay was adopted to measure schwann cells proliferation. Furthermore,CNTF and its mRNA levels were detected in schwann cells cultured in dirrerent media at 48h.
【Results】
1.In vivo experiment
(1) Blood glucose and body weight
—The 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) CNTF expression of sciatic nerve
The integrated option density of CNTF expression in STZ-DM rats was much lower than the normal(P＜0.01).And the levels of CNTF in all the treated groups increased notably compared with model group(P＜0.01).The levels of CNTF in low-dose JMT group,middle-dose JMT group and Neurotropin group were higher than high-dose JMT group(P＜0.01).
(3)CNTF-mRNA expression of sciatic nerve
The levels of CNTF-nRNA expression in STZ-DM rats were much lower than those of the normal rats(P＜0.01).Compared with model group,CNTF-mRNA expression of treated groups up-regulated noticeably(P＜0.01),middle-dose JMT group and Neurotropin group.CNTF-mRNA expression of JMT treated groups were higher than Neurotropin group(P＜0.01).
2.In vitro experiment
(1) MTT colorimetric assay
Schwann cells isolated from newborn rats primary cultured in different medium.The OD values of high glucose group diminished significantly compared with those of normal group 48h later(P＜0.01).The OD values of JMT and Neutrophin group increased greatly compared with 50mMGlu group(P＜0.05) at 48h and there were no significant differences between these two treated groups(P＞0.05).
(2)Confocal laser scanning microscope examination
①The fluorescence intensities of CNTF in schwann cells cultured in high glucose condition were much weaker than those of normal condition(P＜0.01).②The fluorescence intensities of CNTF in schwann cells in JMT and Neurotropin groups reinforced remarkably compared with high glucose group (P＜0.01).③There were no significant differences between these two treated groups(P＞0.05).
(3) QT-PCR
①The expression of CNTF-mRNA in schwann cells cultured in high glucose condition were much weaker than those of normal condition(P＜0.01).②CNTF-mRNA expression in schwann cells of JMT group and Neurotropin group up-regulated compared with high glucose group(P＜0.01)③CNTF-mRNA expression in schwann cells of JMT group was higher than that of Neurotropin group(P＜0.05).
【Conclusion】
1.In vivo experiment
①The significant degressions of CNTF and CNTF-mRNA expression of sciatic nerve in DPN rats attenuated the ability to reparative regeneration.
②JMT could up-regulate the expression of CNTF and CNTF-mRNA in sciatic nerve in DPN rats.
③JMT could not only prevent the progression of nerve dysfunction and degeneration but also to promote regeneration of degenerated nerve fibers. And its efficacy was independent of hypoglycemia.
2.In vitro experiment
①High glucose inhibited the proliferation of Schwann cells remarkably and this action was correlated with the concentration of glucose.
②High glucose depressed the expression of CNTF and its mRNA in schwann cells obviously.
③The medicated serum of JMT could promote schwann cells cultured in high glucose to proliferate and elaborate CNTF and its mRNA effectively.
【Innovation】
To study the effects of Jinmaitong Capsule on CNTF and its mRNA expression in sciatic nerve of STZ-DM rats,as well as to study the effects of medicated serum of JMT on the role of proliferation and elaborating CNTF of schwann Cells 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 basic theory and experimental foundation for the application of JMT to clinical treatment of DPN.
引文
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