中药筋脉通对糖尿病大鼠神经生长因子及对雪旺细胞作用的研究
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摘要
【目的】
1.观察筋脉通对STZ-DM大鼠模型的热水甩尾试验、机械痛阈以及坐骨神经中NGF和NGF-mRNA表达的影响。
2.观察筋脉通含药血清对高糖培养雪旺细胞的增殖及其分泌NGF的影响。
【方法】
1.整体实验:用链脲佐菌素(STZ,60mg/kg)一次性腹腔内注射Wistar大鼠的方法制作糖尿病模型,简单随机分为5组:模型对照组、筋脉通小剂量、中剂量和大剂量组及神经妥乐平组,每组各10只。以体重、鼠龄相匹配的正常大鼠10只作为正常对照组。成模后即开始灌胃给药,筋脉通小、中、大组分别按成人剂量的5倍、10倍和20倍给药;神经妥乐平组按成人剂量的10倍给药。模型组和正常组予灌服蒸馏水1mL/100g/d。所有实验大鼠于灌胃16w后处死。检测各组治疗前及治疗后4w、8w、12w和16w的体重和血糖变化,并进行热水甩尾试验和机械痛阈检测,采用免疫组化染色法测定坐骨神经中NGF的表达,实时荧光定量PCR法检测坐骨神经中NGF-mRNA的表达。
2.细胞实验:从Wistar乳鼠坐骨神经取材,采用反复植块法、差速贴壁法+低浓度胰蛋白酶消化法+G418来培养和纯化雪旺细胞,S-100蛋白免疫组化法进行鉴定。取第3代雪旺细胞,加入DMEM、50mMGlu、75mMGlu培养液以及含葡萄糖50mM的筋脉通未稀释(JMT1:1)、筋脉通1:2稀释(JMT1:2)、筋脉通1:8稀释(JMT1:8)和神经妥乐平(Ntp)含药血清进行培养,设立空白对照组,于培养24h、48h、72h及96h后行MTT比色试验,观察雪旺细胞增殖活性的变化。并采用共聚焦激光扫描显微镜术,进一步检测DMEM、50mMGlu培养基以及含葡萄糖50mM的JMT1:2、Ntp含药血清培养48h后雪旺细胞内NGF表达水平的变化。
【结果】
1.整体实验
(1)血糖和体重监测:STZ-DM大鼠血糖值均显著高于正常组(P＜0.01);各治疗组血糖在各时间点与模型组相比无明显差异(P＞0.05);灌胃前后,各组大鼠的血糖水平无明显变化(P＞0.05)。造模及灌胃前后,各组大鼠之间的体重无明显差异(P＞0.05)。
(2)热水甩尾试验:模型组和各治疗组的甩尾潜伏期均较正常组显著延长(P＜0.01或P＜0.05)。与模型组相比,筋脉通小、中剂量组与神经妥乐平组的甩尾潜伏期显著缩短(P＜0.05);筋脉通大剂量组无明显变化(P＞0.05)。筋脉通各剂量组与神经妥乐平组相比无显著差异(P＞0.05)。
(3)机械痛阈检测:与正常组相比,模型组和筋脉通大剂量组的痛阈值降低非常显著(P＜0.01);筋脉通小、中剂量组与神经妥乐平组无明显降低(P＞0.05)。与模型组相比,筋脉通小、中剂量组与神经妥乐平组的痛阈值显著升高(P＜0.05);筋脉通大剂量组升高不明显(P＞0.05)。筋脉通各剂量组与神经妥乐平组相比无显著差异(P＞0.05)。
(4)坐骨神经NGF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非常(P＜0.01或P＜0.05)。各治疗组的积分光密度值较模型组均有显著增大(P＜0.05或P＜0.01)。各治疗组之间无统计学差异(P＞0.05)。
(5)坐骨神经NGF-mRNA表达:模型组及各治疗组的NGF-mRNA值均较正常组非常显著降低(P＜0.01)。与模型组相比,筋脉通中剂量组和神经妥乐平组的NGF-mRNA值均有明显上调(P＜0.01);筋脉通大、小剂量组无显著变化(P＞0.05)。与筋脉通大剂量组相比,筋脉通中剂量组和神经妥乐平组的NGF-mRNA值明显增高(P＜0.05);筋脉通小剂量组无显著差异(P＞0.05)。
(6)Spearman相关分析显示:坐骨神经NGF-mRNA的表达水平与甩尾潜伏期呈负相关(r=-0.467,P＜0.01);与机械痛阈值呈正相关(r=0.394,P＜0.05)。
2.细胞实验
(1)MTT比色实验:①原代培养的乳鼠坐骨神经雪旺细胞在工作培养基培养的第24h～72h快速增殖、生长旺盛;72h后增殖明显减缓。②在50mMGlu及75mMGlu高糖环境中,雪旺细胞的OD值较DMEM培养液普遍减小,48h后显著降低(P＜0.05或P＜0.01)。③与50mMGlu组相比,75mMGlu组在24h、48h及72h时的OD值无显著性差异(P＞0.05),在96h时显著降低(P＜0.05)。④Spearman相关分析发现雪旺细胞的增殖与葡萄糖浓度呈负相关(r=-0.471,P＜0.01);排除了时间因素后的偏相关分析显示二者仍呈负相关(r=-0.679,P＜0.01)。⑤JMT1:1组的雪旺细胞在24～72h持续增殖,在72h以后衰减;JMT1:2组和Ntp组的雪旺细胞在24～48h增殖明显,在48～72h时减慢,72h后开始衰减;JMT1:8组的雪旺细胞随着时间的延长逐渐递增。⑥在24h时各组之间的OD值无显著性差异(P＞0.05)。在48h时:与50mMGlu组相比,JMT1:2组与Ntp组的OD值显著升高(P＜0.05);与Ntp组相比,JMT1:1组的OD值非常显著降低(P＜0.01)。在72h时:与Control组相比,50mMGlu组及JMT1:1组、JMT1:2组与Ntp组的OD值显著下降(P＜0.01或P＜0.05);JMT1:8组无明显下降(P＞0.05)。在96h时:与50mMGlu组相比,JMT1:2组的OD值显著降低(P＜0.05);其它组则无显著变化(P＞0.05)。
(2)共聚焦激光扫描显微镜检测:①50mMGlu组及治疗组的雪旺细胞表达NGF的荧光强度值较Control组明显降低(P＜0.01)。②与50mMGlu组相比,JMT1:2组与Ntp组的雪旺细胞表达NGF的荧光强度值明显升高(P＜0.01),二者无显著差异(P＞0.05)。
【结论】
1.整体实验:(1)空腹单次腹腔注射STZ 60mg/kg后16周,大鼠出现痛觉过敏和温度觉减退,提示有感觉神经纤维受累,DPN造模成功。(2)DPN大鼠坐骨神经中NGF及NGF-mRNA表达水平显著下降,减弱了受损神经纤维的修复再生能力。(3)STZ-DM大鼠的痛觉过敏和温度觉减退与坐骨神经NGF-mRNA的表达下降有关。(4)筋脉通可显著改善DPN大鼠的痛觉过敏和温度觉减退、上调坐骨神经中NGF及NGF-mRNA的表达,中剂量组效果最好,疗效与神经妥乐平相似。(5)筋脉通具有防止DPN大鼠周围神经组织进一步损伤、促进受损神经纤维修复再生的功能。此作用不是通过降低血糖实现的,且与胰岛素应用无关。
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,经S-100蛋白免疫组化鉴定,纯度可达90%左右。(2)原代培养的乳鼠坐骨神经雪旺细胞于第24～72h生长旺盛;72h后增殖明显减缓。(3)高糖对雪旺细胞的增殖有明显抑制作用,与葡萄糖浓度呈显著负相关。(4)高糖使雪旺细胞分泌NGF的能力明显降低。(5)筋脉通含药血清可有效促进高糖环境培养的雪旺细胞的增殖能力及其分泌NGF的水平,作用与神经妥乐平含药血清相似。
【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的温度觉、痛觉和坐骨神经中NGF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NGF的影响,经检索国内外文献未见报道,为筋脉通临床治疗DPN提供理论基础和实验依据。
[Objective]
1. To study the effects of Jinmaitong Capsule (JMT) on the hydrothermal tail-flick test, pain threshold to mechanical stimulation with Von Frey filament, expression of NGF and NGF-mRNA in STZ-induced diabetic rats.
2. To study the effects of medicated serum of JMT on the role of proliferation and elaborating NGF of Schwann cells cultured in high glucose.
[Methods]
1. In vivo experiment: Fifty SZT-induced diabetic rats ( single intraperitoneal injection, 60mg/kg) 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 (treated with JMT similar to the decuple dose of adult recommended dosage), high-dose JMT group (treated with JMT similar to the twenty-fold dose of adult recommended dosage) and Neurotropin group (treated with Neurotropin similar to the decuple dose of adult recommended dosage). Ten normal rats matching with weight and age served as normal control group. All rats were given intragastric administration for 16 weeks (the normal and model groups were treated with distilled water at dose of 1 mL/100g/d) and then killed. Body weight and blood glucose were detected before and at the 4th, 8th, 12th, 16th week after treatment. The hydrothermal tail-flick and pain threshold to mechanical stimulation with Von Frey filament were carried out before death. The expression of NGF and NGF-mRNA in sciatic nerve were detected by SABC immunohistochemical method and real-time fluorogenetic quantitative PCR respectively.
2. In vitro experiment: Schwann cells were isolated from the sciatic nerves of newborn Wistar rats, then cultivated and purified by methods of repeated explanation, differential velocity adherent technique, low density trypsin digestion and application of G418. And SABC immunohistochemical method with S-100 protein antibody was used to identify them. The 3rd passage Schwann cells were cultured respectively in following conditions including DMEM, high (50 and 75 mM) glucose media supplemented with 20% rat serum, 50mM glucose media containing medicated serum of JMT at a dilution of 1:1, 1:2, 1:8 and Neutrophin at a dilution of 1:1. DMEM served as negative control. After 24, 48, 72 and 96 h cultures, MTT colorimetric assay was adopted to measure Schwann cells proliferation. Furthermore, confocal laser scanning microscope was deployed to determine NGF levels in Schwann cells cultured in DMEM, 50mM glucose media supplemented with 20% rat serum, and 50mM glucose media containing medicated serum of JMT at a dilution of 1:2 and Neutrophin at a dilution of 1:1 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) Hydrothermal tail-flick test: The tail-flick latency of STZ-DM rats were much longer than those of normal rats (P<0.01 or P<0.05). Compared with model group, the time shortened significantly in low, middle-dose of JMT groups and Neutrophin group (P > 0.05), while there was no apparent improvement in high-dose JMT group (P>0.05). There was no significant difference between JMT group and Neutrophin group (P>0.05).
(3) Pain threshold to mechanical stimulation with Von Frey filament: Compared with normal group, the pain thresholds of model group and high-dose JMT group decreased extremely (P<0.01) while low-dose and middle-dose JMT group as well as Neutrophin group didn't lower much (P>0.05). Compared with model group, the threshold values of low-dose, middle-dose JMT group and Neutrophin group raised strikingly (P<0.05), however high-dose JMT group showed no statistical significance (P > 0.05). There were no significant differences among JMT groups and Neutrophin group (P>0.05).
(4) NGF expression of sciatic nerve: The integrated option density of NGF expression in STZ-DM rats was much lower than the normal (P<0.01 or P<0.05). And the levels of NGF in all the treated groups increased notably compared with model group (P<0.05 or P<0.01). There were no significant differences among all the treated groups (P>0.05).
(5) NGF-mRNA expression of sciatic nerve: The levels of NGF-mRNA expression in STZ-DM rats were much lower than those of the normal rats (P<0.01). Compared with model group, NGF-mRNA expression of middle-dose JMT group and Neurotropin group up-regulated noticeably (P<0.01), but low-dose and high-dose JMT group showed no significant changes (P>0.05). There were no significant differences among JMT groups and Neutrophin group (P>0.05).
(6) Spearman correlation analysis showed there was negative correlation between the NGF-mRNA expression of sciatic nerve and the tail-flick latency (r = -0.467 , P<0.01) while positive correlation between the NGF-mRNA expression of sciatic nerve and the pain threshold (r=0.394, P<0.05).
2. In vitro experiment:
(1) MTT colorimetric assay:①Schwann cells isolated from newborn rats primary cultured in DMEM proliferated promptly during 24-72 h and slowed down after 72h.②The OD values of 50mMGlu and 75mMGlu group diminished significantly compared with those of DMEM 48h later (P<0.05 or P<0.01).③The OD values of 75mMGlu group showed no significant difference at 24, 48 and 72 h (P>0.05) but depressed striking at 96h (P<0.01).④Spearman correlation analysis showed there was negative correlation between the proliferation of Schwann cells and the concentration of glucose (r=-0.471, P<0.01) and the similar result when excluding the influence of time (r=-0.679, P<0.01).⑤Schwann cells of JMT1:1 group kept on generating during 24-72 h and attenuated 72h later. While in JMT1:2 and Neutrophin group, Schwann cells grew obviously during 24 to 48 h and slowed down during 48-72 h then faded 72h later. As to JMT 1:1 group, Schwann cells increased progressively along with the time.⑥There were no significant differences of the OD values in all the groups at 24h (P>0.05). However, the OD values of JMT1:2 and Neutrophin group increased greatly compared with 50mMGlu group (P<0.05) and it depressed significantly in JMT1:1 group compared with Neutrophin group (P<0.01) at 48h. Finally, compared with 50mMGlu group, the OD values of JMT1:2 group degraded predominantly (P<0.05) while there were no obvious variances in the other groups (P>0.05).
(2)Confocal laser scanning microscope examination:①The fluorescence intensities of NGF in Schwann cells cultured in high glucose condition were much weaker than those of normal condition (P<0.01).②The fluorescence intensities of NGF in Schwann cells in JMT1:2 and Neurotropin groups reinforced remarkably compared with 50mMGlu group (P<0.01) and there was no statistical difference between them (P>0.05).
[Conclusion]
1. In vivo experiment:
①SZT-induced diabetic rats (single intraperitoneal injection, 60mg/kg) had hyperalgia and thermohypesthesia at 16w, which demonstrated the sensory nerve fibers were injured and the DPN models were established.
②The significant degressions of NGF and NGF-mRNA expression of sciatic nerve in DPN rats attenuated the ability to reparative regeneration.
③Hyperalgia and thermohypesthesia of DPN rats were related to the decrease of NGF and NGF-mRNA expression in sciatic nerve.
④Traditional Chinese Medicine JMT could improve hyperalgia and thermohypesthesia of DPN rats and up-regulate the expression of NGF and NGF-mRNA in sciatic nerve. The middle dose of JMT showed the best effectiveness and it was similar to that of Neurotropin.
⑤Traditional Chinese Medicine JMT 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 and insulin.
2. In vitro experiment:
①The 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.
②Primary cultured Schwann cells isolated from sciatic nerves of newborn rats proliferated rapidly during 24-72 h and retarded after 72h.
③High glucose inhibited the proliferation of Schwann cells remarkably and this action was correlated with the concentration of glucose.
④High glucose depressed the ability of Schwann cells to elaborating NGF obviously.
⑤The medicated serum of JMT could promote Schwann cells cultured in high glucose to proliferate and elaborate NGF effectively and its role was similar to that of Neurotropin.
[Innovation]
To study the effects of Traditional Chinese Medicine Jinmaitong Capsule on thermesthesia, algesthesia and NGF level of sciatic nerve in STZ-DM rats, as well as to study the effects of medicated serum of JMT on the role of proliferation and elaborating NGF 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.
1.观察筋脉通对STZ-DM大鼠模型的热水甩尾试验、机械痛阈以及坐骨神经中NGF和NGF-mRNA表达的影响。
2.观察筋脉通含药血清对高糖培养雪旺细胞的增殖及其分泌NGF的影响。
【方法】
1.整体实验:用链脲佐菌素(STZ,60mg/kg)一次性腹腔内注射Wistar大鼠的方法制作糖尿病模型,简单随机分为5组:模型对照组、筋脉通小剂量、中剂量和大剂量组及神经妥乐平组,每组各10只。以体重、鼠龄相匹配的正常大鼠10只作为正常对照组。成模后即开始灌胃给药,筋脉通小、中、大组分别按成人剂量的5倍、10倍和20倍给药;神经妥乐平组按成人剂量的10倍给药。模型组和正常组予灌服蒸馏水1mL/100g/d。所有实验大鼠于灌胃16w后处死。检测各组治疗前及治疗后4w、8w、12w和16w的体重和血糖变化,并进行热水甩尾试验和机械痛阈检测,采用免疫组化染色法测定坐骨神经中NGF的表达,实时荧光定量PCR法检测坐骨神经中NGF-mRNA的表达。
2.细胞实验:从Wistar乳鼠坐骨神经取材,采用反复植块法、差速贴壁法+低浓度胰蛋白酶消化法+G418来培养和纯化雪旺细胞,S-100蛋白免疫组化法进行鉴定。取第3代雪旺细胞,加入DMEM、50mMGlu、75mMGlu培养液以及含葡萄糖50mM的筋脉通未稀释(JMT1:1)、筋脉通1:2稀释(JMT1:2)、筋脉通1:8稀释(JMT1:8)和神经妥乐平(Ntp)含药血清进行培养,设立空白对照组,于培养24h、48h、72h及96h后行MTT比色试验,观察雪旺细胞增殖活性的变化。并采用共聚焦激光扫描显微镜术,进一步检测DMEM、50mMGlu培养基以及含葡萄糖50mM的JMT1:2、Ntp含药血清培养48h后雪旺细胞内NGF表达水平的变化。
【结果】
1.整体实验
(1)血糖和体重监测:STZ-DM大鼠血糖值均显著高于正常组(P＜0.01);各治疗组血糖在各时间点与模型组相比无明显差异(P＞0.05);灌胃前后,各组大鼠的血糖水平无明显变化(P＞0.05)。造模及灌胃前后,各组大鼠之间的体重无明显差异(P＞0.05)。
(2)热水甩尾试验:模型组和各治疗组的甩尾潜伏期均较正常组显著延长(P＜0.01或P＜0.05)。与模型组相比,筋脉通小、中剂量组与神经妥乐平组的甩尾潜伏期显著缩短(P＜0.05);筋脉通大剂量组无明显变化(P＞0.05)。筋脉通各剂量组与神经妥乐平组相比无显著差异(P＞0.05)。
(3)机械痛阈检测:与正常组相比,模型组和筋脉通大剂量组的痛阈值降低非常显著(P＜0.01);筋脉通小、中剂量组与神经妥乐平组无明显降低(P＞0.05)。与模型组相比,筋脉通小、中剂量组与神经妥乐平组的痛阈值显著升高(P＜0.05);筋脉通大剂量组升高不明显(P＞0.05)。筋脉通各剂量组与神经妥乐平组相比无显著差异(P＞0.05)。
(4)坐骨神经NGF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非常(P＜0.01或P＜0.05)。各治疗组的积分光密度值较模型组均有显著增大(P＜0.05或P＜0.01)。各治疗组之间无统计学差异(P＞0.05)。
(5)坐骨神经NGF-mRNA表达:模型组及各治疗组的NGF-mRNA值均较正常组非常显著降低(P＜0.01)。与模型组相比,筋脉通中剂量组和神经妥乐平组的NGF-mRNA值均有明显上调(P＜0.01);筋脉通大、小剂量组无显著变化(P＞0.05)。与筋脉通大剂量组相比,筋脉通中剂量组和神经妥乐平组的NGF-mRNA值明显增高(P＜0.05);筋脉通小剂量组无显著差异(P＞0.05)。
(6)Spearman相关分析显示:坐骨神经NGF-mRNA的表达水平与甩尾潜伏期呈负相关(r=-0.467,P＜0.01);与机械痛阈值呈正相关(r=0.394,P＜0.05)。
2.细胞实验
(1)MTT比色实验:①原代培养的乳鼠坐骨神经雪旺细胞在工作培养基培养的第24h～72h快速增殖、生长旺盛;72h后增殖明显减缓。②在50mMGlu及75mMGlu高糖环境中,雪旺细胞的OD值较DMEM培养液普遍减小,48h后显著降低(P＜0.05或P＜0.01)。③与50mMGlu组相比,75mMGlu组在24h、48h及72h时的OD值无显著性差异(P＞0.05),在96h时显著降低(P＜0.05)。④Spearman相关分析发现雪旺细胞的增殖与葡萄糖浓度呈负相关(r=-0.471,P＜0.01);排除了时间因素后的偏相关分析显示二者仍呈负相关(r=-0.679,P＜0.01)。⑤JMT1:1组的雪旺细胞在24～72h持续增殖,在72h以后衰减;JMT1:2组和Ntp组的雪旺细胞在24～48h增殖明显,在48～72h时减慢,72h后开始衰减;JMT1:8组的雪旺细胞随着时间的延长逐渐递增。⑥在24h时各组之间的OD值无显著性差异(P＞0.05)。在48h时:与50mMGlu组相比,JMT1:2组与Ntp组的OD值显著升高(P＜0.05);与Ntp组相比,JMT1:1组的OD值非常显著降低(P＜0.01)。在72h时:与Control组相比,50mMGlu组及JMT1:1组、JMT1:2组与Ntp组的OD值显著下降(P＜0.01或P＜0.05);JMT1:8组无明显下降(P＞0.05)。在96h时:与50mMGlu组相比,JMT1:2组的OD值显著降低(P＜0.05);其它组则无显著变化(P＞0.05)。
(2)共聚焦激光扫描显微镜检测:①50mMGlu组及治疗组的雪旺细胞表达NGF的荧光强度值较Control组明显降低(P＜0.01)。②与50mMGlu组相比,JMT1:2组与Ntp组的雪旺细胞表达NGF的荧光强度值明显升高(P＜0.01),二者无显著差异(P＞0.05)。
【结论】
1.整体实验:(1)空腹单次腹腔注射STZ 60mg/kg后16周,大鼠出现痛觉过敏和温度觉减退,提示有感觉神经纤维受累,DPN造模成功。(2)DPN大鼠坐骨神经中NGF及NGF-mRNA表达水平显著下降,减弱了受损神经纤维的修复再生能力。(3)STZ-DM大鼠的痛觉过敏和温度觉减退与坐骨神经NGF-mRNA的表达下降有关。(4)筋脉通可显著改善DPN大鼠的痛觉过敏和温度觉减退、上调坐骨神经中NGF及NGF-mRNA的表达,中剂量组效果最好,疗效与神经妥乐平相似。(5)筋脉通具有防止DPN大鼠周围神经组织进一步损伤、促进受损神经纤维修复再生的功能。此作用不是通过降低血糖实现的,且与胰岛素应用无关。
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,经S-100蛋白免疫组化鉴定,纯度可达90%左右。(2)原代培养的乳鼠坐骨神经雪旺细胞于第24～72h生长旺盛;72h后增殖明显减缓。(3)高糖对雪旺细胞的增殖有明显抑制作用,与葡萄糖浓度呈显著负相关。(4)高糖使雪旺细胞分泌NGF的能力明显降低。(5)筋脉通含药血清可有效促进高糖环境培养的雪旺细胞的增殖能力及其分泌NGF的水平,作用与神经妥乐平含药血清相似。
【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的温度觉、痛觉和坐骨神经中NGF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NGF的影响,经检索国内外文献未见报道,为筋脉通临床治疗DPN提供理论基础和实验依据。
[Objective]
1. To study the effects of Jinmaitong Capsule (JMT) on the hydrothermal tail-flick test, pain threshold to mechanical stimulation with Von Frey filament, expression of NGF and NGF-mRNA in STZ-induced diabetic rats.
2. To study the effects of medicated serum of JMT on the role of proliferation and elaborating NGF of Schwann cells cultured in high glucose.
[Methods]
1. In vivo experiment: Fifty SZT-induced diabetic rats ( single intraperitoneal injection, 60mg/kg) 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 (treated with JMT similar to the decuple dose of adult recommended dosage), high-dose JMT group (treated with JMT similar to the twenty-fold dose of adult recommended dosage) and Neurotropin group (treated with Neurotropin similar to the decuple dose of adult recommended dosage). Ten normal rats matching with weight and age served as normal control group. All rats were given intragastric administration for 16 weeks (the normal and model groups were treated with distilled water at dose of 1 mL/100g/d) and then killed. Body weight and blood glucose were detected before and at the 4th, 8th, 12th, 16th week after treatment. The hydrothermal tail-flick and pain threshold to mechanical stimulation with Von Frey filament were carried out before death. The expression of NGF and NGF-mRNA in sciatic nerve were detected by SABC immunohistochemical method and real-time fluorogenetic quantitative PCR respectively.
2. In vitro experiment: Schwann cells were isolated from the sciatic nerves of newborn Wistar rats, then cultivated and purified by methods of repeated explanation, differential velocity adherent technique, low density trypsin digestion and application of G418. And SABC immunohistochemical method with S-100 protein antibody was used to identify them. The 3rd passage Schwann cells were cultured respectively in following conditions including DMEM, high (50 and 75 mM) glucose media supplemented with 20% rat serum, 50mM glucose media containing medicated serum of JMT at a dilution of 1:1, 1:2, 1:8 and Neutrophin at a dilution of 1:1. DMEM served as negative control. After 24, 48, 72 and 96 h cultures, MTT colorimetric assay was adopted to measure Schwann cells proliferation. Furthermore, confocal laser scanning microscope was deployed to determine NGF levels in Schwann cells cultured in DMEM, 50mM glucose media supplemented with 20% rat serum, and 50mM glucose media containing medicated serum of JMT at a dilution of 1:2 and Neutrophin at a dilution of 1:1 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) Hydrothermal tail-flick test: The tail-flick latency of STZ-DM rats were much longer than those of normal rats (P<0.01 or P<0.05). Compared with model group, the time shortened significantly in low, middle-dose of JMT groups and Neutrophin group (P > 0.05), while there was no apparent improvement in high-dose JMT group (P>0.05). There was no significant difference between JMT group and Neutrophin group (P>0.05).
(3) Pain threshold to mechanical stimulation with Von Frey filament: Compared with normal group, the pain thresholds of model group and high-dose JMT group decreased extremely (P<0.01) while low-dose and middle-dose JMT group as well as Neutrophin group didn't lower much (P>0.05). Compared with model group, the threshold values of low-dose, middle-dose JMT group and Neutrophin group raised strikingly (P<0.05), however high-dose JMT group showed no statistical significance (P > 0.05). There were no significant differences among JMT groups and Neutrophin group (P>0.05).
(4) NGF expression of sciatic nerve: The integrated option density of NGF expression in STZ-DM rats was much lower than the normal (P<0.01 or P<0.05). And the levels of NGF in all the treated groups increased notably compared with model group (P<0.05 or P<0.01). There were no significant differences among all the treated groups (P>0.05).
(5) NGF-mRNA expression of sciatic nerve: The levels of NGF-mRNA expression in STZ-DM rats were much lower than those of the normal rats (P<0.01). Compared with model group, NGF-mRNA expression of middle-dose JMT group and Neurotropin group up-regulated noticeably (P<0.01), but low-dose and high-dose JMT group showed no significant changes (P>0.05). There were no significant differences among JMT groups and Neutrophin group (P>0.05).
(6) Spearman correlation analysis showed there was negative correlation between the NGF-mRNA expression of sciatic nerve and the tail-flick latency (r = -0.467 , P<0.01) while positive correlation between the NGF-mRNA expression of sciatic nerve and the pain threshold (r=0.394, P<0.05).
2. In vitro experiment:
(1) MTT colorimetric assay:①Schwann cells isolated from newborn rats primary cultured in DMEM proliferated promptly during 24-72 h and slowed down after 72h.②The OD values of 50mMGlu and 75mMGlu group diminished significantly compared with those of DMEM 48h later (P<0.05 or P<0.01).③The OD values of 75mMGlu group showed no significant difference at 24, 48 and 72 h (P>0.05) but depressed striking at 96h (P<0.01).④Spearman correlation analysis showed there was negative correlation between the proliferation of Schwann cells and the concentration of glucose (r=-0.471, P<0.01) and the similar result when excluding the influence of time (r=-0.679, P<0.01).⑤Schwann cells of JMT1:1 group kept on generating during 24-72 h and attenuated 72h later. While in JMT1:2 and Neutrophin group, Schwann cells grew obviously during 24 to 48 h and slowed down during 48-72 h then faded 72h later. As to JMT 1:1 group, Schwann cells increased progressively along with the time.⑥There were no significant differences of the OD values in all the groups at 24h (P>0.05). However, the OD values of JMT1:2 and Neutrophin group increased greatly compared with 50mMGlu group (P<0.05) and it depressed significantly in JMT1:1 group compared with Neutrophin group (P<0.01) at 48h. Finally, compared with 50mMGlu group, the OD values of JMT1:2 group degraded predominantly (P<0.05) while there were no obvious variances in the other groups (P>0.05).
(2)Confocal laser scanning microscope examination:①The fluorescence intensities of NGF in Schwann cells cultured in high glucose condition were much weaker than those of normal condition (P<0.01).②The fluorescence intensities of NGF in Schwann cells in JMT1:2 and Neurotropin groups reinforced remarkably compared with 50mMGlu group (P<0.01) and there was no statistical difference between them (P>0.05).
[Conclusion]
1. In vivo experiment:
①SZT-induced diabetic rats (single intraperitoneal injection, 60mg/kg) had hyperalgia and thermohypesthesia at 16w, which demonstrated the sensory nerve fibers were injured and the DPN models were established.
②The significant degressions of NGF and NGF-mRNA expression of sciatic nerve in DPN rats attenuated the ability to reparative regeneration.
③Hyperalgia and thermohypesthesia of DPN rats were related to the decrease of NGF and NGF-mRNA expression in sciatic nerve.
④Traditional Chinese Medicine JMT could improve hyperalgia and thermohypesthesia of DPN rats and up-regulate the expression of NGF and NGF-mRNA in sciatic nerve. The middle dose of JMT showed the best effectiveness and it was similar to that of Neurotropin.
⑤Traditional Chinese Medicine JMT 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 and insulin.
2. In vitro experiment:
①The 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.
②Primary cultured Schwann cells isolated from sciatic nerves of newborn rats proliferated rapidly during 24-72 h and retarded after 72h.
③High glucose inhibited the proliferation of Schwann cells remarkably and this action was correlated with the concentration of glucose.
④High glucose depressed the ability of Schwann cells to elaborating NGF obviously.
⑤The medicated serum of JMT could promote Schwann cells cultured in high glucose to proliferate and elaborate NGF effectively and its role was similar to that of Neurotropin.
[Innovation]
To study the effects of Traditional Chinese Medicine Jinmaitong Capsule on thermesthesia, algesthesia and NGF level of sciatic nerve in STZ-DM rats, as well as to study the effects of medicated serum of JMT on the role of proliferation and elaborating NGF 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.
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