| [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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