Effects Of Jinmaitong Capsule On Ciliary Neurotrophic Factor In Sciatic Nerve Of Diabetic Rats And Schwann Cells

【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 experimentFifty 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 experimentSchwann 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 nerveThe 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 nerveThe 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 assaySchwann 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 examination①The 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-PCR①The 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 experiment①The 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 experiment①High 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.