| [Objective]1.To study the effects of Jinmaitong capsule(JMT) on morphology and expression of nuclear factor kappa B(NF-κB) in sciatic nerves of STZ-induced diabetic rats.2.To study the effects of medicated serum of JMT on ultrastructure, transcription and expression of NF-κB in Schwann cells(SC) cultured in high glucose Medium.[Methods]1.In vivo:Fifty SYZ-induced diabetic rats(single intraperitoneal injection, 60mg/kg) were randomly divided into 5 groups including model group(DM), low-dose JMT group(JL),middle-dose JMT group(JM),high-dose JMT group (JH) and Neurotropin group(Ntp).Ten normal rats matching with weight,age and sex served as normal control group(Con).All rats were given intragastric administration for 16 weeks and then killed.Body weight and blood glucose were detected before and at the 4th,8th,12th,16th week after treatment.The morphologic and morphometric studies of sciatic nerves were investigated by light and transmission electron microscopic techniques.The expression of NF-κB in sciatic nerves were detected by SABC immunohistochemical method.2.In vitro:SC 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 G-418.And SABC immunohistochemical method with S-100 protein antibody was used to identify them.The 3rd passage SC were cultured respectively in different conditions including control gtoup(Con),high glucose group (Glu),JMT group(JMT) and Neutrophin group(Ntp).After 48h cultures,the ultrastructure of SC was investigated by transmission electron microscopy. Furthermore,the expression of NF-κB mRNA and NF-κB in SC were detected by real-time fluorogenetic quantitative PCR and confocal laser scanning microscope respectively.[Results]1.In vivo:(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 the DM 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) Pathological morphology:①Morphology:The morphology of sciatic nerves in the Con group was normal.The DM group showed the loss of mylelinated nerve fibers,axons swelling or atrophy,segmental demyelination,glycogen wthin axons, collagen fiber increasing and basement membrane thickening.All the treated groups improved compared with the DM group.②Morphometric analysis:To compared with the Con group,the density of myelinated fibers and small myelinated fibers,the rate of small myelinated fibers,the size of myelinated fibers and axon cross sections in the DM group were significantly reduced(P＜0.01).However,the density of big myelinated fibers and the size of myelin cross sections were insignificant changes among them compared each other(P＞0.05).And the index above in all the treated groups decreased compared with the DM group, the JM group more significantly improved than the DM group(P＜0.05).The density of myelinated fibers and small myelinated fibers as well as the size of axon cross sections in the JM group were more significantly improved than the JL group(P＜0.05,P＜0.01) (3) NF-κB expression of sciatic nerve:NF-κB expressed in cytoplasm and nuclei of SC,axon and endothelial cells,but not in myelin sheaths.The average area,average optical density and integrated optical density of NF-κB expression in the DM group were much higher than the Con group(P＜0.01).And the levels of NF-κB in all the treated groups decreased compared with the DM group,the JM group notably lower than the DM group(P＜0.05),and average optical density in the JM group decreased compaired with the JS group(P＜0.01).2.in vitro:(1) Ultrastructure:The morphology of SC in the Con group was normal and there was not basement membrane.Some non-nuclear cells were in the Glu group,remained nuclei showed the irregular form.Few organelles and many glycogen,and vacuolar degeneration were in the cells.Other SC showed the ballooning degeneration and broken plasmalemma.Pathological morphology changed in the JMT group,but not notably changed in the Ntp group compared with the Glu group.(2) NF-κB expression of SC:The green fluorescence intensities of NF-κB expressed weekly in cytoplasm of SC in the Con group,but much stronger in cytoplasm and nuclei of SC in the Glu group than it of the Con group(P＜0.01).All treated group showed the weeker green fluorescence intensities in cytoplasm of SC,which compared with the Glu group(P＜0.01).There was no statistical difference between the JMT and Ntp groups(P＞0.05).(3) NF-κB mRNA transcription of SC:The levels of NF-κB mRNA transcription in the Glu group were much higher than those of the Con group(P＜0.01).Compared with the Glu group,NF-κB mRNA expression of the JMT group and the Ntp group down-regulated noticeably(P＜0.01).Between two treated groups showed no significant changes(P＞0.05). [Conclusion]1.In viyo:STZ-induced diabetic rats(single intraperitoneal injection,60mg/kg) had morphology changes at 16w,which included the changes of axon,myelin sheat,vascular and collagen fiber.Mylelinated nerve fibers were lost. Furthermore,the expression of NF-κB significant increased.Traditional Chinese Medicine JMT could improve the pathological morphology of DPN rats and down-regulate the expression of NF-κB in sciatic nerves.The middle dose of JMT showed the best effectiveness.And its efficacy was independent of hypoglycemia and insulin.2.In vitro:SC isolated from the sciatic nerves of newborn Wistar rats and cultivated and purified by methods of repeated explanation,differential velocity adherent technique,low density trypsin digestion and application of G-418. High glucose could change the ultrastructure and up-regulate the transcription and expression of NF-κB in SC.The medicated serum of JMT could down-regulate the transcription and expression of NF-κB and improved the ultrastructure of SC. |
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