| Diabetic neuropathy(DN), one of the complications of diabetes mellitus(DM), which was characterized as a disease of harmful and high morbidity. Its pathogenesis was complicated. Oxidative stress and apoptosis in cells took a great part in the development of diabetic neuropathy. In 2001,Brownlee proposed the unified mechanism theory of the complications of diabetes that the classic way of abnormal glucose metabolism caused excessive oxygen free radicals generated in mitochondrial respiratory chain in high glucose conditions. Studies had shown that Mitochondrion was not only the main site of producing oxygen radicals, but also the center of controlling apoptosis. In recent years, using fluorescence imaging and three-dimensional imaging devices, mitochondrion was considered as a dynamic organelles, which was in the dynamic balance of fission and fusion. Dynamin-related protein 1 (drp-1) was an important molecule mediating mitochondrial fission in mammal. Once Drp-1 was overexpressed, the balance of fission and fusion was disrupted and mitochondrial fragmentation would be increased which promoted cell apoptosis. Tianzheng Yu demonstrated that mitochondrial fragmentation was an upstream factor for ROS overproduction and cell death in prolonged high glucose conditions.So we inferred Drp-1-mediated mitochondrial fission might be more closely related to diabetes complications.The expression and function of Drp-1 in series of tissures was concerned. The above situation enlightened us to consider: Whether the expression of Drp-1 would be changed in diabetic nerve tissure or not? What is the relationship of Drp-1 and Bax, Bcl-2, Caspase-3? Whether Drp-1 would promote the development of diabetic neuropathy or not? What interventions?Resveratrol, which was abundant in cuspidatums, grapes, cassia seeds, peanuts and other natural plants, was a polyphenolic phytoalexin. It involved a wide range of pharmacological role of antioxidant, anti-apoptosis, anti-aging, anti-inflammatory and nerve protection. From retinal cells in vitro,it demonstrated resveratrol down-regulated Bax, up-regulated Sirt1 and Ku70 activities by reducing calcium levels, then inhibited caspase-3 activity, showed anti-apoptotic effect at last.So we took different dosage of resveratrol in this experiment.Objective: To detect the expression of Drp-1 in sciatic nerve of diabetic rats , and to detect the expression of Bax, Bcl-2 and Caspase-3. To observe the relationships among Drp-1 and Bax, Bcl-2, Caspase-3, and to discuss the role of the Drp-1 in diabetic neuropathy. Meanwhile, series dosages of resveratrol were administered, then its effects on Drp-1 were assessed. The ultrastructure of the sciatic nerve from rats was observed by electron microscopy, and the effect of resveratrol on diabetic neuropathy was evaluated in the pathological point, from which we discussed the pharmacal effect of resveratrol on the diabetic neuropathy and its possible mechanisms.Methods: Seventy clean male SD rats were randomly divided into 2 groups: normal group was fifteen and streptozotocin treated group was fifty-five.There have forty-nine successful models in treated group and four models were removed randomly.The remaining forty-five models were randomly divided into diabetic group, low dose of resveratrol treated group (20mg·kg-1·d-1)and high dose of resveratrol treated group(100mg·kg-1·d-1), n =15. After 12 weeks, rats were sacrificed and sciatic nerves were taken. Part I. To observe the general condition of rats in each group, to measure blood glucose by roche blood glucose meter, to detect the expression of Drp-1 by Real-time PCR, and to detect the protein of Bax, Bcl-2 and Caspase-3 by immunohistochemical staining. Part II. With the intervention of resveratrol to Drp-1, the effects were assessed, and the ultrastructure of sciatic nerve was observed by transmission electron microscope.Results:1. Weight and blood glucoseAt the end of the experiment, body weight of normal group increased generally, and blood glucose was normal. Compared with the normal group, body weight of the diabetic group and resveratrol treated group increased slowly, blood glucose increased significantly. The difference was statistically significant (all P <0.05). Compared with the diabetic group, weight and blood glucose of the resveratrol treated group were of no significant difference (P> 0.05). Compared with the low dose of resveratrol treated group, weight and blood glucose of the high dose of resveratrol treated group were of no significant difference,either(P> 0.05).2. Drp-1 mRNA relative expression in sciatic nerve of ratsCompared with the normal group, Drp-1 expression of diabetic group and resveratrol treated group was significantly higher (P<0.05). Compared with the diabetic group, Drp-1 expression of each resveratrol treated groups was significantly decreased (P <0.05). Compared with the low dose of resveratrol treated group, Drp-1 expression of the high dose of resveratrol treated group was lower, the difference was statistically significant(P <0.05).3. Protein Bax, Bcl-2,and Caspase-3 expression in sciatic nerve of ratsCompared with the normal group, protein Bax and Caspase-3 expression increased significantly while protein Bcl-2 expression decreased significantly in diabetic group. Compared with diabetic group, protein Bax and Caspase-3 expression decreased significantly and protein Bcl-2 expression increased significantly in resveratrol treated group, especially in high dose of resveratrol treated group.4. The ultrastructure of rats' sciatic nerves in each group under electron microscopeMyelins were dense and lamellars arrangement were regular in normal group.The mitochondrias were intact, appeared oval-shaped or rod-shaped and showed clear structure.The mitochondrial ridges arranged regularly also.But in diabetic group myelins had loosed and layers had disordered, mitochondria was swelled and appeared dissolved and broken. After the administration of resveratrol, sciatic ultrastructures seemed improving in a certain extent.Conclusions: The overexpression of protein Drp-1 in sciatic nerve of diabetic rats induced apoptosis and degeneration in sciatic nerve. Resveratrol upexpressed protein Drp-1 and reduced apoptosis, which showed a certain protective effect to sciatic nerve of diabetic rats, moreover, the effect was associated with the dosage. |
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