Effects Of Aucubin On Rat Diabetic Encephalopathy Model And Its Action Mechanism

The purpose of this study mainly aims at observation the effects of aucubin and its action mechanism in diabetic encephalopathy rat model and H₂O₂-induced PC12 cells.In this study, we determined the effect and therapeutic mechanism of aucubin on diabetic encephalopathy. With the exception of a control group, all rats were received intraperitoneal injections of streptozotocin (STZ; 60 mg/kg) to induce Type 1 DM. From d 65 after induction of diabetes, rats were treated with various doses and different injective time of aucubin. Cognition function was assayed by Y maze and morphological changes in hippocampal neurons were observed. In diabetic rats, the function of learning and memory was impaired and the neuron density was remarkably decreased. Various dose of aucubin significantly reduced the working errors during behavioral testing, and attenuated neuron loss in hippocampal region in diabetic encephalopathy rats. Aucubin showed a significant neuroprotective effect and improved learning in diabetic encephalopathy rats.Neuroprotection was estimated by the indexes of behavior and histology. Behavioral testing was performed in a Y-maze and the surviving neurons in CA1 to SC of the hippocampus were counted under a microscope. In addition, the apoptotic neurons in the CA1 of the hippocampus were also examined by using the TUNEL staining. In order to clarify the mechanism of aucubin's neuroprotection, the activities of endogenous antioxidants and nitric oxide synthase (NOS) together with the content of lipid peroxide in the hippocampus were assayed. The results proved that aucubin significantly reduced the content of lipid peroxide, regulated the activities of antioxidant enzymatic and decreased the activity of NOS and MDA. All these effects indicated that aucubin was a potential neuroprotective agent and its neuroprotective effects were achieved, at least in part by promoting endogenous antioxidant enzymatic activities. Hippocampi were excised for visual (light and transmission electron microscopic) and immunochemical (Western blot; immunohistochemical) assessments of the CA1 subfield for apoptosis and expression of regulatory proteins Bcl-2 and Bax. Treatment responses to all the parameters examined (body weight, plasma glucose, Y-maze error rates, pyramidal cell ultrastructure, proportions of apoptotic cells, levels of expression of Bcl-2 and Bax, and survivability of neuronal cells) were identical: These findings confirm the association of apoptosis with the encephalopathic effects of diabetes mellitus, and suggest a major role of the expression levels of Bcl-2 and Bax in the regulation of apoptotic cell death. All of the results suggest that aucubin could effectively inhibit apoptosis by modulating the expressions of Bcl-2 and Bax genes.In this study, the effect of aucubin on H₂O₂-induced apoptosis (0.25 mM) was studied by using a rat pheochromocytoma (PC12) cell line. Hoechst 33258 assays revealed the appearance of a collection of multiple chromatin and fragmented apoptotic nuclei upon H₂O₂ treatment. however, the incidence of apoptotic nuclei was significantly reduced when cells were treated with aucubin. Transmission electron microscopy was found preferable to alternatives for distinguishing between nuclei and organelles. Obvious apoptotic morphological changes were observed in the H₂O₂ induced cells. Addition of aucubin to the H₂O₂ clearly enlarged the apoptotic nuclei, which were also smoother and resembled those of the control cells. When the apoptotic cells were analyzed quantified using flow cytometry, when PC12 cells were concurrently treated with aucubin, the percentage of apoptotic cells decreased from 35.1±3.0 to 23.1±1.1%. Bcl-2 protein increased and Bax protein declined in aucubin treatment. Western blot analysis confirmed the changes in anti-apoptotic and pro-apoptotic proteins in PC12 cells, as did Bcl-2 and Bax levels, which coincided with the flow cytometry results. H₂O₂ treatment caused a time-dependent increase in caspase-3 proteolytic activity, when aucubin was added caspase-3 activity declined. On the basis of these findings, we speculate that the neuroprotective effects of aucubin may be achieved by the regulation of Bcl-2 family members, as well as modulating the caspases cascade activation.