The Protective Effect And Mechanism Of Adapentpronitrile On Diabetic Neuronal Injury

Objective:To investigate the protection and potential mechanisms of adapentpronitrile?APPN?,a DPP-?inhibitor synthesized by our laboratory,on diabetic rats neuronal injury.Methods:1.Diabetic rats were established by fed with high fat diet?HFD?for 4weeks,after that rats were received a singly intraperitoneal injection of streptozotocin?STZ??30mg/kg?followed by continuous HFD for 4 weeks.Then SD rats were divided into 4 groups:control group?CMC-Na?,diabetes mellitus group?CMC-Na?,high dose group?APPN,4.5mg/kg?and low dose group?APP,1.5mg/kg?.APPN were intragastrically administered once a day,for a month.HE staining was used to observe histopathological changes in rat brain tissue.The biochemical method was adopted to detect SOD activity and MDA content in rat cortex and hippocampus.The expression of APP,A?,GLP-1R,BAX,Bcl-2,cytochrome c,caspase-9 and caspase-3was measured by Western blotting.2.Four SD rats?180-200g?were injected adapentpronitrile?50 mg/kg?via tail vein after fasting for 12 hours,blood and brain tissue was collected after 30min administration.The concentration of adapentpronitrile in plasma and brain tissue was detected by HPLC-UV method.3.The effect and mechanism of APPN on HT22 cell injury induced by high glucose?HG?or Al?mal?₃:Establishment of HT22 injury model induced by HG or Al?mal?₃ in vitro:To determin the optimal concentration and time point of HG or Al?mal?₃for HT22 cell injury.The cell viability,LDH leakage rate,cell apoptosis and morphology induced by HG/Al?mal?₃ were detected by MTT assay,LDH assay,flow cytometry and inverted microscope,respectively.To investigate the protective effect of APPN on neuronal injury induced by HG/Al?mal?₃,the cell viability,LDH leakage rate,cell apoptosis and morphology were detected by MTT assay,LDH assay,flow cytometry and inverted microscope,respectively.To investigate the protective mechanism of APPN for neuronal injury,mitochondrial membrane potential?MMP?,apoptosis and reactive oxygen species?ROS?were stained by Mitochondrial Membrane Potential Assay Kit with JC-1,TUNEL FITC Apoptosis Kit and Reactive Oxygen Species Assay Kit with DCFH-DA,respectively,finally observed under inverted microscope.Morphological change was observed under inverted microscope and transmission electron microscopy?TEM?.The expression of BAX,Bcl-2,cytochrome c,caspase-9 and caspase-3 was measured by Western blotting.DPP-?activity was measured by the method of Gly-Pro-MAC and detected by microplate reader equipment of fluorescence.Results:1.Our results showed that adapentpronitrile significantly ameliorated the neuronal injury and decreased the APP and A?expressions in the hippocampus and cortex of diabetic rat caused by HFD/STZ,and that adapentpronitrile significantly attenuated oxidative stress,down-regulated pro-apoptotic proteins cytochrome c,BAX,caspase-9 and caspade-3,and up regulated anti-apoptosis protein Bcl-2 in diabetic rat hippocampus and cortex.However,the GLP-1R expression had no significant difference among all groups.2.Thirty minutes after injection of adapentpronitrile?50 mg/kg?via tail vein,its concentration in the brain tissue of rat was 0.2034±0.0094?g/g while it was 28.002±5.691?g/ml in the blood.3.Our experimental results showed that compared with the control group,the cell viability was significantly decreased,LDH leakage rate and cell apoptosis were significantly increased in 75mM HG or 200?M Al?mal?₃-overload HT22 cells for 36h.Mitochondrial membrane potential and the expression of Bcl-2 significantly decreased,accompanied with a significant increase of apoptosis,reactive oxygen species generation,and the expressions of pro-apoptotic proteins in HT22 cells exposed to HG/Al?mal?₃.APPN treatment significantly increased the cell viability,decreased LDH leakage rate and apoptosis.Treatment of adapentpronitrile also significantly protected neuron against injury and suppressed ROS over generation and mitochondrial apoptosis induced by HG/Al?mal?₃.However,the DPP-?activity was not detected in HT22 cells.Conclusion:DPP-?inhibitor adapentpronitrile could through the blood-brain barrier to improve the neuronal injury of diabetic rats,and to ameliorate the HT22 cells damage induced by high glucose-or maltol aluminum-overload.The neuroprotective mechanism of adapentpronitrile may be related to the reduction of oxidative stress,improvement of mitochondrial membrane potential function and inhibition of mitochondrial apoptosis pathway.