The Study Of Mechanism Of Icariine On The Repairing Of Mitochondrial Fission-Fusion Dynamics Imbalance

To explore the relationship between pathological changes of Alzheimer disease and Drp1 and its phosphorylation regulation pathway and mitochondrial fission- fusion imbalances, and study nerve protective effect of ICA on mechanism of mitochondria.Choose 3×Tg AD model mice and the same genetic background of the NTg mice as experimental object,(1) to cultivate their newborn rat cortical neurons, with 20μM ICA, using Western Blot to compare the regulation of ICA on PDHE1α, COXⅣ and Drp1 and the expression level of its phosphorylated protein, studies the correlation between Drp1 and Aβ and the synapses markers.(2) using immunofluorescence staining and mitochondrial specificity fluorescent protein technology, laser confocal observation and time-lapse imaging technology, to study the changes of distribution and transportation and mitochondria index of mitochondria in neurons, explore the effect of ICA on abnormal mitochondria transfer dynamics.(3) using the ICA mixed feed to feed the 3×Tg AD model mice from 2 month to 8 month,at the same time to feed 3×Tg AD model mice of 4 month and 12 month and nontransgenic mice, extracting mitochondria, detecting the effect of age on Drp1 gene expression and phosphorylation modification, and the effects under the condition of normal and AD pathology, evaluate the intervention effect of ICA.(4) measure and compare mitochondrial functions such as cytochrome c oxidase activityThe results of experiences are the follows.(1) ICA can lower the expression of APP, BACE1, Tau46 and p S396 in primary neurons, improve the expression of PSD95, PDHE1α, and COX Ⅳ, reduce the level of ROS.(2) ICA can reduce the aggregation of Aβ in axon and call back the expression and distribution of PSD95 and PDHE1α.(3) ICA can significantly improve the level of p-Drp1S637, increase the expression of Mfn2(P< 0.05), reduce the expression of Drp1 and CNAα in primary neurons.The results of 8 month mice with ICA are basically identical with the primary neurons, and ICA can increase the mitochondrial cytochrome c oxidase activity in mice cortex and hippocampus(P< 0.05).(4) The expression of Drp1 is growing with age and Mfn2 is decreasing with age in 3×Tg AD mice(P< 0.05). The expression of p-Drp1S637 was rise in the cytoplasm and decrease in mitochondria; PDHE1α decreased with age and COX Ⅳ decreased, too(P<0.05).(5) Mitochondrial tracer study revealed that ICA can increase mitochondrial anterograde and retrograde transport, promote the mitochondria index, effectively repair AD pathology caused by abnormal distribution of mitochondria, fragmentation, and movement speed.So, we get three conclusions.(1)ICA can inhibitate the expression of BACE1, adjust the distribution of Aβ and reduce the abnormal aggregation in neurons, repair pathological damage of Alzheimer’s disease.(2) the mitochondrial fission- fusion imbalance increases with age gradually, AD pathology worsens mitochondrial transport dynamic imbalances.(3) ICA can increase the mitochondria in the axonal transport, improve the imbalance of mitochondrial fission- fusion caused by AD, repair damaged mitochondrial function.