Studies On The Mechanism Of Stachydrine Anti-oxidative Stress Damage In Neuronal Cells Of Alzheimer’s Disease

Alzheimer’s Disease（AD）is a common dementia that affects memory,cognitive function and behaviour,offen accompanied by neuroinflammation,oxidative stress and other pathological features,its pathogenic mechanism is not yet clarified,and therapeutic strategies targeting Aβdeposition and tau hyperphosphorylation have not yielded satisfactory results.At present,more and more studies have found that oxidative stress also plays an important role in the pathogenesis of AD,which can lead to neurological dysfunction,neuroinflammation,and synaptic plasticity damage,these symptoms can in turn promote Aβoligomerization,resulting in a vicious circle in the body,thus inhibiting oxidative stress may be a potential direction for treating AD.Stachydrine is a natural alkaloid with high content in motherwort,which has biological activities such as improving microcirculation,anti-fibrosis,anti-inflammatory and neuroprotection.Meanwhile,it was detected that stachydrine can reduce the generation of reactive oxygen species（ROS）and inhibit the level of oxidative stress in cells through NOX2/ROS/m TOR pathway.However,the effect and mechanism of stachydrine for nerve cell damage and oxidative stress in AD are not clarified yet.In this study,Aβ_25-35is used to induce PC12 cells to be an AD injured cell model.By detecting cell viability,lactate dehydrogenase（LDH）release rate,ROS,malondialdehyde（MDA）and pro-inflammatory factor（IL-1β）of PC12 cells treated with Aβ_25-35alone or in combination with stachydrine,the protective effect of stachydrine on neural cells was evaluated.Our results showed that Aβ_25-35induced oxidative stress and inflammatory response,which led to the damage of PC12 cells,however,stachydrine can down-regulate the increase of ROS,MDA and IL-1βinduced by Aβ_25-35,significantly inhibit the damage of PC12 cells induced by Aβ_25-35,thus increase the viability of cells and reduce the release of LDH.Second,molecular docking and molecular dynamics were used to simulate the docking of stachydrine with different domains of NOX2,which is to predict the binding mode and action mechanism of stachydrine targeting NOX2,the results showed that stachydrine stably binds with p47phox PX,and p47phox SH3,and its binding site is the groove surface of PX domain,which is required for the PX domain to bind to phosphatidylinositol on the cell membrane.At the same time,stachydrine also occupies the position in p47phox that binds to p22phox.Then,Western blot technology is used to explore the effect of stachydrine on the NOX2 subunit protein expression and the cytoplasmic subunit translocation,the results showed that stachydrine could reduce the increase of NOX2 cytoplasmic subunit p47phox expression induced by Aβ_25-35,and inhibit the translocation of p47phox to cell membrane.Neverthless,the membrane subunit gp91phox was not affected.At last,we also investigated the microglia（HMC3 cell）activity and the pro-inflammatory factors’level of HMC3 cell treated with Aβand stachydrine alone or in combination,the result shows that stachydrine could alleviate the damage of HMC3 cells by Aβ_25-35,and inhibit the expression of inflammatory factors IL-1β.In conclusion,stachydrine could hinder the complete assembly of NOX2 by down-regulating the expression of NOX2 cytoplasmic subunit p47phox protein and preventing the translocation of p47phox protein from cytoplasm to cell membrane,thereby reducing the generation of ROS,inhibiting the occurrence of oxidative stress and inflammatory response,and protecting nerve cells.Meanwhile,stachydrine can also reduce the expression of inflammatory factors in microglia and inhibits the occurrence of neuroinflammation,alleviate the further damage to nerve cells.The experimental results of this study preliminarily elucidated the potential mechanisms of stachydrine’effect on neuroprotection,anti-oxidative and anti-inflammatory abilities,and lays a theoretical and experimental foundation for the development of therapeutic drugs of AD.