| Background and ObjectiveAlzheimer’s disease(AD)is a common,progressive neurodegenerative disease of the nervous system.It is the single biggest cause of dementia,accounting for 50%-75%Senile dementia.It is now the fourth leading cause of death in the world,after cardiovascular disease,malignancy and stroke.At present,the problem of population aging is becoming more and more serious,and the incidence of Alzheimer’s disease increases with age,with roughly doubling in prevalence every 5 years after age 65.Epidemiological data show that 46.8 million people worldwide are now suffering from dementia,which is expected to rise to nearly 65 million by 2030 and will rise to about 150 million by 2050.Typical clinical manifestations of Alzheimer’s disease include impaired memory,decreased executive function,progressive impairment of daily life and personality disorders.The histopathological features of Alzheimer’s disease are characterized by the deposition of anyloidβ-protein(Ap)in specific brain regions and the presence of neuritic plaque(NP)/senile plaques(SP).Excessive phosphorylation of tau protein aggregates in neurons to form neurofibrillary tanglesnies,resulting in neuronal death,glial cell proliferation and loss of synaptic links.In 2018,the Alzheimer’s Association recommended redefining Alzheimer’s disease.which is defined by by its underlying pathologic processes in the brain rather than cognitive symptoms such as memory loss.According to statistics,a third of those over 70 years of age who have no cognitive impairment actually have a pathological change in the brain of Alzheimer’s disease.In patients with Alzheimer’s disease,the corresponding pathological changes such as Ap,tau protein have appeared in the brain 15 to 20 years before the onset of clinical symptoms.Therefore,it is necessary to study the early biomarkers and pathogenesis of Alzheimer’s disease.So far,the exact pathogenesis of Alzheimer’s disease is not clear.There are many theories,such as Ap-protein cascade hypothesis,oxidative stress hypothesis,mitochondrial cascade hypothesis,cholinergic damage hypothesis,immune inflammation abnormality hypothesis,pathogen hypothesis,metal ion disorder hypothesis,neurovascular hypothesis,etc.The hypothesis of Aβ protein cascade is still the mainstream theory,which holds that Aβ plays an important role in the pathogenesis and progression of Alzheimer’s disease.The imbalance of Aβ production and clearance is the initial event leading to neuronal degeneration and dementia.The accumulation of Aβ oligomer triggers a series of cellular changes,including tau protein hyperphosphorylation、mitochondrial oxidative stress、mitochondrial dysfunction、synaptic connection disorder、inflammatory response and so on.Mitochondria are the energy factory of eukaryotic cells and the main site of ROS production,involving many important biological processes in the cells.Studies have shown that mitochondrial dysfunction and oxidative stress are early important pathophysiological changes in Alzheimer’s disease.Mitochondrial dysfunction resulted in decreased cytochrome oxidase activity、increased production of reactive oxygen species(ROS)in neurons、altered intracellular calcium homeostasis、decreased mitochondrial ATP and activate the mitochondrial pathway apoptosis.Mitochondrial pathway apoptosis is an important endogenous pathway of apoptosis,which plays a key role in the process of apoptosis.Its regulation is mainly regulated by a variety of pro-apoptotic/anti-apoptotic proteins and their various complexes.Thus,it can promote or inhibit the apoptosis of mitochondria.The transmembrane potential between the mitochondrial intima and the outer membrane constitutes the mitochondrial membrane potential(Mitochondrial Membrane Potential,MMP),which is maintained by the consumption of ATP pump by the proton pump of the mitochondrial intima.After mitochondrial pathway apoptosis was initiated,mitochondrial membrane potential decreased or disappeared,which led to the transfer of apoptotic proteins such as cytochrome C(Cytochrome c,Cyto C)、Bax between mitochondria and cytoplasm and the activation of cysteine aspartate-specific proteinases(Caspase).Then apoptosis was initiated,which resulted in programmed cell death.Autophagy is responsible for the degradation and recycling of unnecessary or dysfunctional cytoplasmic components and organelles by relying on lysosomes or vacuoles(yeast),such as misfolding or accumulating proteins and damaged organelles(e.g.mitochondria).By this way,the aggregation of abnormal proteins and damaged organelles can be reduced,and the balance of the cells can be maintained under the adverse conditions,and the normal survival of the cells can be ensured.Studies have shown that autophagy protects degenerative neurons by clearing toxic proteins and damaged organelles.The selective clearance of damaged dysfunction mitochondria by autophagy in time,the reduction of oxidative stress response,the maintenance of mitochondrial quality control and the stability of mitochondrial structure and function are of great significance for the normal growth and metabolism of cells.Autophagy disorder is closely related to many biological processes in the pathogenesis and progression of neurodegenerative diseases such as Alzheimer’s disease.Currently,the main treatments for Alzheimer’s disease include the control of accompanying mental disorders and the improvement of cognitive function by acetyl-cholinesterase inhibitors of acetylcholinesterase(AChEIs)、N-methyl-Daspartate receptor antagonists and other supportive therapies.There is no modified therapy that can alter/reverse the pathology and course of Alzheimer’s disease.In view of the complexity of the pathogenesis of Alzheimer’s disease,the development of drugs targeting different targets of Alzheimer’s disease is still in an experimental stage and have all failed.The results suggested that the strategy of Alzheimer’s disease treatment should re-examine its core pathogenesis and focus on the choice of "multi-target coordinated intervention".Therefore,it is imminent and significant to study the effective drugs targeting the factors of Alzheimer’s disease,and the traditional Chinese medicine may be explored and studied as a therapeutic direction.Polydatin(PD)is an effective monomer component separated from the dry root and rhizome of Polygonum cuspidatum,a traditional Chinese herb.It is a derivative of resveratrol and has a similar pharmacological effect with resveratrol.Polydatin has multiple pharmacological effects of anti-platelet aggregation n inhibiting thrombosis,anti-atherosclerosi、anti-oxidative stress、improving microcirculation、protecting cardiomyocytes、vascular smooth muscle cells、endothelial cells、anti-shock、protecting liver、reducing lung injury、neuroprotection、antitussive、antiasthmatic、immunomodulatory and antitumor activities.At present,the research on the neuroprotective effect of PD has gradually become the focus of neurology.The research direction is mostly focused on ischemic cerebrovascular disease.There is little research on neurodegenerative diseases such as Alzheimer’s disease.Therefore,in this study,we observed the protective effect of polydatin on Aβ25-35 induced neuronal injury and its related mechanisms,and provided experimental and theoretical basis for further exploring the potential treatment of Alzheimer’s disease drugs.Objective:In this study,we observed the effect of polydatin on the damage of SH-SY5Y cells induced by Aβ25-35,and explored the protective effect of polydatin on neurons and its possible mechanism.Methods:1.SH-SY5Y cells were treated with polydatin and Aβ25-35,and the cell viability were determined by MTT.Then we determine the concentration and time of action of Aβ25-35 in constructing AD cell models,the concentration of polydatin in the subsequent experiments.2.SH-SY5Y cells in each group were treated with AP25-35 and polydatin.With Annexin V-FITC/PI staining,the apoptotic cell rates were quantified by using flow cytometric analysis.The cytoplasm and mitochondria of SH-SY5Y cells were separated,and the expression of apoptosis-related protein cleaved caspase-3、caspase-9、Cyto C and Bax were determined by western blot method in cytoplasm/mitochondria.3.SH-SY5Y cells in each group were treated with AP25-35 and polydatin,then the expression of autophagy associated protein LC3 Ⅱ/Ⅰ and Beclin 1 were detected by western blot method.4.SH-SY5Y cells in each group were treated with autophagy inhibitor Bafilomycin A1、Aβ25-35 and polydatin,respectively.With Annexin V-FITC/PI staining,the apoptotic cell rates were quantified by using flow cytometric analysis.The cytoplasm and mitochondria of SH-SY5Y cells were separated,and the expression of apoptosis-related protein cleaved caspase-3、caspase-9、Cyto C and Bax were determined by western blot method in cytoplasm/mitochondria.5.SH-SY5Y cells in each group were treated with autophagy inhibitor Bafilomycin A1、Aβ25-35 and polydatin.The changes of mitochondrial membrane potential were detected by JC-1 staining and flow cytometry.The level of ATP was measured by CellTiter-Glo and the content of ROS was measured by DCFH-DA.Results:1、The results of MTT assay;when SH-SY5Y cells were only treated with Aβ25-35,compared with the normal control group,the cell viability of SH-SY5Y cells induced by AP25-35 at 10μM decreased by 64.28 ± 4.73%after 24 hours of injury,which was suitable for the establishment of AD cell model.When SH-SY5Y cells were only treated with different concentrations of polydatin for 24 hours,the cell activity did not change significantly.Compared with Aβ group,the viability of SH-SY5Y cells increased significantly after pretreatment with 50μM、100μM、200μM、3 00pM polydatin for 24 hours in combination with Aβ25-35,and there was no significant difference between the 100μM polydatin group and the 200μM、3 00μM polydatin groups.2.Polydatin inhibits apoptosis of SH-SY5Y cells induced by Aβ25-35 by inhibiting mitochondrial apoptosis pathway.The results of flow cytometry showed that polydatin treatment could significantly inhibit the apoptosis of SH-SY5Y cells induced by Aβ25-35,and the number of apoptosis of SH-SY5Y cells in group Aβ25-35 was significantly higher than that in control group,while the number of apoptosis in polydatin+Aβ25-35 group was significantly lower than that in Aβ group.The results of western blot showed that the expression of cleaved caspase-3.cleaved caspase-9、Cyto C protein in cytoplasm of Aβ group was upregulated,and the expression level of Cyto C protein in mitochondria was significantly down-regulated,compared with the normal control group.The expression of cleaved caspase-3、cleaved caspase-9、Cyto C in cytoplasm of polydatin+AP25-35 group decreased significantly compared with Aβgroup,while the expression of Bax protein was up-regulated compared with Aβ group;the expression of Cyto C protein in mitochondria significantly increased,while Bax protein decreased.These results suggest that polydatin can inhibit mitochondrial pathway apoptosis induced by Aβ25-35 in SH-SY5Y cells.3.Polydatin promotes autophagy of SH-SY5Y cells.The results of western blot assay showed that the expression of LC3 Ⅱ/Ⅰ、Beclin 1 protein in the cytoplasm of Aβ group was significantly up-regulated than that of normal control group.Compared with Aβ group,the expression of LC3 Ⅱ/Ⅰ Beclin 1 protein in cytoplasm of 50μM polydatin+Aβ25-35 group and 100μM polydatin+Aβ25-35 group were significantly up-regulated.These results suggest that polydatin can promote autophagy of SH-SY5Y cells.4.Polydatin inhibits apoptosis of SH-SY5Y cells induced by Aβ25-35 in by promoting autophagy.The results of flow cytometry showed that compared with Aβ25-35 group,polydatin pretreatment could significantly inhibit the apoptosis of SH-SY5Y cells induced by Aβ25-35.When the autophagy inhibitor Baf A1 was pretreated,the number of apoptotic cells in the Baf Al + Aβ25-35+ polydatin group was significantly higher than that in the Aβ25-35+ polydatin group.The results of western blot showed that the expression of cleaved caspase-3、cleaved caspase-9、Cyto C in cytoplasm of polydatin+AP25-35 group decreased significantly compared with Aβgroup,while the expression of Bax protein was up-regulated compared with Aβ group;the expression of Cyto C protein in mitochondria significantly increased,while Bax protein decreased.Compared with Aβ25 35+ polydatin group,the expression levels of cleaved Caspase-3、cleaved caspase-9、Cyto C protein in the cytoplasm of Aβ25-35+polydatin+Baf group were obviously up-regulated,and the expression level of Bax protein decreased;the expression level of Cyto C protein in mitochondria was significantly reduced and the expression level of Bax protein was increased.These data suggest that polydatin inhibits mitochondrial apoptosis in SH-SY5Y cells by inducing autophagy.5、Polydatin reduces oxidative stress and improve mitochondrial dysfunction in SH-SY5Y cells through autophagy.The change of mitochondrial membrane potential detected by flow cytometry showed that the number of JC-1 monomers was significantly decreased and the decrease of mitochondrial membrane potential was significantly inhibited after cells were pretreatment with polydatin,compared with Aβ25-35 group.When the autophagy inhibitor Baf A1 was added,the number of JC-1 monomers in the Baf Al + Aβ25-35+ polydatin group was significantly higher than that in the Aβ25-35+ polydatin group,the inhibitory effect of polydatin on mitochondrial membrane potential decrease was partially reversed.Polydatin significantly increased the content of ATP in SH-SY5Y cells induced by Aβ25-35,while autophagy inhibitor Baf partially inhibited the increase of ATP content in SH-SY5Y cells induced by polydatin.Polydatin could significantly decrease the level of ROS in SH-SY5Y cells induced by Aβ25-35,while autophagy inhibitor Baf partially reversed the effect of polydatin and increased the content of ROS in SH-SY5Y cells.These results suggest that polydatin can reduce oxidative stress and mitochondrial dysfunction in SH-SY5Y cells by promoting autophagy.Conclusion:1、Polydatin can inhibit apoptosis of SH-SY5Y cells induced by Aβ25-35 by inhibiting mitochondrial dependent apoptotic pathway.2、Polydatin promotes autophagy of SH-SY5Y cells.3、Polydatin inhibits apoptosis of mitochondrial pathway in SH-SY5Y cells induced by Aβ25-35 by promoting autophagy.4、Polydatin reduces oxidative stress and mitochondrial dysfunction in SH-SY5Y cells induced by Aβ25-35 by promoting autophagy. |
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