Effect Of Emodin On The Mechanism Of The Neurotoxicity Induced By Nitric Oxide

Objective: Alzheimer's disease(AD)is a chronic neurodegenerative disorder characterized by gradual memory decline and cognitive dysfunction,accompanied by abnormal personality and behavior.It has been a serious threat to the physical and mental health of the elderly.Neuronal death is one of the major pathological characteristics in AD brains.Reactive oxygen species(ROS)and Nitric Oxide(NO)free radicals generated from oxidative stress play an important role in the pathogenesis of neurodegenerative disease and cardiovascular and cerebrovascular diseases.Excessive NO production can cause free radical damage and induce neuron death.Some natural antioxidants can play an active role by oxidative-stress resistance in the prevention and treatment of senile degenerative diseases.As one of the traditional Chinese medicines,Emodin has already been widely applied to clinical practice.Recently,Emodin has been reported to have antioxidant,immunomodulatory,antibacterial,anti-inflamatory and other functions,which is more and more widely used in treating many diseases such as intestinal diseases,nephrosis,angiocardiopathy and pancreatitis,etc.Forkhead transcription factor O1(FOXO1)is a vital member of the Forkhead family of transcription factors,known to regulate the transcription of genes involved in cell cycle arrest and DNA repair in response to oxidative stress or apoptosis.However,the pathogenesis and therapeutic strategies of AD have not yet been fully identified.furthermore,Whether NO free radicals can regulate FOXO1 and apoptosis induced by it,and What natural antioxidants play an neuroprotective role through FOXO1 signaling pathway,are still not quite clear.This study will be conducted with its focus on the correlation of AD-related NO,natural antioxidants and FOXO1,so to detect the possible interactive mechanism of them and its effectiveness in attenuating neurotoxicity and neuronal apoptosis,which will eventually put forward a new perspective and clue to prevent and treat neurodegenerative diseases like AD.Methods: This study took C57BL/6 mouse primary cortical neurons and HT22 cells as the cell model,which were transfected with the 3×IRS-luciferase,Fas L-luciferase,or Bim-luciferase reporter system.On DIV 7,the neurons were treated with different reagents and drugs at certain concentration,including NO donor Nitrosoglutathione(GSNO)and L-Arginine(L-Arg),58 natural Chinese herbal extracts composed of Emodin,Berberine hydrochloride,Deoxyschizandrin and others,and also the composition of L-Arg and Emodin.Then,the neurons were subjected to dual luciferase reporter gene assay to observe the transformation of FOXO1 transcriptional activity and its downstream proapoptotic genes(Fas L,Bim)expression,subjected to CCK-8 cell activity assay to observe the activity of cells,subjected to immuno-staining to image using Metamorph software and a Nikon,or subjected to Western blot analysis for the indicated antibody to detect FOXO1,AKT and related proteins.Through these experiments,we study the influence and mechanism of NO on neuronal apoptosis induced by FOXO1,the impact of natural antioxidants upon the transcriptional activity of FOXO1,the influence and mechanism of Emodin obtained by screening on neuronal apoptosis induced by FOXO1,and the influence of Emodin upon NO and FOXO1.Graph Pad Prism 6 and Adobe Illustrator CS4 were applied to data processing and image display,T test and variance analysis were used to data analysis(?=0.05).Results: In these experiments,we found that NO donor GSNO or L-Arg significantly activated the transcriptional activity of FOXO1 in both mouse primary cortical neurons and HT22 cells,further increased the expression of FOXO1 downstream proapoptotic genes(Fas L,Bim),decreased neuronal activity,and induced neuronal impairment.When the concentration of L-Arg reached 50mmol/L,neuronal apoptosis occurred.In primary neuronal cells,we screened 58 natural Chinese herb extracts that target to regulating FOXO1 activity.It showed that Moroxydine hydrochloride and Deoxyschizandrin increased the transcriptional activity of FOXO1,whereas Emodin,Berberine hydrochloride and 2,3,5,4-tetrahydroxyl diphenylethylene-2-O-?-Dglucoside contributed to decrease its activity.In particular,Emodin had obvious suppressive effect(P=0.0038),and was widely used in clinic almost without side effects.In HT22 cells,Emodin significantly inhibited the transcriptional activity of FOXO1,further decreased the expression of FOXO1 downstream proapoptotic geneBim,whereas didn't affect the morphology of neurons.Although Emodin reduced the activity of AKT to a certain extent and regulated FOXO1,the decisive effect was that it significantly reduced the expression level of the protein and inhibited the transcriptional activity of FOXO1.Furthermore,Simultaneous use of emodin in L-Arg significantly inhibited the nuclear accumulation and transcriptional activity of FOXO1 induced by the excessive activation of NO,and showed a quantitative correlation.Conclusion: The main conclusions of the study are: 1.In vitro neurons,NO can significantly increase the transcriptional activity of FOXO1,and upregulate the expression of FOXO1 downstream proapoptotic genes(Fas L,Bim).2.The NO induced activation of apoptotic pathway regulated by FOXO1,can decrease neuronal activity,and finally induce neuronal impairment and apoptosis.3.The effects of 58 natural Chinese herbal extracts on FOXO1 transcriptional activity are not the same: Moroxydine hydrochloride and Deoxyschizandrin can increase the transcriptional activity of FOXO1,whereas Emodin,Berberine hydrochloride and 2,3,5,4-tetrahydroxyl diphenylethylene-2-O-?-D-glucoside contribute to decrease its activity.In particular,Emodin is worthy of more attention because of its better clinical application effects.4.In vitro neurons,Emodin can significantly inhibit the transcriptional activity of FOXO1,further decrease the expression of FOXO1 downstream proapoptotic gene Bim.What's more,it doesn't cause damage to neurons.5.Emodin can inhibit the transcriptional activity of FOXO1,which is mainly related to the significant reduction of the expression level of FOXO1 protein 6.Emodin can dramatically alleviate the excessive activation of NO to FOXO1,thus attenuate the oxidative damage of neurons induced by NO.This current study first demonstrated the new mechanism of NO in inducing the neuronal damage,which will benefit to deep understanding the neurotoxicity of NO free radical.Secondly,these findings will make FOXO1 as a potential therapeutic target so to screen out drugs which can restrain the activity of FOXO1 to attenuate neurotoxicity caused by oxidative stress.Thirdly,by Chinese herb extracts and antioxidants screening,we identified Emodin as a natural antioxidant that can decrease FOXO1 activity,and provided new experimental statistics to show the neuroprotective effect and molecular mechanism of Emodin as a natural antioxidant.The study was conducted with the focus on the correlation of AD-related naturalantioxidants,NO and FOXO1.A preliminary description of signaling pathway and combinative mechanism of Antioxidants-NO-FOXO1 was provided.All these will certainly provide significant reference to the clinical application of Emodin and other traditional Chinese herbs.This study will also offer a new insight and set of methods to the pathomechanism and treatment of AD and other neurodegenerative diseases.