Study Of Effect Mechanism Of NADH On Cell Model Of Alzheimer's Disease

Alzheimer's disease (AD) is the most familiar one of braindenaturalization diseases, with main clinic manifestation of progressivedementia. Researches are said that excessive cell apoptosis may be the causeof neural degeneration in AD. A great deal of β-amyloid protein(Aβ)is found in the brain tissue and cerebrovessels sediment of ADpatients, and Aβplays an important role in the formation ofsenile plaque (SP) and occurrence and development of AD. Tillnow, being lack of ideal AD model, studies on mechanism andtreatment of AD are confined to some extent. Primary culturedneuron model was adopted in former researches. Although modelof this kind is a representative and differential one, itsapplication is limited for high technique, unstable culturingsystem and long cycle of experiment. It has been proved to betrue that Clonal Rat Pheochromocytoma Cell (PC12) has nature of neuralorigin and neuron and can be cultured in passage with steady growthbackground.NADH is one of main media in tricarboxylic acid cycle and betweeninner membrane of mitochondrion, and an important assistant enzyme takingpart in cell metabolism and energy reaction. It is key to start biologicalantioxidation and anti-injury of DNA. Studies in vitro showed that NADHcould promote repair of DNA and synthesization of endogenous cell factors,and could rescue cells from apoptosis. However, as far as protection of NADHfor neural injury is concerned, especially apoptosis due to Aβ, there is fewreport domestically and overseas.ObjectiveThe purpose of this study is to build AD cell model through treating withAβon cultured PC12 with neural growth factor (NGF), and toevaluate toxicity of Aβ. Meanwhile, NADH joins in and itsinfluence on injury by A βis valued. The study suppliesprimarily theoretical and experimental proof for application ofantioxidator——NADH in AD treatment in the future.Methods 1.PC12 is cultured in complete DMEM liquid with 50 ng·ml-1( about2nmol·L-1)NGF. About a week later, neural fiber comes up, andafter 10 days of continual cultivation, neuron-like cell comes intobeing. 2.Aβ25-35 of different concentration is added to neuron-likecell and standard indexes are measured on different point. 3.Cell activity of model is evaluated by MTT pre-andpost-treatment with NADH. 4.Concentration of calcium in model cell is measured byFura-2/AM. 5.Changes of DNA in model cell including committingapoptosis is displayed with agarose gel electrophoresis (AGE). 6.Ratio of apoptosis and death in model cell is measured withacridine·orange-ethidium Bromide (AO-EB) staining. 7.Morphological changes of model cell are observed through optic andelectronic microscope pre-and post-treatment with NADH. 8.Ratio of apoptosis and death in model cell is evaluated with HEstaining pre-and post-treatment with NADH. 9.Nuclear injury of model cell is evaluated with TUNEL staining pre-and post-treatment with NADH. 10.LDH releasing rate of model cell is measured to evaluate membranepenetrability pre-and post-treatment with NADH. 11.Degree of lipid peroxidation of model cell membrane is evaluatedthrough ameliorated thiobarbituric acid (TBA) method pre-and post-treatmentwith NADH. 12.Antioxidation ability of model cell is evaluated by measuring activityof SOD,CAT,GSH-PH pre-and post-treatment with NADH. 13.Degree of reactive oxygen species (ROS) injury of model cell isevaluated by measuring NO content pre-and post-treatment with NADH. 14.DNA content of model cell is evaluated through flow cytometry(FCM) pre-and post-treatment with NADH.Results 1.Results of MTT show that cell activity of cultured PC12 with NGFdecreases after treatment with Aβ25-35 and negative relation withperiod and dosage (Repeated Measures One-way, ANOVA,F=11.86, P<0.01); After treatment with 300~400μg·ml-1NADH,rate of living cell is 74.77±2.54%,87.65 ±1.98% respectively,remarkably higher than that of model (52.38±4.63%). 2.Results of calcium concentration show that 36~48 hr later, aftertreatment of 30~40 μmol·L-1A β25-35 , [Ca2+]i is 177.22 ±3.60nmol·L-1,184.39±2.44 nmol·L-1 and 197.55±7.29 nmol·L-1,206.59±3.91 nmol·L-1 respectively, remarkably higher than that of model(92.49±3.65 nmol·L-1,95.16±3.14 nmol·L-1). 3.Result of AGE shows that bigger molecular weight DNA is observednear sample in control and 5μmol·L-1,10μmol·L-1 Aβ25-35 groups;On the other hand, typical trapezium strip——DNA ladder isobserved in 20μmol·L-1 ,30μmol·L-1,40μmol·L-1 groups. 4.Result of AO-EB staining shows that 36hr later, withtreatment of 30μmol·L-1Aβ25-35, ratio of apoptosis is 38.6%;48hr later, 22.8%. Ratio of death rises to 36.2% from 23.4%.Typical trait of apoptosis comes up 6hr later in 40μmol·L-1Aβ25-35, both apoptosis peak and death peak takes on earlier than 30μmol·L-1Aβ25-3 group. 5.Result of HE staining shows that cell morphology inNADH group of lower concentration has few changes withoutstatistical meaning. With treatment of 300~400μg·ml-1NADH,cell count of death and apoptosis decreases remarkably. 6.Result of TUNEL staining shows that rate of positive cellis 20.16±3.22% and 16.51±3.64% in NADH group of 300～400μg·ml-1, decreased remarkably than that of model(42.88±2.35% ). 7.Result of LDH releasing shows that releasing rate inNADH group of 300～400μg·ml-1 is 10.69±2.06% and 6.78±1.38% respectively, decreases remarkably than that of model(25.74±1.42% ). 8.Result of assessment of antioxidation enzyme activityshows that enzyme activity of SOD, CAT and GSH-PH increasesremarkably than that of model. 9.Result of TBA shows that TBARS in NADH group of300～400μg·ml-1 is 64.84±6.19 nmol/g(protein) and 31.27±4.91 7...