| Alzheimer's disease (AD) is a progressive neurological degenerationdisease, which the most common clinical manifestations are disorders ofrecent memory, learning and other cognitive functions, and the major patholo-gical changes exhibit the formation of senile plaque(SP), neurofibrillarytangles (NFTs) and amyloid degeneration of cortex arteries and arteriole, adecrease in numbers of cortex nerve cells. The causes and mechanisms of ADare very complicated. At present, it's considered to be related to the coopera-tion of heredity, metabolism and environmental factors. Neurologists haveraised some hypothesis including loss of cholinergic neuron, deposition ofβ-amyloid protein (Aβ), phosphorylation of tau protein and gene mutation, etc.Recently, many researches have focused on oxidative stress, free radicaltoxicity and cell apoptosis. When Aβ contributing to neurodegeneration in ADis deposited and formed to plaque, reactive oxygen species (ROS) includingH2O2 and ·OH, which damage protein, lipids and DNA, can be produced. AndAβ can activate nuclear factor kappa B (NF-κB), trigger Caspase cascadereaction and induce cell apoptosis. Aβ can lead to AD through neurotoxicitycaused by mechanism of oxidative stress and cell apoptosis, so major scholarsdeem Aβ plays an important role in the pathogenesis of AD. MCI-186, a novelfree radical scavenger, can inhibit lipid peroxidation and cell apoptosis throughclearing up ·OH so that protect vascular endothelial cells and nerve cells. Inthis paper, Aβ25-35, as an inductor, acted on PC12 cells to make fine cell modelof AD. Oxidative damage and apoptosis of Aβ25-35 were studied. This paperintroduced MCI-186 into AD cell model first, and mechanisms of MCI-186 onanti-apoptosis were investigated firstly through discussing the relationshipbetween MCI-186 and expression of NF-κB, Caspase-3 mRNA and protein, inorder to supply theory basis for exploring pathogenesis and exploiting newdrugs of AD.1 Set up AD cell model and the anti-apoptosis effect of MCI-186 on PC12cells induced by Aβ25-351.1 Set up AD cell modelPC12 cells derived from pheochromocytoma of adrenal possess charac-teristics of easy obtainment, long survival time and neuroendocrine, and theyhave been applied to the research of nervous system diseases. Effect of Aβdepends on state of peptide aggregation and concentration of protein, and itstoxic segment is a peptide of 25-35 amino acid. Therefore, Aβ25-35 inducedPC12 cells to set up cell model of AD in this paper.In this paper, analysis of the relationship between time and effect revealedthe maximal inhibitory rate of Aβ25-35 at different concentrations appeared at24-36hr after Aβ25-35 was added into PC12 cells. At 36hr after Aβ25-35 inducedPC12 cells, the relationship between dosage and effect was analyzed, resultsshowed inhibitory rate of PC12 cells gradually increased with increasedconcentrations of Aβ25-35 and IC50 of Aβ25-35 induced PC12cells was 29.76μmol·L-1. Therefore, Aβ25-35 at concentration of 30μmol·L-1 induces PC12 cellsfor 36 hours to develop an ideal AD cell model.1.2 Study on cell apoptosis resulting from PC12 cells induced by Aβ25-35PC12 cells at logarithmic growth phase were divided into 5 groupsincluding the normal control group and Aβ25-35 groups at the concentrations of10, 20, 30, 40μmol·L-1. Concentration of intracellular free calcium([Ca2+]i)and relationship between cell apoptosis and cell death were determined,respectively, at 6hr, 12hr, 24hr, 36hr, 48hr and 60hr after Aβ25-35 at differentconcentrations induced PC12 cells. Results showed apoptosis rate and deathrate of PC12 cells induced by Aβ25-35 was positive correlation with action timeand dosage of Aβ25-35, and appearance of death peak was later 12-24hr thanthat of apoptosis peak. [Ca2+]i of PC12 cells induced by Aβ25-35 was increasedin a time-and dosage-dependent manner. Time of the highest level of [Ca2+]iwas about 12hr earlier than that of cell apoptosis. So, it's concluded thatAβ25-35 can induce cell apoptosis and cell death, but cell apoptosis is dominantand cell death is secondary;cell apoptosis probably results from intracellularcalcium overload.Results of flow cytometry analysis(FCA)showed: numbers of cells at G0or G1 stage increased prominently, numbers of cells at S and G2 or M stagedecreased, cell proliferation index lowered and changes of cell cycle had adosage-dependent relationship with the concentration of Aβ25-35, meantime, thesubdiploid apoptosis peak could be seen in the image of FCA. Therefore, it'sconcluded that differentiation of PC12 cells arrested the cell cycle at theG0/G1-phase by Aβ25-35, thus leading to cell apoptosis.By means of hematoxylin and eosin(HE) staining, TdT-mediated dUTPnick and labeling(TUNEL) staining, and electron microscope, cell apoptosisform of PC12 cells induced by Aβ25-35 was observed. Many apoptosis cellsappeared in the early course of induce. Apoptosis cells with swollen cell,shorten ecphyma, pyknotic nucleus and condensed chromoplasm, could beseen with HE staining. Apoptosis cells, which were characteristic of buffypyknotic and condensed nucleus, could be seen with TUNEL staining. Withelectron microscope, apoptosis cells had the following morphological andbiochemical characteristics: intact cell membrane flattened with disappearingmicrovillus, cytoplasm condensed, and chromatin aggregated to becomecrescent or patching shape and located on the internal surface of nuclearmembrane, but cell organs had fewer changes and electron density of cyto-plasm was near to the normal. Apoptotic bodies were formed at mid-late stageof apoptosis. Except for apoptosis cells, necrosis cells also could be seen andtheir features as followed, cell membrane was destructed, mitochondria andendoplasmic reticulum swelled into vacuole, nucleus disappeared, chromatinbecame into pieces and electron density of cytoplasm significantly decreased.1.3 Study of MCI-186 on resisting apoptosisAfter MCI-186 of different concentrations (10, 20, 30, 40μmol·L-1),respectively, was incubated with PC12 cells for 24hr, 30μmol·L-1Aβ25-35 wasadded. Meanwhile, normal control group and treatment group with Aβ25-35 wasestablished. Then PC12 cells in above six groups went on incubating for 36hr.After that, 5mg·mL-1MTT was added into medium. Result showed 20μmol·L-1MCI-186 had the most powerful protection to PC12 cells.[Ca2+]i of PC12 cells in pre-protection group with 20μmol·L-1MCI-186was lower than that in treatment group with 30μmol·L-1Aβ25-35 (P<0.01), thussuggesting MCI-186 could inhibit intracellular calcium overload.Numbers of cells at G0 or G1 stage decreased, numbers of cells at S and G2or M stage increased, and cell proliferation index elevated in the pre-protection group with 20μmol·L-1MCI-186. There was difference in cell cyclebetween the pre-protection group with 20μmol·L-1MCI-186 and treatmentgroup with 30μmol·L-1 Aβ25-35 (P<0.001), but no difference between MCI-186group and normal control group (P>0.05), so it's concluded that MCI-186 canpromote cells convert G0/G1-stage into S-stage, increase composition of DNA,and induce PC12 cells proliferate. Numbers of apoptosis PC12 cells in thepre-protection group with MCI-186 decreased significantly, thus suggestingMCI-186 can inhibit cell apoptosis.1.4 Effects of Aβ25-35 and MCI-186 on ·OHScavenging effect of ·OH was determined with CuSO4-PHEN-VitC-H2O2biochemiluminescence system. Results showed Aβ25-35 could promote genera-tion of ·OH in the Aβ25-35-PHEN system, but MCI-186 could quench ·OHgenerated by Aβ25-35 in this systems and the highest scavenging rate ofMCI-186 was 20μmol·L-1. Therefore, MCI-186 is characteristic of protectionthrough inhibiting oxidation damage evoked by Aβ25-35.2 Study of mechanisms on oxidative damage resulted from PC12 cellsinduced Aβ25-35 and antioxidation damage of MCI-186·OH can induce oxidative reaction with protein, carbohydrate, lipids andDNA freely, and also promote non-enzymatic glycosylation reaction, thusleading to the formation of toxic carbonyl compounds including proteincarbonyl, advanced glycosylation end products(AGEs)and lipid peroxidationend products, which play an important role in occurrence and development ofAD.In this paper, content of protein carbonyl, AGEs and MDA and score ofDNA injury in the treatment group with Aβ25-35 were higher than those in thenormal group(P<0.001), suggesting that Aβ25-35 can result in effects ofoxidation and glycosylation through ·OH produced by Aβ25-35 acting onmacromolecule substance such as protein, carbohydrate, lipids and DNA. Butabove index in the pre-protection group with MCI-186 were lower than thosein the treatment group with Aβ25-35 (P<0.001 or P<0.01), and those index inthe pre-protection group with MCI-186 were near to the normal level,suggesting that MCI-186 could restrain effects of oxidation and glycosylationby scavenging ·OH produced by Aβ25-35.3 Expression of NF-κB, Caspase-3 mRNA and protein in the PC12 cellsinduced Aβ25-35 and effects of MCI-186 on themAβ and ROS produced by Aβ activate nuclear factor kappa B (NF-κB),which contributes to cell apoptosis and development of AD. NF-κB, as aregulatory factor of cell apoptosis, also acts on Caspase-3 which is anapoptosis effector to cause cell apoptosis in the process of execution. Caspase-3 also plays an important role in AD by participating in the decomposition ofAPP, PS-1 and PS-2.There were many contradictory concepts about effects of NF-κB on cellapoptosis. Some specialists thought NF-κB was an inducing-apoptosis factorwhich could be activated to promote cells apoptosis, the others believed thatNF-κB could inhibit cell apoptosis through inducing expression of apoptosisinhibitor protein to blockade cascade reaction of Caspases and conduction ofapoptosis signals. Even someone referred that effects of NF-κB had double-sides in the neuron, that is, both resistance and promotion to cell apoptosis,which resulted from difference in activated degree and expression level ofNF-κB.In this paper, mRNA expression of NF-κB P65 and Caspase-3 weredetected by RT-PCR, protein expression of NF-κB P65 and Caspase-3 P20were detected by Western blot. Results showed Aβ25-35 could increase theprotein and mRNA expressions of NF-κB P65 and Caspase-3, suggestingAβ25-35 could induce cell apoptosis of PC12 cells. Otherwise, MCI-186 coulddecrease the protein and mRNA expressions of NF-κB P65 and Caspase-3,suggesting MCI-186 could inhibit activation of NF-κB P65 and Caspase-3 toprotect PC12 cells. MCI-186 exerts a protection effect through different targetsin the activation pathway of NF-κB, including lowering expression of NF-κBP65 mRNA, blocking nucleic localization of NF-κB and combination NF-κBwith DNA, and interrupting phosphorylation and degradation of IκB. In thispaper, result showed that appearing of calcium overload was earlier 12hr thanthat of typical cell apoptosis features. Intracellular calcium overload canpromote mitochondria release cytochrome C, activate Caspase-9 and Caspase-3, ultimately lead to cell apoptosis. MCI-186 can cut down calcium overloadand apoptosis pathway caused by Caspase-3 through scavenging ·OH torealize inhibit cell apoptosis and protect nerve cells.Key words: Alzheimer's disease;β-amyloid protein;Pheochromocytomaderived cell line(PC12 cells);MCI-186;Oxidative stress;Cell apoptosis;Nuclear factor kappa B(NF-κB);Cysteinyl aspartate specific protease-3(Caspase-3)...
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