Acid amyloid Lithium inhibition of protein secretion and the protective effects of neuronal.doc

Acid amyloid Lithium inhibition of protein secretion and the protective effects of neuronal Of: Hou Jun Lu Zhang Xiaoyu Luo Huanmin on behalf of Road Abstract Objective To study the acid lithium on human amyloid precursor protein in early old hormone 1 (M146L cells secrete -amyloid protein (A1 42 and its effects on cultured mouse cortical neurons of newborn rats. Methods AD in vitro gene transfection may be secreted A1 42 of the M146L cells in the culture medium by adding low, medium and high dose (3.1,5,8 mmol / L lithium tungsten or positive control, cell activity and by M146L medium A1 42 to the determination of acid lithium cells A1 42 M146L effect. serum-free culture in vitro neonatal mouse cortical neurons by NSE immunocytochemical staining for neuronal identification, observation of neuronal neurite growth status, analysis of acid on cerebral cortex neurons of lithium neuroprotection. Results low, medium and high doses of lithium tungstate had no cytotoxicity on the M146L, low, medium and high doses of lithium inhibit the M146L W cells A1 42, primary cultures of neonatal mouse cortical neurons by NSE immunocytochemistry the cells were stained brown majority, indicating that the cultured cells into neurons basically, In addition to low concentrations ( 0.008 mmol / L, the final concentrations of 0.2,0.04 mmol / L lithium tungsten could significantly increase the number and length of processes, may promote the survival of neurons. Conclusion lithium can inhibit the amount of acid secreted A1 42 M146L cells , cortical neurons of newborn mice have neuroprotective effects. Key Words lithium tungsten, Alzheimers disease, M146L cells, amyloid, neuroprotection Abstract Objective To explore the effects of lithium tungstate (LT) on the secretion of amyloid beta protein 1 42 (A1 42of M146L and the neuropretection on neurons from cerebral cortex of newborn mouse in vitro. Methods M146L cells were culture by low, middle and high dose of LT or positive control drug and the cell activity and the content of A1 42 were detected. The neurons from cerebral cortex of newborn mouse were cultured in B27 supplemented neurobasal, a new serum free medium combination, and identified by NSE staining; MTT assay was carried out to investigate the effect of the LT on development of neurons. Results Different concentrations of LT had no influence on the survival of M146L cells, Different concentrations of LT could inhibit the secretion of A1 42 by M146L cells, Most of the neurons were stained to be positive with NSE, which suggested them being neurons; The results showed that LT (0.04 mmol / L, or 0.2 mmol / L) could increase the number and the length of neurites as well as the number of survival neurons. Conclusions Adequate LT could inhibit the secretion of A1 42 by M146L cells and has a neuroprotective effect on the neurons from cerebral cortex of newborn mouse. Keywords: Lithium tungstate; Alzheimers disease; Amyloid beta protein; M146L cells; Neuroprotective effect amyloid protein (A1 42 generation inhibitors is Alzheimers disease (AD important direction for drug development, most of these drugs is still in experimental research stage, the individual clinical studies. Recent studies show that lithium has neuroprotective role in a variety of factors could reverse neuronal apoptosis induced, to a certain extent, the promotion of neurogenesis (1). Phiel such study found that lithium can inhibit GSK 3 reduced A secretion (2). the same time, found that sodium tungstate is an inhibitor of GSK 3 (3), and combined with the synergistic effect of lithium chloride (4). Therefore, envisaged by the lithium chloride and sodium tungstate tungsten by chemical synthesis of lithium, The effect may be better. the present study, several tungsten lithium reagents with a comparison of neuronal function to verify this hypothesis and to explore a better A1 42 generation inhibitors. tungsten first lithium-based laboratory second synthesis, but also of acid and abroad for the first time the expression of lithium on A, with independent intellectual property rights (patent number: ZL200710027721.1. 1 Materials and methods 1.1 Materials 1.1.1 Experimental Animals Kunming mice (Experimental Animal Center of Guangdong Province to provide, Certificate of Conformity: 2003A003 1.1.2 Reagent M146L cells (Harvard Medical School Dr. Zhang Jimin as gifts, acid lithium (Jinan University, Laboratory of Neuropharmacology, Institute of Brain Science to provide, batch number: 061230, lithium chloride (Tianjin Chemical Reagent Kermel Company, batch number: 00060529, sodium tungstate (Sinopharm Chemical Reagent Co., Ltd., batch number: F20060419, DMEM cell culture medium (Gibco U.S. company, No: 12100046, human A1 42 ELISA kit (Japan WAKO company, approved No: 4008, recombinant human basic fibroblast growth factor (rhbFGF, R & D, batch number: AU824031, other reagents were of analytical grade. 1.1.3 Instrument 680 automatic microplate reader (Bio rad U.S. companies, 3K18 low-temperature high-speed centrifuge (Sigma, USA, cell culture plates (Corning, USA. 1.2 Experimental Methods 1.2.1 Acid Lithium M146L cell activity and A secretion in cell culture of M146L, M146L cell recovery, with 15% FBS DMEM culture medium. The medium containing G418 (200 g / ml, dobutamine Luo adriamycin (puromycin, 25 g / ml anti-eukaryotic cells of two antibiotics, the resistance screening .37 , saturated humidity, 5% CO2 conditions, culture, medium was changed every 2 d subculture, exponential growth of cells taken experiment. (1) cell activity was measured. logarithmic growth phase of the M146L cells with culture medium adjusted to a single cell suspension, the concentration of 2.5 105 / ml.96 hole circle at the end of each hole cell culture plate by adding 200 more than l suspension after 8 h culture, abandoned washing liquid, take 195 l DMEM medium and low, medium and high acid lithium of the 5 l, the final concentrations of 3.1,5,8 mmol / L. The other set control group, lithium chloride group, sodium tungstate group, lithium chloride + sodium tungstate mixed group, and add the appropriate liquid 5 l, after the three final concentrations of 20,5, (5 +5 mmol / L, Repeat 8 holes each dose group. After 24 h culture, the culture medium aspiration, each well add fresh medium 180 l DMEM and 5 mg / ml MTT 20 l. continued after 4 h culture supernatant, add 150 l dimethyl sulfoxide (DMSO, 10 min to a crystal oscillator is completely dissolved, set on a microplate reader, 490 nm wavelength absorbance was measured (OD value. (2) A1 42 content (enzyme-linked immunosorbent assay). Standard protein concentrations were prepared (in pmol / ml: 100,50, 25,10,2.5,1,0, activity was measured with the same cell, cultured for 24 h, each well 100 l medium access. reagent blank control group added buffer 100 l, the blank sample group, dosing group and the standard group, each adding 100 l corresponding reagents, 4 incubated overnight buffer rinse thoroughly and discard the blank samples of the control group, dosing group and standard products are added in each hole labeled antibody 100 l, 4 incubated for 1 h, washed five times repeated, by adding dyes, dark place at room temperature 30 min, each well add 100 l Stop Solution and mix, yellow liquid. with a wavelength of 450 nm detection. Links to free paper download http:/eng.com Figure 1 in cultured cortical neurons of the identification (NSE, 250) (slightly 1.2.2 Tungsten neuroprotective effect of lithium (1) newborn mouse cortical neurons and the identification of serum-free culture: newborn Kunming mice (<24 h cerebral cortex, with neurons into single cell suspension culture medium solution, inoculated into pre-coated 0.012 5% L polylysine 24 well culture plate, seeded 12 for every 130,000 cells, inoculated cultured 4 h to start the experiment. using neuron-specific enolase (NSE immunocytochemical staining for neuronal identification, shall be stained dark brown neurons (Figure 1. (2) Acid Lithium mouse cortical neurite length and number of processes: The small 24-well plates cultured neonatal rat cortical neurons 4 h. medium aspiration, each well add the NeurobasalTM containing 1% B27 medium 390 l, were divided into 8 groups: control group (adding 10 l PBS solution, positive control group: recombinant human basic fibroblast growth factor (rhbFGF group (adding rhbFGF, a final concentration of 20 ng / ml, lithium chloride group (lithium chloride, the final concentration of 1.0 mmol / L, sodium tungstate group (the final concentration 0.3 mmol / L, lithium chloride + sodium tungstate group (final concentrations: 0.1 mmol / L +0.1 mmol / L, lithium tungsten low (0.008 mmol / L, in (0.04 mmol / L, high (0.2 mmol / L three groups, the final volume in each group were 400 l. cultured for 48 h, in the inverted microscope ( 250 under the observation of the growth to cell body was obvious flare, the neurons grow processes for recording object 5 randomly selected for each hole horizons, each of three duplicate wells, with an eyepiece micrometer were measured in each field of vision of 15 neurite length and neurite number, take the mean to compare experiment was repeated 3 times. 1.3 Statistical data are x +-s that the use of SPSS 10.0 statistical software for analysis of variance. 2 Results 2.1 Acid Determination of lithium toxicity test M146L concentration, OD values of each group were: control group 0.891 + -0.038, 0.882 + -0.028 lithium chloride group, sodium tungstate group 0.880 + -0.038, sodium tungstate + lithium chloride, 0.867 + -0.041, low, medium and high concentrations of acid lithium group were: 0.974 + 0.886 + -0.039,0.861 + -0.029 and -0.034. Compared with the control group, low, medium and high concentration of tungsten lithium group and the other positive control group showed no significant difference (P> 0.05, that the group had no significant effect M146L cell activity, no cytotoxic effect. 2.2 Acid Lithium M146L cells A1 42 of Table 1. Lithium chloride, sodium tungstate, sodium tungstate and lithium chloride + lithium group of tungsten acid secretion was inhibited by A1 42. Acid Lithium inhibition of A1 42 secretion dose-effect relationship was certain. Table 1 Acid Lithium M146L cells of A1 42 (slightly Compared with the control group: 1) P <0.05,2) P <0.01, the same below Table 2 Acid 48 h of lithium in cultured cortical neurons (abbreviated 2.3 Acid Lithium mouse cortical neurite length and number of processes shown in Table 2, rhbFGF group compared with the control group, neurite number, neurite length were significantly increased, lithium chloride and sodium tungstate group compared with the control group the number of processes no significant difference, but the lithium group compared with the control neurite length was significantly increased W lithium chloride + sodium group compared with the control group, the number of processes, neurite length were significantly increased. low concentration of acid lithium group compared with the control group, neurite number, neurite length was no significant difference in the concentration of acid in the lithium group compared with the control group, no significant number of processes differences in neurite length was significantly increased, while the high concentration of acid lithium group compared with the control group, neurite number, neurite length increased significantly, but high concentration of tungsten is not the role of lithium and rhbFGF, but better than rhbFGF group, the other groups. newborn mouse cortical neurons by acid after 48 h of lithium treatment, lithium tungsten low concentration group and control group similar to the poorer growth to unipolar, bipolar neurons Lord, processes short, the formation of cross-network less dense, the concentration of acid in the lithium group, significantly more than cells protruding out of the control group and the low concentration of acid lithium group, and there three and multi-level neurons, high concentration of acid lithium group, more neurons survived to three and the main multi-stage neurons, neurite longer, faster growth, and thus the formation of local networks. Figure 2. Figure 2 inverted microscope ( 250 mice in each group were observed in cultured cortical neurons 48 h growth (slightly 3 Discussion Now that the pathogenesis of A in AD plays a very important role. Thus preventing the synthesis and deposition of A and promote its metabolism of drugs in the treatment of AD has great potential (5). A inhibitors inhibit the generation and inhibition of A two types of A aggregation (6). apoptosis in AD brain abnormalities is the main performance, and also the main cause neuronal loss (7). The cell lines used in the experiments while M146L is two transfected genes in human AD (APP and mutant PS1 in CHO cells over-produce it A1 42, is an ideal mechanism of A expression in the cell model. Lithium non-essential trace elements for the human body has a broad role in regulating physiological functions, involving neuropsychological, immune, endocrine and blood system (8). Study found that lithium can inhibit the phosphorylation of tau protein in stable microtubule p p stable catenin and anti-apoptosis (9), can promote in vitro proliferation of neural progenitor cells, glutamate antagonist, glucocorticoid inhibition of proliferation. Another experiment found that lithium could promote small adult rat dentate gyrus cell proliferation and neurogenesis, and the newborn neurons and glial cells was basically unchanged. This result shows that the role of lithium chloride is multifaceted, it can not only increase neurogenesis , but also promote the occurrence of glial cells. study found that lithium chloride, sodium tungstate and the like are GSK 3 inhibitor, lithium chloride and the combined and synergistic effect. but also has been reported, with low sodium tungstate the characteristics of toxicity (3,4). Acid synthesized in our laboratory with the lithium salt of lithium and sodium tungstate their respective advantages. This study also showed that the acid concentration range of lithium in the test, and other similar positive effect of drug group, and M146L cells without toxicity, and M146L cells inhibited the secretion of A1 42. Meanwhile, the cerebral cortex of newborn mice can promote neurite number, neurite length and the number of surviving cells to increase, decrease neuronal death. Therefore, lithium tungsten is expected to become neurodegenerative diseases such as AD, treatment. References 1 Richard SJ.Lithium and GSK 3: one inhibitor, two inhibitory actions, multiple outcomes (J). Trends Pharmacol Sci, 2003,24 (9): 441 3. 2 Phiel CJ, Wilson CA, Lee VM, et al.GSK 3alpha regulates production of Alzheimers disease amyloid beta peptides (J). Nature, 2003,423