| Alzheimer’s disease (Alzheimer disease, AD), characterized by progressive cognitive dysfunction and the pathological loss of neurons, is one of the major neurodegenerative diseases in the current world. The clear mechanism associated with AD remains unclear. Although a variety of hypothesis have been put forward, such as the Tau protein related theory, inflammatory mechanisms, the doctrine of free radical damage or cholinergic factors, the theory of Aβ toxicity is still the mainstay hypothesis.AD is mainly due to the formation of senile plaques, composing of Aβ (β-amyloid protein), and the aggregation of hyperphosphorylated Tau protein, causing neurons filament winding and the lost of neurons. In the studies of neurodegenerative diseases, Aβ exits in two different states:oligomers or fibrils. These two states exert different intensities of damage to neurons. It is generally believed that the toxicity of Aβ oligomers is stronger than Aβ fibrils. Nevertheless, because Aβ oligomers are less stable and easily convert into fibrils, the time-window for vitro studies is relatively short. As a consequence, the identification of Aβ oligomers is particularly important. Otherwise it is likely to lead to false-positive results for the testing drugs. Unfortunately, there is few reports on the identification method for the aggregation state of Aβ. To this end, the present experiment firstly prepared different states of Aβ1-42, and then test the possibility of identification through comparing morphology under an atomic force microscope.During the development of AD, excessive deposition of Aβ forms senile plaques due to the dynamic imbalance between Aβ production and clearage. As a result, a large number of activated microglia cells are attracted to gather around senile plaques in AD. The aggregation of Aβ induces microglia cells to produce inflammatory cytokines, including TNF-α and IL-1β, leading to the loss of neurons and other kinds of cells. After identification of the state of Aβ, the present study then used the more toxic Aβ oligomers to establish AD microglia models in vitro and based on that, investigated the inflammatory mechanisms of AD.It has been found that the complement system plays an important role in the inflammatory pathogenesis of Alzheimer’s disease. Aβ, microglia, astrocytes, abnormal upregulated complement and cytokines together form a complex network of inflammatory injury, resulting in a series of inflammatory response, eventually leading to neuronal apoptosis. During this process, the damage mediated by the complement system is a very important aspect:Aβ specifically binds the classical complement pathway C1q and after activation, C1q further activates C3, C4, C5sequentially, while decomposing respective complement factors and generating various biologically active fragments C3a, C4a, C5a. These active fragments eventually form the membrane attack complex (MAC, C5b-9complex) and cause the dissolve and apoptosis of neurons in the brains of AD patients. In this process, the role of C5a is not very clear. Recently, studies have found that in C3-/-mice, IgG immune complexes was still able to produce C5a-dependent acute lung injury, and the strength was comparable to the wild-type mice. This finding suggests that the generation of C5a could be independent of C3, indicating a brand-new activation pathway of the complement system. This also implies that the generation of C5a will produce a similar inflammatory injury induced by the classical activation pathway of the complement system.Therefore, the current study aimed to investigate the possible effects and related mechanism of C5a on AD microglial activation and the release of inflammatory mediators. Further, the study also aimed to clarify the treatment effect of the administration of the C5a receptor antagonist (PMX205) in this process.[Objective]To establish the method to prepare and indentify Aβ1-42oligomers and fibrils. After the establishment of microglia lesions model of Alzheimer’s disease (AD) inducing by Aβ1-42oligomers, the study further aims to investigate the possible effects and related mechanism of C5a and its receptor antagonist (PMX205) on AD microglial activation and the release of inflammatory mediators.[Materials and Methods]1. Materials:Mice BV2microglia cell line was purchased from Wuhan University Type Culture Collection Centre, Australia fetal bovine serum (GIBCO), DMEM/F12high glucose medium (GIBCO), pancreatin (containing EDTA,0.25%, the biochemical companies of jitai), synthetic biocrystals Aβ1-42(ENZO Company), hexafluoroisopropanol (HFIP, Sigma), dimethyl sulfoxide (DMSO, and MP Biomedicats), complement C5a (HBT Company), C5a receptor antagonist (PMX205) hydrocinnamate-[OP (D-Cha) WR](GL Biochem Limited synthesis), PE-labeled anti-mouse CD88stream-the antibody (Biolegend company), mouse TNF-a ELLSA kit (Biolegend), CCK-8kit (Japan&Associates, Inc.).Instrument:Spotless workbench (China AIRTECH), cell incubator (U.S. Thermo FOMA), inverted microscope (Japan OLYMAPUS,), flow cytometry (Germany, Becton Dickinson), low-temperature high-speed centrifuge (Germany, Beckman The), SynerTM HT multi-channel microplate reader (United States of America, BIO Tek), atomic force microscopy (the Primitive Nano Instruments, Inc., Model CSPM5500).2. methods:2.1BV-2cell culture BV-2cell lines were inoculated into the medium, consisting of high glucose DMEM and10%fetal bovine serum. Cells were incubated in an incubator at37℃+5%CO2and were passaged once every three days, with0.25%trypsin+0.02%EDTA digestion.2.2Preparation of Aβ1-42oligomers and fibrils lmg Aβ1-42powder was dissolved in220μL hexafluoroisopropanol (HFIP), and incubated at room temperature for60min until Aβ1-42fully dissolve. Then the Aβ peptide-HFIP was placed in the ice for5～10min in the fume hood until the complete volatile of HFIP. After air-drying, Aβ peptide-HFIP formed a transparent film and then was dissolved in44μL of freshly anhydrous100%DMSO to get the5mmol/L Aβ1-42synthetic peptide. Next, the peptide was diluted with phenol red-free F12medium to a final concentration of50nmol/L, which was incubated for24h at4℃. The solution was then centriguated with14000r/min at4℃for10min. And the supernatant was just the Aβ1-42oligomers, which was stored under-80℃for future use. If5mmol/L Aβ1-42-DMSO synthetic peptide was dissolved with10mmo/L hydrochloric acid and then diluted to a final concentration of50nmol/L, placed under37℃for24hours, Aβ1-42fibrils were therefore made, which was stored under-80℃for future use.2.3Identification of different states of β1-42using an atomic force microscope. Aβ1-42products were drooped on the surface of freshly dissociated mica and the mixture was allowed to stand at room temperature for5minutes, followed by gently rinsing with deionized water and being dried for10minutes. After that, the product were put in a drying dish placed in the observed under an atomic force microscope. Use the contact mode and then the tapping mode to scan the products, respectively. Parameters for the tapping mode:force constant40N/m, resonance frequency300kHz, scan frequency1Hz. The images were processed using the software Imager4.60.2.4Comparison of the viability of BV-2cells treated with Aβ1-42oligomers and fibrils by the method of CCK-8. BV-2microglia cells were seeded in96-well plates with a density of105/ml, and were cultured with serum-free DMEM/F12high glucose medium for24hours. After that, different concentrations (0.01um/L,0.1um/L,1um/L10um/L) of Aβ42oligomers and fibrils were then added to the medium. After24h, the supernatant was sucked and then180ul medium+20ul CCK-8were then added to each well and the cells were incubated in darkness for2hours. Finally, the OD value in450nm of wavelength for each group was measured with a microplate reader. This experiment was repeated for three times.2.5Establishment of AD microglia disease model, stimulated by Aβ1-42oligomers. The experimental groups included the groups treated by Aβ1-42, Aβ1-42+C5a, Aβ1-42+C5aRA, Aβ1-42+C5a+C5aRA, respectively, with a group cell without any intervention as a control.2.6Levels of TNF-α in supernatant detected with ELISA. Twenty-four hours after treatment, the supernatant was collected from different groups and the TNF-α levels was measured with ELISA, as instructed by the manufacture. The absorbance values (A) was then measured by a96-well microplate reader under450nm of wavelength. Finally, the levels of TNF-α were calculated according to the standard curve.2.7The expression of CD88using flow cytometry. Twenty-four hours after treatment, cells in different groups were digested and resuspended into single cell suspension and incubated with PE-labeled anti-mouse CD88antibody for30min at4℃. Then the cells were washed3times with PBS, followed by detecting the expression of CD88with flow cytometry.StatisticsAll measuremental data were expressed as means+s. Statistical analysis were conducted with SPSS13.0for Windows, comparing continuous variables of groups used One-way ANOVA,LSD methods was used as multiple comparison for heterogeneity, and Dunnett’s T3method was used as multiple comparison for heterogeneity of variance. Significance was defined as P<0.05.[Results]1. By AFM, the oligomeric form of Aβ1-42appeared as small globular or oval structures that were about5nm in height. In contrast, the fibrillar form of Aβ1-42appeared as long threads about5nm in height and>1OOOnm in length.2. Aβ1-42oligomers and fibrous could both damage BV-2cell with a dose-effect relationship at the same time and the same concentration level.The survival rates of BV-2cell treated with Aβ1-42oligomers were significantly lower than those treated with fibrillar form of Aβ1-42, since the concentration of0.0um/L levels (P<0.05).3. Aβ1-42oligomer significantly stimulated BV2cells to release TNF-a and CD88, compared to the control group ((t=16.60, P<0.05and t=4.81, P<0.01, respectively). C5a further increased the release of TNF-a (F=24.50, P<0.05). In contrast, C5aRA inhibited remarkably the release of TNF-a, stimulated by Aβ1-42(F=280.12, P<0.01).50nmol/L C5aRA significantly inhibited the release of TNF-α in AD microglia cell group treated by5nmol/L C5a (t=28.06, P<0.01), and100nmol/L C5aRA significantly reduced the concentration of TNF-α treated by10nmol/L C5a (t=5.73, P<0.01). Nevertheless, C5a or C5aRA didn’t show a significant effect on the expression of CD88in BV2cells, treated with Aβ1-42(P>0.05).[Conclusions]AFM offers a simple, direct and accurate way to differentiate between the oligomeric and fibrillar states of AP1-42. Oligomeric Aβ1-42is more cytotoxic than the fribrilar one. C5a enhances the effect of releasing TNF-a from Microglial cells, stimulated by Aβ1-42and this effect can be significantly inhibited by the application of C5aRA. |
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