The Influence Of Complement Component C1q On Aβ1-40 Fibers-induced BV-2 Microglia Inflammation

Alzheimer's disease(AD) is a progressive neurodegenerative disorder that affects the elderly.The neuropathological hallmarks of the disease include diffused brain atrophy, intracellular neurofibrillary tangles and extra cellular amyloid plaques. Considering the still growing number of the elderly, it has become an increasing burden for the society. At present, the etiology of AD is still not fully understood. However, a numbers of evidence indicate that brain inflammation induced byβ-amyloid(Aβ) deposition is closely associated with the pathogenesis of this disease. The complement system as an important part of humoral immunity, are also involved in the inflammation. In AD brains, researchers found that the early classical pathway complement components C1, C2, C4 and the end Membrane attack complex (MAC). In addition, Strohmeyer found alternative complement pathway is also associated with AD, because he showed that factors B and D, the key factor in the alternative pathway, exist in the AD brain by immunohistochemistry. Although CSF levels of MBL have been found to be reduced by 44% in AD patients compared to control subjects, there is no evidence for direct in-volvement of the lectin pathway in activation of complement in AD. However, It's still unclear which role complement may play in AD brain. Recent studies found complement Clq may have a dual role in pathogenesis and progression of AD. Some scholars find it has neurotoxicity:It's report that multivalent binding of complement protein C1q to the amyloid beta-peptide (A beta) promotes the nucleation phase of A beta aggregation. Domestic research also found that C1q may lead to oxidative toxicity in brain neurons and neuronal death. And some scholars believe that C1q has neuroprotection:In vitro, Pisalyaput confirmed that C1q enhances the phagocytosis of A beta by microglia, and can protect neurons from Aβinduced neurotoxicity. In addition, some study found that C1q inhibits beta-amyloid-and serum amyloid P-induced neurotoxicity. And Deborah recently found that Clq enhances microglial clearance of apoptotic neurons and neuronal blebs and modulates subsequent inflammatory cytokine production.However, whether did Clq advance intracranial chronic inflammation led by Microglia? This is possible, because C1q may promotes the combination of Aβand Microglia. First, complement C1q can combine with Aβ(as described above), and also Microglia had C1qR, C1q receptor, which can bind to C1q. Second, it has also been found that C1q is associated with amyloid deposits in brain. Therefore, C1q, Aβand microglial are very close to the location in AD brain. In summary, it's possible that the complement C1q as an intermediate assists Microglial bind to Aβand thus promotes inflammation led by Microgial.After BV-2 microglial cells treated with 100 mg/L beta- Amyloid fibers (fAβs), some were given C1q, others were given C1q and C1qA. Then, interleukin-6 (IL-6) and tumor necrosis factorα(TNF-α) in the Supernatant and Cell lysate were determined by the sandwich ELISA respectively. And also their mRNA were measured by qPCR. Activated microglial cells are characterized by a round, amoeboid morphology and higher expression of the CD45 marker. So, we also assessed the expression level of CD45 by flow cytometry.METHODS1.1. BV-2 cells were grown and maintained in Dulbecco's modified Eagle's medium(DMEM) supplemented with 4 mM glutamine, penicillin G (100 U/ml), and streptomycin (100 mg/liter). And the medium contained 10% fetal calf serum (FCS) and 4.5 g/liter glucose. Then Incubations were carried out at 37℃in a 90% relative humidity atmosphere of 5% CO2.1.2. The solutions of 1.0 mg/ml Aβ1-40 were incubated at 37℃for 7 days in triple-distilled water. Each sample was placed on Formvar-carbon coated 200 mesh copper grids for 1 min and then were negatively stained with 2% uranyl acetate for 1 min. The grids were examined on a electron microscope at 80 kV.1.3. The viability of the cells was measured by the (CCK-8) test, as described previously. BV-2 cells were seeded in 96-well culture plates containing sera-free meium at an initial density of 50,000 cells per well. After a 12-hr incubation, Aβ1-40(100mg/L), aggregated for 7 days at 37℃(and detected by thioflavin-S), and Clq(10nmol/L or 50nmol/L) and/or C1qA(10nmol/L or 50nmol/L) were added to some of the wells for another 24-hr.Next, fresh sera-free medium containing 10% CCK-8 was administered to the cells, which were incubated for 2 hr at 37℃. The colorimetric reaction was measured by absorption at 450 nm. Because the concentration of the colored product is proportional to the number of viable cells, cell viability was calculated as the percentage of untreated cells that are considered 100% viable.1.4. Ten thousand BV-2 cells per well were cultured for 12hr in 6-well culture plates containing sera-free medium or growth medium. All drugs(as described above) was then added to some of the wells and the cells were incubated for an additional 24 hr. The cells were then fixed for 30 min at 4℃with 4% paraformaldehyde in PBS and permeabilization was carried out by washing the cells with 1% NH4Cl diluted in PBS and adding 0.1% Triton X-100 diluted in PBS for 2 min.After incubation with blocking buffer (3% skim milk in PBS), rat anti-mouse CD45 antibody was added to the cells for 1 hr at room temperature. After thorough washes, rhodamine red-X (RRX)-conjugated donkey anti-rat IgG antibody or fluorescein-isothiocyanate (FITC)-conjugated donkey anti-rat IgG antibody was incubated for 1 hr at room temperature. Finally, the cells were mounted by by flow cytometry.1.5. Microglial cells were plated on 6-well tissue culture plates 50000 cells per well and incubated under the same conditions and with the same treatments. Culture supernatants from microglia were collected 24h after the start of the treatment,centrifuged to remove cells,aliquoted and stored at -20℃, until assayed for the presence of cytokines. To determine intracellular cytokine levels, cells remaining in the wells were washed in PBS and lysed in PBS containing 0.5% of the nonionic detergent Nonidet (NP)-40 (100μl/well).Then the supernatants and Cell lysate were antibated 2 hours at room temperature. After this,100μl of biotin anti-TNF(2μg/ml of the blocking solution) was added to each well and incubated for 2 hours at room temperature,followed by a 20 min incubation with 100μl(2 drops) per well of streptavidin-HRP ready-to use solution.Finally,100μl/well substrate solution (0.05% 3,3',5,5'-tetramethylbenzidine and 0.012% H2O2 in 0.05M citrate buffer, pH5.0) was added and incubated for 30 minutes at 37℃.The reaction was stopped with 1M H2SO4,50μl/well,and the optical density atλ=450 nm was measured in a microplate reader.Concentrations of TNF-a in the samples were calculated from the calibration curve constructed using known amounts of rat TNF-a standards.The same to IL-6.1.6 Ten thousand BV-2 cells per well were cultured for 12hr in 6-well culture plates containing sera-free medium or growth medium.After all treat as above, investigate the mRNA expession of TNF-αand IL-6 by quantitate -PCR.2.Statistical AnalysisAll measurement data were expressed as mean±SD. Statistical analyses were conducted with SPSS13.0 for Windows, comparing continuous variables of groups used One-way ANOVA, LSD method was used as multiple comparison for homoscedasticity, and Dunnett's T3 method was used as multiple comparison for heterogeneity of variance.Significance was difined as P<0.05.[Results]1.Comparison of BV-2 cells survival in each groupIn all treatment, there is no significant difference in survival (F=2.020,P=0.103).2. Comparison of CD45 expression of BV-2 cells in each groupDifferent CD45 expression in each group BV-2 microglia cells are detected by Flow cytometry. In all treatment, there is very significant difference in expressing CD45(F=59.031,P=0.000). And by the treatment of LPS (1μg/L), BV-2 microglia cells express CD45 significantly increased compared with other groups, the difference was statistically significant (P<0.05); treated by Aβ1-40 (100mg/L)+ Clq (50nmol/L), BV-2 microglia cells express CD45 less compared with LPS group, more compared with other group, the differences were statistically significant (P<0.05).3. Comparison of IL-6 and TNF-αin Supernatant and cell lysate of each group3.1 IL-6 concentration of cell lysate and supernatan in each group:In all treatment, there is very significant difference in IL-6 concentration of supernatants and cell lysate (supernatan F=77.838, P=0.000; cell lysates F=24.038, P=0.000). After treating with LPS, IL-6 levels of BV-2 cell supernatants and cell lysates measured significantly increased compared with other groups, the difference was significant (P<0.05), while those of the other groups in the supernatant and cell lysate were no significant difference between each other (P> 0.05)3.2 TNF-αconcentration of the cell lysate and supernatant in each group:there is very significant difference in TNF-αconcentration of supernatants and cell lysate (supernatan F=157.891, P=0.000; cell lysates F=166.897, P=0.000). by LPS, TNF-αconcentrations of he supernatant and cell lysate increased compared with the other groups, the differences were statistically significant (P<0.05). in addition, with the treatment of Aβ1-40 (100mg/L)+C1q (50nmol/L), TNF-αin the supernatant significantly increased compared with the other groups but LPS group(P<0.05).4. IL-6 and TNF-αmRNA expression in comparison4.1 IL-6 mRNA expression levels:In all treatment, there is very significant difference in IL-6 mRNA expression levels(F=66.743, P=0.000). with the treatment of LPS, BV-2 microglia IL-6 mRNA expression was significantly higher than other groups, the difference was statistically significant (P<0.05); and no significant difference between other groups(P> 0.05).4.2 TNF-αmRNA expression levels:In all treatment, there is very significant difference in TNF-αmRNA expression levels(F=65.695, P=0.000). with the treatment of LPS, levels of TNF-αmRNA was significantly higher than other groups, the difference was statistically significant (P<0.05), and treating with Aβ1-40 (100mg/L)+C1q (50nmol/L), BV-2 microglia TNF-αmRNA expression levels were lower than LPS group, higher than other groups, the differences were statistically significant (P<0.05).[Conclusion]1.complement C1q enhanced Aβon microglia activation.2.complement Clq enhanced Aβ-induced microglia inflammation, that is under the intervention of complement Clq and Aβ, TNF-αlevels secreted by BV-2 microglia were significantly increased.