| Neurodegeneration diseases including Alzheimer's disease (AD), Parkinson's disease(PD), Multiple Sclerosis and the AIDS dementia complex are the most common of old age, the cause of which is very complicated. At present, most of scholars think that activation of neuroglial cells which trigger the chronic inflammatory reactivity play an important role in neurodegeneration diseases brain. A lot of cells such as microglia, astrocyte and neuron are involved in the process of in neurodegeneration diseases, microglial cells are the most important cells in in neurodegeneration diseases brain.Microglia, the resident innate immune cells in the brain, plays a pivotal role in the pathogenesis of in neurodegeneration diseases characterized by chronic inflammation. Microglia adaptes to the central nervous system (CNS) environment and exhibiting different morphological aspects and functional specializations. Two principal forms of microglia have been described:the ramified resident form (resting) and the so-called ameboid microglia (activated). "Resting" microglia presents in the healthy adult CNS and responds to a variety of pathological events by distinct morphological and functional alterations. The ameboid microglia- a transient population detected in the immature brain or in the adult brain after injury and infections. During the resting state microglia express several key surface receptors at low levels- major histocompatibility complex (MHC) I; these include the tyrosine phosphatase (CD) 45 (also known as leukocyte common antigen), CD-14, and CD11b/CD18 (Mac-1). Such "activated" microglia shows enhanced expression of MHCⅡand adhesion molecules and several metabolic characteristics, such as the production of large amounts of oxygen and nitrogen radicals. In neurodegeneration diseases such as AD and PA, the level of MHCⅡin microglia is increased.While microglial function is beneficial and mandatory for normal CNS functioning, microglia become toxic to neurons when they are over-activated and unregulated in AD or PA. Microglia are activated in response to specific stimuli (such asβ-amyloid protein, LPS, ATP, Interferonγ(IFNγ), and advanced glycation end products) to produce pro-inflammatory factors (for example, tumor necrosis factor (TNF)α, prostaglandin E, and IFNγand reactive oxygen species (ROS), which might induce inflammatory responses in neurodegeneration diseases brain and contribute to neuronal injury and death. Moreover direct or indirect interactions between these pro-inflammatoy factors may impair neuronal death, affect microglial activatioin, or activate inflammatory pathways in neurodegeneration diseases. Therefore, identification of molecular targets involved in the initiation and maintenance of microglial activation may lead to a better understanding of inflammatory processes leading to neurodegeneration diseases.Epidemiological data favor that a diet rich in saturated fatty acids (SFA) is considered an increased risk factor for the development of AD. For example, in a 21-year follow-up study, it was found that abundant SFA intake from milk products and spreads at midlife was associated with poorer global cognitive function and prospective memory. Other studies demonstrated that greater intake of saturated fat increased the risk for impaired cognitive function in middle-aged or aged populations. This notion has been supported by animal studies. In this connection, it has been reported that rodents feeding high levels of SFA also show impaired learning and memory performance and develop AD-like pathophysiological changes in their brains.Fatty acid is free to transport across the blood-brain barrier. Therefore, brain fatty acid homeostasis may be dependent on its levels in the periphery. It is therefore conceivable that diets rich in SFA may increase brain uptake of intact free fatty acids from the plasma through the blood-brain barrier. In addition, the fatty acid profile of neurofibrillary tangles in AD brain is rich in palmitic acid (PA) and stearic acid (SA), and the white matter in AD brain is characterized by high total fatty acid contents. PA and SA were reported to increase hyperphosphorylation of tau, and upregulateβ-secretase, the rate-limiting enzyme in production of amyloid beta (Aβ) peptides in primary rat cortical neurons. These actions were mediated by these two SFA on astrocytes, possibly through enhanced astrocytic synthesis of ceramide. Several fatty acids were reported to stimulate the aggregation of tau protein and Aβin vitro. Despite the accumulating data, the basic mechanism behind the causal relationship between SFA and the pathogenesis of AD has not been well established.However, it remains uncertain whether SFA can initiate microglial activation and whether this response can cause neuronal death. Using BV-2 microglial cell line and primary microglial culture, we showed that PA and SA could activate microglia via Toll-like receptor (TLR)4/nuclear factorκB (NF-κB) signaling, as assessed by reactive morphological changes and significantly increased secretion of pro-inflammatory cytokines, nitric oxide and reactive oxygen species, which trigger primary neuronal death.1. The role of SFA in the microglial activationBV-2 cells were maintained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) in a 5% CO2 incubator. For all experiments, BV2 cells were used at 75% to 80% confluency. Primary microglia was prepared from the whole brains of 1-2 days old mouse as described previously. Briefly, mice were sacrificed by decapitation and whole brains excluding cerebelli and olfactory bulbs were isolated. The meninges were removed, tissues were enzymatically digested using trypsin-DNAse, mechanically dissociated, and the cell suspension was passed through 150μm cell strainers before centrifuging at 1000 rpm/min for 5 min. After triplicate counts with hemocytometer, cells were plated into 60 mm tissue culture flasks at a density of 1×106 viable cells/ml in DMEM/F12 supplemented with 10% FBS. Media was changed every 3 days after plating. On day 14, the mixed glial cultures were shaken on on an orbital shaker at 500 rpm for 2 h to dislodge microglial cells. The separate microglial cells were plated into 12-well plates at a density of 1×106 cells/well. The purity of microglia cultures was assessed using CD11b antibody and more than 97% of cells were stained positively. Cells were cultured for 7 days before treatment. Before experiment, plated cells were incubated with serum-free DMEM for 1 h. After this, the medium was replaced with serum-free DMEM containing either PA (C16:0, saturated fatty acid), SA (C18:0, saturated fatty acid), lipopolysaccharide (LPS), pyrrolidine dithiocarbamate (PDTC) or anti-TLR4 neutralizing antibody (anti-TLR4 Ab) for indicated times. Quantitative data were presented as the mean±SD. of at least three independent experiments. Statistical analysis of data was done by Student's t-test or by one-way ANOVA using Dunnett's test in multiple comparisons of means. Differences were considered statistically significant if the P value was< 0.05.Next we prepared fatty acid-album in complexes. PA or SA was solubilized in ethanol at 70℃. Then PA or SA was combined with fatty acid-free and low endotoxin bovine serum albumin at a molar ratio of 10:1 (fatty acid:albumin) in serum-free medium at 50℃for 6 h for a final PA or SA concentration of 25-200μM as described previously. Fatty acid-albumin complex solution was freshly prepared prior to each experiment. The final concentration of ethanol was< 0.5%. In most of the experiments, BV-2 cells or primary microglial cells were treated with individual SFA at 25-200μM concentration, and the controls received BSA and vehicle only.To evaluate the possible contamination of PA or SA with LPS, the endotoxin content was determined by the chromogenic Limulus amebocyte lysate test, following the manufacturer's instructions. The endotoxin content in the 100μM PA and 100μM SA solution was≤3.45×10-3 pg/ml (1.77×10-5 EU/ml), which is far below the concentration required to induce microglial activation under our assay conditions.In order to confirm that incubation with SFA would not induce microglia death, cell viability was assessed at 48 h after PA treatment by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). BV-2 cell viability following treatment with PA at 25μM (94.78±4.34%),50μM (101.09±14.61%), and 100μM (89.49±6.51%) was not significantly different from the control (100.08±5%) (P>0.05). However, exposure of microglial cells to PA at 200μM resulted in significantly fewer viable cells (66.82±4.91%) as compared to cells in the control (P<0.05). And primary microglial cell viability following treatment with PA at (25μM-100μM) was not significantly different from the control (P>0.05). Moveover, SA (25μM-100μM) showed no cytotoxicity on BV-2 cells. In view of this and because PA is common in the diet and constitutes a large proportion of circulating free fatty acids, we have used PA (25μM,50μM and 100μM) as a representative SFA in most of the subsequent experiments.Microglial activation is associated with changes in morphological types and surface antigen. It is interesting to note that primary microglia and BV-2 cells were activated following treatment with PA for 6 h and 24 h. This is manifested by light microscopic imaging which showed that after 6 h and 24 h incubation with PA (100μM), cells assumed a round outline or appeared ameboid form compared to the control cells treated with BSA and vehicle which were mostly ramified with numerous, long extending processes; very few ameboid cells are observed in the control. Microglial activation is associated with marked increase in CD11b expression. At 24 h after treatment with PA (100μM), or LPS (500 ng/ml), our results showed that there was dramatically increased CD11b expression in primary microglial cells.Microglia activation results in pro-inflammatory cytokine secretions, which are toxic to neuronal injury and death. Next we determined the effect of SFA on pro-inflammatory cytokines secretion in microglial cells. RT-PCR analysis showed that in BV-2 cells exposed to different concentrations of PA(25μM,50μM and 100μM) or LPS (500 ng/ml) for 4 h, the levels of TNF-α, IL-1βand IL-6 mRNA expression were significantly increased compared to the control. By ELISA, we then determined the production of TNF-α, IL-1βand IL-6 in the medium of BV-2 cells treated with PA at different concentrations (25μM,50μM and 100μM) for 12 h,24 h, and 48 h. As a positive control, BV-2 cells were stimulated with LPS (500 ng/ml) for 24 h. The results showed both LPS and PA, at either low or high concentrations, stimulated microglia to produce increased amounts of cytokines. In addition, PA stimulated the release of TNF-α, IL-1βand IL-6 in BV-2 cells from 12 h onward at all the concentrations (25μM,50μM and 100μM); the maximum production was observed at 24 h. In view of, we have used PA (100μM) at 24 h as a representative dose in the molecular mechanism experiments. Remarkably, IL-1βsecretion of the PA-treated microglia (409.47±54.54 pg/ml) was higher than those in LPS-treated cells (306.93±45.10 pg/ml) at 24 h. Nonetheless, the levels of TNF-αand IL-6 were 1～2 fold higher in LPS (TNF-α,451.14±40.95 pg/ml; IL-6,209.88±22.60 pg/ml) than those in the PA-treated microglia (TNF-α,344.06±18.38 pg/ml; IL-6,65.19±8.49 pg/ml) at 24 h.Activated microglia is known to release nitric oxide that is capable of causing neuronal damage in CNS.We first tested the effect of PA on NO release by measuring nitrite quantities in BV-2 cells culture supernatants. It was found that upon different concentrations of PA (25μM,50μM and 100μM) treatment for 12 h,24 h, and 48 h or LPS (500 ng/ml) for 24 h, NO release was significantly increased from the cells in all three treatment groups in a dose-dependent and time-dependent manner compared to the control. At 24 h, the levels of NO were higher in 100μM PA (9.44±1.07μM) than those in control (2.58±0.42μM), wherase less than those in LPS (14.11±2.13μM). The cells exposed to different concentrations of PA (25μM,50μM and 100μM) for 4 h also exhibited increase in iNOS mRNA expression in a dose-dependent manner. In conjunction with nitrite quantification, iNOS expression responsible for NO production was evaluated by Western blot and immunocytochemisty. Western blot results showed that PA dose-dependently increased iNOS expression in BV-2 cells. Moreover, in 100μM PA (0.649±0.048) treated group, iNOS expression was higher than that in LPS treated cells (0.503±0.085). By immunofluorescence, iNOS expression was induced after 6 h of treatment with PA (100μM) in primary microglia cells; at 24 h after treatment, the expression was visibly more intense.Microglia activation results in elevation of intracellular ROS levels in them. We next examined whether SFA treatment could affect intracellular ROS levels in BV-2 cells. The cells were treated with PA (100μM) for 12h and 24 h, or LPS (500 ng/ml) for 24 h, following addition with ROS fluorescent probe- H2DCFDA and DHE to detect hydrogen peroxide (H2O2) and superoxide (O2-) production, respectively. It was observed that both 100μM PA (5.88±0.65) and LPS (7.25±0.19) increased H2O2 in BV-2 cells compared to the control (1.01±0.04) at 24 h. At the same time, both 100 μM PA (4.59±0.28) and LPS (4.44±0.67) increased O2- in BV-2 cells compared to the control (1.00±0.04) at 24 h.In order to investigate whether above effects are PA specific, we then tested other SFA-SA, which is present in the serum accounting for close to 13% of the total fatty acids. We found that treatment with 100μM SA for 4 h also induced a marked morphological change and increased expression of pro-inflammatory cytokine mRNA and iNOS mRNA in BV-2 cells. These results suggest that SFA play a rather general role in microglial activation.2. The molecular mechanism of SFA in microglial activationPrevious studies have demonstrated that SFA can induce NF-κB activation in a macrophage. NF-κB is an essential transcription factor for the expression of cytokine and iNOS expression in microglia. We therefore investigated the potential nuclear translocation of NF-κB following the stimulation of microglia with PA. For these experiments, BV-2 cells were treated with or without PA (100μM) or LPS (500 ng/ml) for 1 h, and the p65 subunit of NF-κB in the nuclear fraction was assessed by using immunofluorescence. It was observed that either PA or LPS was capable of activating NF-κB, as demonstrated by the increased levels of the NF-κB subunit, p65, in the nucleus, whereas p65 was localized primarily in the cytosol during the resting state.It has been demonstrated that phosphorylation of serine residues 529 and 536 of the RelA/p65 subunit leads to a transactivation of NF-κB. We next investigated whether PA regulates the phosphorylation of p65. The proteins harvested from the cells after 1 h treatment with or without PA (25μM,50μM and 100μM) were processed for Western blot to detect intracellular level of phospho-p65 (ser536). The result showed that phospho-p65 levels were significantly elevated in all three treatment groups. PA (25μM,0.202±0.028; 50μM,0.342±0.066; and 100μM, 0.456±0.66) and LPS (0.478±0.042) exposure induced phospho-p65 levels in a dose-dependent manner compared to the control (1.00±0.09).In order to observe the effect of PA on the transcriptional activity of NF-κB, cells were transfected with a plasmid construct containing 3×NF-κB binding sites associated with the luciferase reporter plasmid and a control vector. It was observed that PA (25μM,1.70±0.31; 50μM,2.78±0.43; and 100μM,4.06±0.64μM) and LPS (6.03±0.24) exposure induced NF-κB-driven luciferase activity in a dose-dependent manner compared to the control (1.00±0.09).The role of NF-κB in PA-induced proinflammatory cytokines and NO production was examined using specific NF-κB pathway inhibitor-PDTC. In BV-2 cells treated with PDTC (100μM, a non-toxic concentration), for 4 h, PA-induced gene expression of iNOS and pro-inflammatory cytokines were significantly suppressed. In addition, PDTC reduced PA-induced NO, TNF-a and IL-1βsecretion. However, PDTC did not exert a significant effect on PA-induced IL-6 (54.60±10.09 pg/ml) production.These findings indicate that SFA is capable of inducing a rapid response of NF-κB in microglia, triggering the expression of cytokines (e.g., TNF-αand IL-1β) and inflammatory mediators such as NO.We wondered whether PA-induced activation of NF-κB was regulated via TLR4. The results showed that 1 h of PA treatment significantly increased the p65 translocation of NF-κB. Notably, incubating the cells with anti-TLR4 Ab (10μg/ml, a non-toxic concentration) prevented the PA-induced activation of NF-κB, suggesting an involvement of TLR4 in the activation of the transcriptional factors. Moreover, the treatment of BV-2 cells with anti-TLR4 Ab inhibited PA-induced pro-inflammatory mediators production and anti-TLR4 Ab reduced PA-induced NO, TNF-α, IL-1βand IL-6.3. Activation of microglia by PA treatment leads to bystander neuronal deathActivated microglia is known to produce an array of cytokines and other inflammatory mediators that are in turn deleterious for surrounding neurons in the CNS. We wonder whether culture supernatants derived from microglia treated with PA could actually affect neuronal survivality. First we prepared the primary neuron. Primary cultures of mouse cortical neurons were prepared as previously described. Briefly, cortices of 1-2 days old BALB/c mouse were dissected aseptically in calcium-magnesium-free (CMF)-Tyrode solution following decapitation. The meninges were removed, tissue were chopped into smaller pieces and collected in CMF-Tyrode. These were treated with trypsin DNAse and then dissociated in the same solution by triturating to make a single cell suspension, pelleted and resuspended in the serum-free Neurobasal medium with B27 supplement system. Cortical cells were plated at a density of 1.0×106 cells per well and allowed to differentiate for 7 days. Arabinoside was used for the inhibition of astrocyte multiplication. At day 7, the mice neuronal medium was removed and replaced by a conditioned medium from microglial cells.BV-2 cells were incubated in the absence or presence of PA (25μM-100μM) for 12 h and the medium was changed with fresh serum-free DMEM, After 12 h, supernatants were collected and filtered. To check whether PA-induced microglia activation causes bystander neuronal death, we treated primary neurons with the medium mentioned above and served as PA-CM. The control comprised of culture supernatants from BSA and vehicle-treated BV-2 cells and served as control-CM. Neurons were incubated with microglia-conditioned medium for 2 days, and then were analyzed by MTT and Hoechst 33342 nuclei staining. The results demonstrated a significant induction of cell death and apoptosis in PA-CM treated neuron compared to the control. To determine the mechanism of neurotoxicity effect of PA-induced microglial activation, we detected the expression of Bcl-2, Bax and Caspase3. The RT-PCR analysis showed that in PA-CM treated neuron, the expression of Bax mRNA was increased while the expression of Bcl-2 was decreased compared to control-CM. Western blot showd the expression of Bcl-2 was decreased in PA-CM treatment, which suggested that they were involved in the neurotoxicity effects induced by PA. These results indicate that microglia produced inflammatory mediators in response to PA and that the mediators accumulated in the medium were capable of inducing neuronal death.The present results have shown that SFA can induce microglial activation as manifested by its actions on BV-2 cells and primary microglial cells. We have shown that SFA treatment induced microglial activation, as shown by changes in cell morphology consistent with a reactive phenotype, and caused significantly higher production of ROS, NO, and pro-inflammatory cytokines including TNF-α, IL-1βand IL-6 in microglia, resulting in bystander neuronal death. Moreover, PA treatment induced a marked expression of IL-1βand iNOS comparable to that with LPS. Additionally, we have shown that PA treatment activated NF-κB. It is striking that inhibition of NF-κB activation, with its inhibitor PDTC resulted in inhibition of iNOS, TNF-α, IL-1βand IL-6 mRNA expression, and production of TNF-α, IL-1βand NO except for IL-6. Another major finding was that in cells treated with anti-TLR4 Ab repressed PA-induced NF-κB activation and pro-inflammatory mediator productions. These results suggest that SFA could activate microglia and stimulate TLR4/NF-κB pathway to trigger the production of pro-inflammatory mediators, which may contribute to neuronal death by increased in the ratio of Bcl-2/Bax and caspase-3 activation. The present data suggest the potential implications for the mechanisms by which SFA activated microglia could link nutrition with inflammatory diseases of the CNS. Moreover future basic research and experimental animal studies are necessary to clarify the exact mechanisms.
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