Study On The Mechanisms Of Neuroinflammation Regulated By Phosphatidylcholine-Specific Phospholipase C

ObjectiveMicroglia are innate immune cells in the central nervous system, it plays a key role in a variety of nervous system diseases associated with the inflammatory response. Thus it is very important for us to study the injury and repair of neuroinflammatory response related to activated microglia. However, the exact mechanism of microglia activiatation is not well understood.Phosphatidylcholine-specific phospholipase C (PC-PLC) might play a key role in the signal transduction. The PC-PLC hydrolyses phosphatidylcholine to DAG. DAG is the second message in the cells and activates protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Under various physiological and pathological conditions, the signal transduction via PC-PLC is important in cell proliferation, differentiation and apoptosis. Previous studies indicated that PC-PLC was both involved in neural development and inflammatory response. Furthermore, PC-PLC plays an important role in the LPS-mediated signal transduction. D609, a specific inhibitor of PC-PLC, be used to study the process of microglia activation in this study. The study would provide a theoretical basis for studying the molecular mechanisms of microglia activation. Our data would provide a theoretical basis for the neurological diseases theraphy in clinic.Methods1. Observation of cell morphological changes by Phase Contrast Microscope.2. Detection of activated microglia:1) Observation of cell morphological changes by Phase Contrast Microscope.2) Analysis of inflammatory cytokines TNF-αand IL-1βby ELISA assay.3) Nitrate reductase method to detect inflammatory mediators NO content.3. Analysis of the effect of PC-PLC in activated microglia using its specific inhibitor D609. 1) Cell viability was determined by MTT-assay.2) PC-PLC activity assay.3) Analysis of inflammatory cytokines TNF-αand IL-1βby ELISA assay.4) Nitrate reductase method to detect inflammatory mediators NO content.4. Analysis of the mechanism in activated microglia mediated by PC-PLC.1) Detection the phosphorylation levels of p38 MAPK, JNK, ERK by Western blot method.2) Nitric oxide synthase (NOS) activity assay was performed by the NOS detection kit .Results1. BV2 microglial cell culture Morphological changes of BV2 cells could be observed on phasecontrast microscopy. BV2 cells clustered distribution, smaller cell body, cytoplasm concentration, refractive index and strong, clear and bright.2. LPS could activate microglia.2.1 Morphological changes of BV2 cells stimulated by LPS Morphological changes of neuron could be observed on phasecontrast microscopy. When the cells were exposed to LPS 1μg/ml for 24h, cell morphology changed significantly. Cell body shaped from the normal amoeba-like into flat cell bodies become larger, more spreading, processes significantly reduced or no processes, the majority of suspended cells.2.2 LPS could effectively induce the activation of microgliaThe results from ELISA assay experiments showed that when the BV2 cells were exposed to LPS 1μg/ml for 24h, the activities of inflammatory cytokines TNF-αremarkably increased, 10 times more than normal control cells (P<0.01). Meanwhile, inflammatory factor IL-1βwas increased significantly (P <0.01).2.3 LPS could induce the inflammatory mediators NO releaseThe results from Nitrate reductase assay experiments showed that when the BV2 cells were exposed to LPS 1μg/ml for 24h, the content of NO was 28.14μmol/L twice more than the content of NO which in normal control cells (P<0.01).3. PC-PLC involved in the activation of BV2 microglia3.1 The cytotoxicity of D609 on microgliaThe PC-PLC activity assay showed that after treatment with D609 50-200μM, the activities of PC-PLC decreased significantly in a dose dependent manner (P<0.01). The results from MTT assay experiments showed that when the cells were exposed to D609 10-100μM for 12-48h, there was no significant difference in the viability between normal and D609 treated groups. However, following the treatment with D609 200μM for 24-48h, the cell viability declined remarkably (P<0.01). In subsequent experiments, 100nM D609 was used.3.2 PC-PLC involved in the regulation of inflammatory cytokine release on activated microgliaThe results from ELISA assay experiments showed that following the pretreatment with D609 100nM for 30min, the production and release of inflammatory cytokines TNF-αand IL-1βinduced by 1μg/ml LPS was inhibited significantly.3.3 PC-PLC activation in microglial release of NO Following the pretreatment with D609 100nM for 30min, the LPS induced intracellular NO level was decreased significantly. 4. The related mechanism of microglia activation mediated by PC-PLC. 4.1 The signal transduction of microglia activation regulated by PC-PLC. Western blot analysis revealed that when the BV2 cells were exposed to LPS 1μg/ml for 30min, phosphorylated SAPK/JNK and phosphorylated p44/42 were increased significantly. Following the pretreatment with D609 100nM for 30min, the LPS induced phosphorylation could be inhibited obviously. These results suggest that PC-PLC contributed to the microglial activation through the SAPK/JNK and p44/42 signaling pathway. 4.2 PC-PLC regulated the activation of microglia by mediating the activition of iNOS. After treatment of LPS 1μg/ml for 24h, the activation of TNOS and iNOS was significantly increased in cell culture medium (P <0.01), while the activity of cNOS did not significantly differ between normal or treated groups. Following the pretreatment with D609 100nM for 30min, the LPS induced iNOS level could be inhibited obviously (P <0.01). conclusion 1. LPS could activate microglia, the inflammatory cytokines TNF-αand IL-1βincreased significantly. 2. PC-PLC involved in the activation of BV2 microglia 3. PC-PLC contributed to the microglial activation through the SAPK/JNK and p44/42 signaling pathway. 4. PC-PLC regulated the activation of microglia by mediating the activition of iNOS.