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Study on the Mechanisms of Neuroinflammation Regulated by Phosphatidylcholine-Specific Phospholipase C

Author: FuJiHua
Tutor: LiFu
School: Taishan Medical College
Course: Oncology
Keywords: Lipopolysaccharide Microglia Phosphatidylcholine -specific phospholipase C Inflammatory
CLC: R741
Type: Master's thesis
Year: 2011
Downloads: 26
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Abstract

Purpose of microglia in the central nervous system of innate immune cells in a variety of neurological disorders associated with inflammatory response plays a key role. Microglial activation and thus research on the nervous system inflammatory response and repair the damage is significant. Experiments, we used lipopolysaccharide activated microglia activation process to study the release of inflammatory cytokines and their signal transduction mechanisms. Phosphatidylcholine-specific phospholipase C (PC-PLC) is prevalent in a variety of cells is an important enzyme that specifically hydrolyzes phosphatidylcholine and di-esters of phosphoric acid acyl glycerol choline, the latter as a cell the second messenger can further activation of protein kinase C, and then transmits the signal to the mitogen protein kinase, causing cell proliferation, differentiation and apoptosis, and many other functional changes. PC-PLC in nervous system development and inflammation signaling pathway plays an important role. We study the use of its specific inhibitor D609 PC-PLC regulate microglial activation role in the process, in order to clarify the PC-PLC in the process of microglial activation mechanism of action provides experimental evidence, as well as the clinical treatment of nervous system theoretical basis for the disease. Methods 1. Inverted microscope morphological changes (2) the detection of activated microglia 1) cell morphology was observed under an inverted microscope changes. 2) ELISA detection of inflammatory cytokines TNF-а, IL-1β activity, evaluation of microglia activation. 3) the use of nitrate reductase method to detect changes in NO levels of inflammatory mediators. 3 Use the PC-PLC inhibitor D609, PC-PLC study microglial activation in the role of a) using MTT method to detect D609 cytotoxicity. 2) PC-PLC activity was detected. 3) ELISA detection of inflammatory cytokines TNF-а, IL-1β activity to evaluate PC-PLC in the role of activated microglia 4) the use of nitrate reductase method to detect changes in NO levels of inflammatory mediators. 4. PC-PLC in microglial activation in the mechanism of a) Western blot was used to detect p38 MAPK, JNK, ERK phosphorylation levels. 2) the use of nitric oxide synthase (NOS) kit to detect iNOS activity. Results 1. BV2 microglial cell cultures was observed by inverted phase contrast microscope, BV2 cells clustered distribution, smaller cell body, cytoplasmic concentration, refractive index and strong, clear and bright. 2. LPS activates microglia 2.1 LPS stimulated BV2 morphological changes observed results BV2 cells treated with 1μg/ml LPS 24h, significant changes in cell morphology, cell body morphology changes from the normal amoeba-like flat, soma larger than spreading, projections significantly reduced or no protrusions, cell suspensions majority. 2.2 LPS can effectively induce microglial activation was detected by ELISA in this study found 1μg/ml LPS stimulated BV2 cells after 24h, treated cells produce and release large amounts of inflammatory cytokines TNF-α, and the normal control cells was increased by about 10-fold difference was significant (P lt; 0.01). Inflammatory cytokines IL-1β was increased by 2-fold difference was significant (P lt; 0.01). 2.3 LPS can activate microglia, release of inflammatory mediators NO In this study, nitrate reductase assay BV2 NO content in culture medium and found that when stimulated with 1μg/ml LPS BV2 cells after 24h, the culture medium was 28.14 NO content μmol / L, than the normal control group cells 12.35μmol / L increased twice as highly significant difference (P lt; 0.01). 3. PC-PLC in BV2 microglial activation D609 3.1 PC-PLC inhibitor microglia cells of toxic effects under the conditions of serum ,50-200μM of D609 can effectively inhibit the activity of PC-PLC, and showed concentration-dependent manner. MTT assay was found in the presence of a serum ,10-100μM D609 processing BV2 cells 12-48h no effect on cell viability, and 200μM D609 BV2 cells treated 24h and 48h can inhibit cell survival. Therefore, this study using the D609 PC-PLC activity blocking effect concentration of 100μM. 3.2 PC-PLC activation of microglial cells involved in the regulation of inflammatory cytokine release ELISA assay used in this study can be found 1μg/ml LPS activation of BV2 cells after 24h, produce and release large amounts of inflammatory cytokines TNF-α and IL- 1β. 100μM D609 pretreatment 30min blocked PC-PLC activity, can effectively inhibit the inflammatory cytokines TNF-α and IL-1β release. 3.3 PC-PLC activation of microglial cells involved in NO release this study, nitrate reductase assay BV2 NO content in culture medium and found that when stimulated with 1μg/ml LPS BV2 cells after 24h, the culture medium with the normal activity of NO the control group was significantly higher compared to cells. D609 can inhibit LPS-induced NO release. 4. PC-PLC regulate microglial activation mechanism of action 4.1 PC-PLC by regulating the SAPK / JNK and p44/42 signaling pathway mediated by the activation of microglia. Use 1μg/ml LPS activated BV2 cells after 30min, p38 protein phosphorylation was not obvious, but SAPK / JNK and p44/42 protein phosphorylation levels were significantly increased. D609 pre-blocked with PC-PLC activity, can inhibit SAPK / JNK and p44/42 phosphorylation. 4.2 PC-PLC by regulating the activity of iNOS in mediating the activation of microglia. The results show that 1μg/ml LPS activation of BV2 cells, cell culture medium total TNOS and inducible iNOS activity was significantly higher (P lt; 0.01), while the structural cNOS activity did not change significantly. Blocked by pretreatment D609 PC-PLC activity, can inhibit iNOS activity increased (P lt; 0.01). Conclusion 1. LPS activates BV2 microglial cells in the experimental group of inflammatory cytokines IL-1β, TNF-α levels were significantly higher. 2. PC-PLC in BV2 microglial activation, the use of PC-PLC inhibitor D609, can effectively inhibit the inflammatory cytokines TNF-α, IL-1β, NO release. 3. PC-PLC by regulating iNOS activity, in mediating the activation of microglia. 4. PC-PLC by regulating the activation of SAPK / JNK and p44/42 signaling pathway mediated by the activation of microglia.

Study on the Mechanisms of Neuroinflammation Regulated by Phosphatidylcholine-Specific Phospholipase C

Abstract

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