Study On The Neurotoxic Effects Of Microglial Cells Activated By Tryptase Via PAR2

BackgroundMicroglia is the main resident immunological cell in the central nervous system (CNS). Activation of microglia and the neuroinflammation mediated by it play a critical role in the onset and progression of degenerative disease of CNS. Further research work about the activation and regulation of microglia is of great significance, which might be therapeutically applicable in treatment of these diseases.Protease-activated receptor2is a member of G protein-coupled receptors (GPRS), which is activated by trypsin and tryptase. Recently, many studies have shown that PAR2is expressed widely in the CNS and associated with diverse cellular functions including the pathogenesis of ischemia and neurodegeneration. However, both protective and degenerative role of PAR2has been reported. Recent studies suggest that stimulation of PAR2induced astrocytes activation and increased NO production through MAPK-NF-κB pathway. However, reports about physiological and pathological roles of PAR2in microglia are relatively scarce and remain to be largely unresolved. Previously, we detected the expression of PAR2on the microglial cells and mast cell-derived tryptase would promote the activation of microglia via PAR2. Taken together, we hypothesized that microglia activated by tryptase would increase the release of inflammatory mediators via PAR2.In the present study, we aimed to investigate time-dependent changes in inflammatory and neurotrophic response of microglia activated via PAR2, which will lay the foundation for the further research of activated microglia-mediated neurologic disease.Methods1. Time-dependent changes in inflammatory and neurotrophic response of microglia activated by tryptaseCultured microglia cells were incubated with tryptase. Microglia conditioning medium (MCM) was collected at0,0.5,1,2,12,24and72h and used in the experiment. The amount of BDNF, IL-1β, TNF-α in MCM was measured by ELISA. The concentration of nitrite was determined by the Griess assay as described under Materials and methods.2. Specific agonist, antagonist and shRNA for PAR2were used to confirm the role of PAR2in inflammatory response of microglia activated by tryptaseCultured microglia cells were divided into four groups. Group1:control group; Group2:microglia+SLIGRL-NH2; Group3:microglia+FSSRY-NH2+Tryptase; Group4:microglia+PAR2shRNA+Tryptase. MCM was collected at24h later and the concentration of nitrite was determined.3. Neurotoxic effects of microglia according to varying time periods after activation of microglia via PAR2Cultured neurons were incubated using a fresh medium with MCM, which was collect at1(Early group) and72h (Late group) after tryptase stimulation or was unstimulated (Control group). The apoptotic cells were determined by fluorescence activated cell sorting (FACS) and terminal dUTP nick-end labeling (TUNEL) at48h.4. Statistical analysisStatistical analysis was performed by spss13.0software. Data are expressed as means±Standard Deviation (x±S) for each group. The differences among experimental groups were detected by one-way analysis of variance. A P-value of less than0.05was considered to be statically significant.Results1. Tryptase exerts a time-dependent change in neurotrophic and inflammatory responses of microgliaThe concentration of BDNF rose significantly during the first1h of tryptase treatment, but returned to near baseline within72h. In contrast, the concentrations of the pro inflammatory factors IL-1β and TNF-α did not increase until12-24h after the start of tryptase treatment and remained elevated for at least72h. Similarly, nitrate concentration, a proxy for NO release, also rose after about12h of tryptase treatment and continued to increase for the duration of the measurement period.2. Activation of PAR2is involved in inflammatory response of microglia activated by tryptase PAR2activating peptide, SLIGRL-NH2, increased the NO production significantly(11.24±1.09VS2.25±0.26, p<0.05). PAR2antagonist FSLLRY and specific shRNA oligonucleotide for PAR2prevent tryptase induced NO production, which suggest that the tryptase induced NO production is mediated by PAR2activation (2.53±0.40VS2.25±0.26, p>0.05;2.25±0.27VS2.25±0.26, p>0.05)3. Neurotoxic effects of microglia according to varying time periods after activation of microglia via PAR2The percentage of early apoptotic and late apoptotic cells was determined by fluorescence activated cell sorting (FACS). Three populations of neurons were distinguished by this dual staining method: viable (no staining), early apoptotic (Annexin V+PI-), and late apoptotic (Annexin V+PI+). Flow cytometry demonstrated that the percentage of early apoptotic and late apoptotic neurons incubated in72h MCM was significantly higher than in cultures treated with1h MCM or MCM from untreated microglial cultures (13.60%+1.51%VS3.33%±0.51%, p<0.05;13.60%±1.51%VS3.27%±0.42%, p<0.05)The number of TUNEL-positive cells was significantly higher in neuronal cultures incubated in72h MCM than in cultures incubated in media from unstimulated microglia (control group) and the group incubated with1h MCM (17.67%±0.45%VS5.57%±1.35%, p<0.05;17.67%±0.45%VS6.67%±0.47%, p<0.05).ConclusionIn conclusion, the present study revealed that activation of microglia by tryptase via PAR2triggered the sequential release of neuroprotective and neurotoxic factors, with early transient release of BDNF and delayed sustained release of NO, IL-1β, and TNF-α. However, the physiological and pathological roles of PAR2in CNS should be further studied in animal experiments.