The Role And Mechanisms Of SPLA₂in Neuroinflammation

BackgroundNeuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. One of the main characters of CNS inflammation is the generation of the pro-inflammatory mediators, such as arachidonic acid (AA). The main enzymatic process responsible for AA metabolism is catalyzed by phospholipase A2(PLA2). AA released mainly by PLA2from the Sn-2position of phospholipids is metabolized by cyclooxygenases (COX) and lipooxygenases (LOX) to its eicosanoid metabolites such as prostanoids (PG) and leukotrienes (LT) that damage brain cells. The most abundant isoforms of PLA2enzymes family include cytosolic PLA2α (cPLA2α) and secretory PLA2(sPLA2). However, the influence of sPLA2on endotoxin such as lipopolysaccharide (LPS) Induced neuroinflammation has not been reported. Acetylpuerarin is a newly modified isoflavone based on puerarin. Although current studies indicate that acetylpuerarin produces neuroprotection against ischemia-reperfusion injury, the precise mechanisms for its beneficial effects are still not fully understood.ObjectivesThe objective of the study is to investigate the role of sPLA2and anti-inflammatory effect of acetylpuerarin on eicosanoid signaling pathway.Materials and MethodsThis study includes two parts:1. The role of sPLA2-IIA in LPS-induced neuroinflammation was investigated using primary rat astrocytes. RT-PCR analysis was performed to examine the mRNA level of sPLA2-IIA in primary rat astrocytes and microglia. ELISA-based assay was used to measure the PGE2concentration of the supernatant of astrocytes. Western blot determined the protein expression related to AA signaling. In vivo induced by the intracerebroventricle microinjection (i.c.v.) of LPS was employed to explore the regulation of MAPK-cPLA2a-PGE2pathways by sPLA2-IIA in mice cerebral cortex. ELISA-based assay was used to determine the PGE2concentration of the supernatant of mice cerebral cortex. Protein expression of sPLA2-IIA, phosphor-ERK1/2, phospho-p38, phospho-cPLA2a, COX-2and mPGES-1were evaluated by western blot.2. The pharmacokinetic properties of puerarin after oral or i.v. dosing of acetylpuerarin was measured by HPLC analysis. We used primary rat astrocytes to simulate inflammatory situation to study the role of acetylpuerarin in LPS-stimulated route of eicosanoid biosynthetic pathway. We used MTT solution to examine the cytotoxicity of acetylpuerarin in primary rat astrocytes. Western blot and immunofluorescence staining were used to determine the protein level of group V sPLA2, PGE2and LTC4concentration was measured by using ELISA-based assay kit. Western blot examined the activation of ERK1/2, cPLA2a, NF-kB and protein expression of COX-2and LOX-5.Results1. LPS exposure led to a time-and dose-dependent increase of sPLA2-IIA mRNA expression in murine primary astrocytes but not induce increase of sPLA2-IIA mRNA or protein expression in the primary microglia, we used astrocytes to simulate similar inflammatory situation in vitro to study the role of sPLA2-IIA in LPS-stimulated route of PGE2biosynthetic pathway. To investigate the effect of sPLA2-IIA on PGE2release, a highly specific sPLA2-IIA inhibitor, SC-215, was presented to the medium of astrocytes together with LPS. SC-215treatment lowered the elevated PGE2level induced by LPS in a dose dependent manner in astrocytes. In vivo study we found that the addition of SC-215(1.218μg) with LPS (2.5μg) showed a nearly90%decrease in sPLA2-IIA expression. As expected, PGE2production were significantly inhibited (>75%) in the cortex of SC-215-treated mice at the same time point. Our study found that LPS caused a time-dependent induction of phospho-cPLA2α, phosphorylation of ERK1/2preceded that of cPLA2α and increased to a maximum at10minutes in LPS treated brain tissue. The residual phosphorylation was still detectable30minutes after stimulation and returned to baseline by60minutes. In subsequent experiments, changes in the activation of these downstream molecules in cortex after SC-215treatment were examined. The phosphorylation of cPLA2a at1hour and phosphorylation of ERK1/2at10minutes were significantly prevented in mice treated with SC-215prior to LPS challenge, but the induction of phospho-p38at10minutes were not affected upon SC-215pretreated and remained similar to LPS challenge values. Mice were i.c.v. injection of an inhibitor of cPLA2α activity (AACOCF3) or MEK1/MEK2inhibitor (U0126) and then exposed to LPS for1hour. AACOCF3and U0126infusion did decrease the brain concentrations of PGE2by3.5-and4-fold, respectively. The constitutive COX-1enzyme was expressed only at low levels in cortex and was not significantly altered by LPS treatment. In contrast, the COX-2enzyme was not expressed under basal conditions in mice cortex but was induced by LPS stimulation and was downregulated in the presence of SC-215. The mPGES-1is the final enzyme in the cascade to ultimately generate PGE2. In mice cortex, mPGES-1was found to be constitutively expressed and was upregulated upon LPS treatment, the change was significantly prevented by sPLA2-IIA inhibition.2. The time-concentration curve of puerarin was accord with two-compartment model. The exposure profile of puerarin in plasma after i.g. administration acetylpuerarin were as follows: Cmax (12.07±0.15) μg/mL,Tmax (1±0.33) h, t1/2(2.52±0.21) h, AUC(0-∞)(44.76±4.13) mg·L-1·h. The AUC(0-∞) of puerarin after iv administration is (36.67±5.3) mg·L-1·h. The absolute bioavailability of puerarin is48.12%. Acetylpuerarin exerted no significant cytotoxicity on primary rat astrocytes. To examine the anti-inflammatory effect of acetylpuerarin on primary astrocytes, we analyzed the LPS-induced generation of PGE2and LTC4. The pretreatment of acetylpuerarin inhibited LPS-indcued production of PGE2and LTC4dose-dependently. To investigate the effect of acetylpuerarin on group V sPLA2, the protein level and immunoreactivity were examined by double immunofluorescence labeling and western blot. The results showed that group V sPLA2expression was noticeably enhanced by LPS stimulation, pretreatment with acetylpuerarin resulted in a dramatically dose-dependent decrease in group V sPLA2expression. To investigate the possible molecular mechanism by which acetylpuerarin prevents release of eicosanoids, we examined the LPS-induced phosphorylation and activation of cPLA2α, ERK1/2and p65subunit of NF-κB. Our data showed that acetylpuerarin partially blocked the induction of phosphorylation of NF-κB as well as phosphorylation of ERK1/2and prevents subsequent activation of cPLA2α in a time-dependent manner during LPS exposure and further decrease formation of inflammatory lipid mediators. Furthermore, stimulation of primary rat astrocytes with LPS caused approximately4-fold and2-fold increase in the protein levels of COX-2and LOX-5, respectively. Pre-incubation of astrocytes with acetylpuerarin prevented the LPS-induced protein expression of COX-2and LOX-5dose-dependently.Conclusions1. LPS evokes marked PGE2generation in primary astrocytes in a time-dependent fashion accompanied by release of sPLA2-IIA. The inhibition of sPLA2-IIA alleviates the release of PGE2following the neuroinflammation induced by LPS. ERK1/2, not p38is required for the sequential activation of cPLA2α and further release of PGE2induced by LPS. The elevated signaling of the sPLA2-IIA-ERK1/2-cPLA2a-COX-2-mPGES-1pathway contributes to PGE2overexpression and secretion in primary astrocytes and mice cortex.2. The established method could be used for the analysis of pharmacokinetics of the acetylpuerarin in rats. According to the results in the literature, the absolute bioavailability of puerarin administered intragastricly in rats was only5%-6%, our results showed that the exposure profile of puerarin in plasma after i.g. administration acetylpurearin was markedly improved (the absolute bioavailability of puerarin was up to48.12%). The ability for acetylpuerarin to attenuate the expression of group V sPLA2and the activation of ERK1/2/cPLA2α loop and phospho-p65levels in astrocytes is an indication that acetylpuerarin can prevent the activation of signaling molecules and downstream pathways leading to eicosanoids generation. Acetylpuerarin significantly prevented the LPS-induced expression of COX-2and LOX-5in a dose-dependent manner. Thus, inhibition of COX-2and LOX-5appears to be responsible for the decreased biosynthesis of pro-inflammatory PGE2and LTC4.