| AbstractObjective:To investigate the role of phosphatidylcholine-specific phospholipase in early brain damage caused by subarachnoid hemorrhage and its mechanism of action.Methods:Firstly, 72 rats were randomly divided into four groups: sham group(n=18),SAH+vehicle group(n=18), D609-L(low-dosage, 5mg/kg)group(n=18) and SAH +D609-H(high-dosage, 10 mg/kg) group(n=18). D609 was purchased from Sigma(M-5250)and dissolved in normal saline as the vehicle. D609 or vehicle at equal volume was injected intraperitoneally at 0.5 h after SAH. In all of the groups describe above, the blood and brain tissue samples were obtained separately from rats at 12 h, 24 h and 48 h after SAH(n=6 for each time point) for the time course study of PC-PLC activity and the effect of D609 on PC-PLC activity. Secondly, 96 rats were randomly divided into four groups: sham group, SAH + vehicle group, D609-L group and SAH + D609-H group(n =24 per group).Based on the results of the time course study, the blood and brain tissue samples were obtained from rats at 48 h after SAH. Among the 24 rats per group, 12 rats per group were separately used for blood – brain barrier(BBB) integrity assay(n=6) and brain edema evaluation(n=6), while brain tissues of six rats per group were embedded in paraffin and cut into paraffin sections at 4um thickness for terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) staining and immunofluorescence analysis, and brain tissues of another six rats per group were homogenized and lysed in ice-coldRIPA lysis buffer(Beyotime, P0013) for Western blot analysis. The blood of rats was collected by retro-orbital puncture using glass Pasteur pipettes, and subsequently, the blood serum was obtained and stored at- 80 ° C until used for PC-PLC activity and inflammatory cytokines analysis. Then, all the rats were deeply anesthetized by chloralhydrate(36 mg/100 g body weight), and the brain tissues, except that for brain edema evaluation, were collected after transcardially perfusion with ice-cold PBS.Results:General observationNo significant changes in body weight, mean arterial blood pressure, temperature, or injected arterial blood gas data were detected in any of the experimental groups(data not shown). The mortality rate of rats in the sham group was 0%(0/42 rats), and it was 11.8 %(17/143 rats) in the SAH group. The injection of blood would lead to absence of spontaneous respiration for about 10 s and return to normal respiration shortly.Upregulation of PC-PLC activity in the brain tissue and serum of rats after SAHCompared with sham group, SAH group showed significantly higher PC-PLC activity in the brain tissue of rats. The increase of PC-PLC activity in the brain tissue was more prominent at 24 h and 48 h after SAH induction. PC-PLC enzymatic activity was also measured in all serum samples. In sham group, PC-PLC activity was at a low level in the serum, and nor were changes found in 24 h post-SAH induction. However, at 48 h after SAH, significant increase in PC-PLC activity was detected in the serum of rat. Remarkably,administration of D609 at 10mg/kg effectively inhibited the increased PC-PLC activity in the brain tissue and serum induced by SAH, while D609 at 5 mg/kg only inhibited the increased PC-PLC activity in the brain tissue at 24 h and 48 h after SAH, suggest high dose of D609 had a more potent capacity to resist PC-PLC activity following SAH.Inhibition of PC-PLC activity attenuated neurological behavior impairment induced by SAHAt 48 h after induction of SAH, the rats showed severe neurological behavior impairment relative to the sham group(Table 2).When given of intraperitoneal injection of D609 at both dosages, the neurological behavior impairment was significantly reversed.Additionally, as compared with low-dosage D609, high-dosage D609 had a greater effect on improvement of neurological deficits of rats undergoing SAH.Inhibition of PC-PLC activity reversed SAH-induced brain injuryTo evaluate the effects of D609 treatment on BBB integrity following SAH, Evans blue extravasation assay was performed(Fig. 4A). Compared with Sham group, significant increase in Evans blue extravasation was detected in SAH group. The mean value of Evans blue extravasation was decreased by D609 treatment at both dosages. The resultssuggested that D609 treatment could attenuate BBB injury in this rat SAH model.Then, we performed brain water content assay to evaluate the influences of D609 treatment on brain edema following SAH. As shown in Fig. 4B, SAH caused a significant increase in brain water content, which was significantly reduced by D609 treatment. The results indicated that SAH aggravated brain edema, which was significantly attenuated by D609 treatment.Inhibition of PC-PLC activity attenuated SAH-induced neuron and brain microvascular endothelial cell apoptosisTo investigate the underlying mechanisms of D609-induced brain protection after SAH, changes in apoptotic index of neurons and brain microvascular endothelial cells were quantified by TUNEL/Neu N and TUNEL/v WF immunofluorescent double-labeling(Fig.5A and B). Compared with sham group, the apoptotic index of neurons and brain microvascular endothelial cells in SAH group were significantly higher. Remarkably,compared with SAH group, D609 treatment restored the percentage of TUNEL-positive neurons and brain microvascular endothelial cells in the brain. Consistently, Western blot assay showed that D609 treatment blunted the increased protein levels of apoptosis proteins, including active-caspase 3 and bax, induced by SAH. All the results corroborated the anti- apoptotic effects of D609 on neurons and brain microvascular endothelial cells following SAH.PC-PLC mediated SAH-induced inflammatory responseAs accumulating evidence indicated an important role of PC-PLC in the cross talk between apoptosis and inflammation, we then tested the effects of D609 on plasma concentration of the inflammation related factors, including IL-1β, IL-6 and TNF-α. As shown in Fig. 6, therewere increased concentrations of IL-1β, IL-6 and TNF-α in SAH group as compared to sham group. Remarkably, D609 treatment significantly reduced theplasma concentrations of IL-6 and TNF-α, suggesting the anti-inflammatory action of D609. However, D609 treatment could not significantly reduce the level of IL-1β.Oxy Hb upregulated PC-PLC activity in cultured neuronsWe further analyzed whether Oxy Hb may alter the activity of PC-PLC on cultured primary-cultured rat cortical neurons in vitro. On 24 h of stimulation of cultured primary-cultured rat cortical neurons with Oxy Hb, an increased activity of PC-PLC was found. In addition, Oxy Hb mediated PC-PLC enzymatic activation was significantly abrogated in neurons treated with D609.Inhibition of PC-PLC activity improve the survival of neurons exposed to Oxy Hb treatmentTo further determine the potential role of PC-PLC on neuron apoptosis induced by SAH, the effects of D609 on the survival of primary-cultured rat cortical neurons exposed to Oxy Hb were tested. Firstly, morphology assay was performed and the results showed that, compared with normal group, Oxy Hb-treated neurons exhibited a loss of condensation of the soma and neuronal arborization, which is consistent with the morphological features of neuron death. Remarkably, D609 at both dosages significantly alleviated the morphological changes induced by Oxy Hb. In addition, low-dosage D609 slightly and nonsignificantly increased the survival rate of neurons, but high-dosage D609 significantly increased the survival rate as compared with control group... |
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