N-SMase2/Ceramide Activation In Astroglia Induce Neuron Damage Through Inflammation Stress In Rat Hippocampi Postischemia

Objective:To investigate the molecular mechanism underlying accumulation of ceramide in astrocytes after cerebral ischemia reperfusion and its effects on the function of neurons.Methods:Rats in the experimental groups were subjected to four-vessel occlusion, endured10min ischemia followed by reperfusion. The N-SMase2inhibitor GW4869, N-SMase2siRNA, TNFa inhibitor R-7050, p38MAPK inhibitor SB-203580, PP2B inhibitor, PKCS inhibitor Rottlerin, A2BAR antagonist MRS-1754and N-SMase2agonist DNR were administered to rat lateral ventricle to observe the changes of N-SMase, A-SMase and N-SMase2activity. Morphological localization of ceramide and other related proteins were detected by Immunofluorescence or immunohistochemistry. Neuron lesion was observed with thionine stain, and the changes of N-SMase2, P38MAPK and p-P38MAPK levels were detected with Western blot. Besides, the combination of N-SMase2, RACK1and EED was detected by co-immunoprecipitation. The mRNA level changes of inflammatory cytokines such as IL-1β, IL-6, TNFa were detected and analysed by Realtime PCR. Additionally, in vitro primary astrocyte cell culture, primary neuron cell culture and transwell co-culture were introduced and oxygen glucose deprivation was established. After administering N-SMase2agonist DNR, NF-kB inhibitor PDTC to stimulate primary astrocyte, we observed the effect of astrocyte on neuron by detecting neuron MAP-2protein with immunofluorescence and detecting neuronal apoptosis with TUNEL.Results:At ischemia/reperfusion30min, ceramide accumulation was notable in rat hippocampus astrocyte, N-SMase and N-SMase2activity was enhanced obviously while A-SMase activity remains unchanged. Ceramide was produced and accumulated in astrocytes of the rat hippocampi. The activity of N-SMase, ceramide production and neuron lesion can be inhibited by N-SMase2inhibitor GW4869, which indicated that N-SMase2may involve in ceramide accumulation in astrocytes of rat hippocampus and neuron lesions. SB-203580and MRS-1754can remarkably depress the rising of N-SMase2activity, while TNFa inhibitor R-7050can partly suppress the rising of N-SMase2activity, which indicated that A2bAR-p38MAPK signaling pathway involved in N-SMase2activation induced by brain ischemia while TNFa may regulate N-SMase2activity in some degree. Furthermore, N-SMase2inhibitor GW4869can decrease expression of IL-1β, IL-6and TNFα, and then protect neuron. It suggested that activated N-SMase2involved in inflammatory factors releasing and the apoptosis of neurons.Conclusion:In early stage of cerebral ischemia injury, N-SMase2, which is activated by p38MAPK and upregulated partly by TNFa, participate in the regulation of ischemic injury of central nervous system. The production of ceramide induces the releasing of inflammatory factors in astrocyte, resulting in the neuron injury.