The Role Of Diacylglycerol Kinase E In Experimental Cerebral Ischemia-reperfusion Injury

Ischemic stroke is a neurological disease caused by thromboembolic artery occlusion,which is characterized by rapid onset and development with high fatality and disability rate in China.Currently the major clinical treatment methods for stroke are drug and surgical thrombolysis.These methods can save some dying brain cells to a certain extent,however,they may also induce a further damage to the ischemic tissue,which result in a state named "cerebral ischemia-reperfusion injury,CIRI".But the mechanisms of cerebral ischemia-reperfusion injury is unclear and therefore there is no satisfactory intervention means till now..Diacylglycerol Kinases(DGKs)are a family of important regulators of inositol phospholipid signal system which composed by several members.Diacylglycerol Kinase E(DGKE)is the only member of Ⅲ subclass of DGK family that exhibits unique structure and biological function compared with other members.Recent clinical studies reported that the recessive mutation of DGKE is a novel pathophysiological mechanism of atypical hemolytic uremic syndrome(aHUS).Our previous study also found that DGKE played a protective role in acute kidney injury caused by renal ischemia/reperfusion,but the role of diacylglycerol kinase E in ischemic brain injury has not been reported.Research ObjectiveThe experimental cerebral ischemia-reperfusion injury mode in mice was established by middle cerebral artery occlusion(MCAO),and the hypoxic environment in vitro was simulated by oxygen-glucose deprivation(OGD).By means of morphology,molecular biology,flow cytometry and other methods,the expression pattern and role of DGKE in cerebral ischemia-reperfusion injury in vivo and in vitro were studied by using model animals and RNA interference techniques.Experimental methods and results1.The expression pattern of DGKE in various tissues of miceThe expression levels of mRNA and protein of DGKE in various tissues of mice were detected by real-time quantitative PCR and Western blotting(WB)methods,respectively.The results showed that DGKE was expressed in mouse brain as well as many other tissues.2.The expression levels of DGKE in experimental cerebral ischemia-reperfusion injuryWild type C57BL/6J mice aged 8-12 weeks(weighing 23±2g)were randomly divided into sham-operation(sham)and cerebral ischemia reperfusion injury group(CIRI).The mouse model of experimental cerebral ischemia was established by occlusion of the middle cerebral artery,reperfusion was achieved after two hours of ischemia,which was confirmed by neurological score,calculation of cerebral infarction area,morphological staining and TUNEL staining.The mRNA and protein expression levels of DGKE in the cerebral cortex of mice in different groups were detected by real-time quantitative RT-PCR,Western blotting and immunohistochemistry(IHC)methods.The results showed that compared with the sham operation group,the expression level of DGKE in the cerebral cortex of the ischemic hemisphere was significantly decreased after cerebral ischemia-reperfusion injury.Furthermore,the co-localization relationship between DGKE and different neuronal cells was detected by immunofluorescence(IF)staining.3.The role of DGKE in experimental cerebral ischemia-reperfusion injuryDgke overexpression(Rosa26-Dgke+/+)mice and Wide Type(WT)mice were used in the study.Through methods of neurological score,cerebral infarction area calculation,HE staining,Nissl staining and TUNEL staining,it was found that compared animals in WT group,DGKE overexpression could significantly improve the neurobehavioral defects,reduce the infarct area,alleviate the pathological changes of cerebral cortex on the ischemic hemisphere,increase the number of surviving neurons and reduce the number of apoptotic cells in the ischemic penumbra.Using markers of astrocytes and microglia respectively,we found that there are a large number of activated astrocytes and microglia appeared in the penumbra of cerebral cortex after ischemia-reperfusion in wild group,while overexpression of DGKE could significantly reduce the activation of astrocytes and microglia after cerebral ischemia-reperfusion.These results suggest that overexpression of DGKE has a protective effect on experimental cerebral ischemia-reperfusion injury.4.Inflammatory response of brain tissue after the overexpressed DGKE attenuates ischemia-reperfusion injury.Real-time quantitative RT-PCR and IF staining were used to detect the expression level of inflammatory cytokines and inflammatory cell infiltration in brain tissue.The results showed that the overexpressed DGKE could significantly reduce the overexpression of inflammatory cytokines caused by ischemia-reperfusion injury and reduce the infiltration of neutrophils and macrophages,indicating that the neuroprotective effect of DGKE may be related to the reduction of inflammatory reaction.5.The neuroprotective mechanism of DGKE in cerebral ischemia-reperfusion injury is related to the inhibition of PKC pathway.Western blot method was used to detect the level of PKC phosphorylation in the penumbra of cerebral cortex after cerebral ischemia-reperfusion injury.The results indicated that compared with the sham operation group,PKC in the cerebral cortex penumbra of the CIRI group was activated significantly after ischemia-reperfusion,while overexpressed DGKE could significantly reduce the activation of PKC in the ischemic brain tissue,based on the biochemical effect of DGK,we speculated that the neuroprotective mechanism of DGKE may related to the inhibition of DAG-PKC pathway.Similar results were achieved in in vitro experiments.Conclusion and innovationConclusionThe expression of DGKE in cerebral cortex was significantly decreased after cerebral ischemia-reperfusion injury,and the overexpression of DGKE played a neuroprotective role against focal cerebral ischemia-reperfusion injury,which might be through a inhibiting of DAG-PKC pathway.InnovationThis study confirmed the protective role of diacylglycerol kinase E in cerebral ischemia reperfusion injury for the first time,which may provide a research basis for expanding the biological function of diacylglycerol kinase E,enrich the pathophysiological mechanism of cerebral ischemia/reperfusion injury,and provide an idea for the clinical development of effective intervention strategies for ischemic cerebrovascular diseases.