The Mechamisms Of Cystatin C In Cerebral Ischemic Tolerance Induced By HBO Preconditioning

Background: Cerebrovascular diseases have become the second leading cause for deathand disability of adults. However, the effective treatments for those diseases are still verylimited. Recently, we have reported that hyperbaric oxygen (HBO) preconditioning cansignificantly reduce the infarct size and improve the neurological behavior afterischemia-reperfusion injury in animals. We found that HBO preconditioning elevatesautophagic activity and then autophagy elicits a neuroprotective effect against ischemicinjury in the brain. How the HBO preconditioning, however, elevates autophagic activityis still unclear. To further investigate the mechanisms of HBO preconditioning-inducedtolerance against transient focal cerebral ischemia will be helpful for finding a new tartgetin clinical management of stroke.Cystatin C is an important endogenous inhibitor of cysteine protease that is targetedto the lysosomal system where the cystatin C works as an effectively inhibitor of cathepsinactivities. It has been reported that Cystatin C is neuroprotective in cultured neuronsexposed to cytotoxic challenges, including nutritional-deprivation and oxidative stress,through protection of cell membrane structure and promotion of autophagic activity. We also found that cystatin C was significantly increased in serum and brain in HBOpreconditioning group after ischemia-reperfusion injury. However, whether the increasedexpression of cystatin C is involved in neuroprotection induced by HBO preconditioningis still unclear.So, we assumed that HBO preconditioning upregulates cystatin C and inhibits itsdownstream signal protein mTOR, in turn promotes autophagy, and as a consequence,induces a tolerance against transient focal cerebral ischemia. In current project, we usedcystatin C-siRNA or cystatin C protein to down-regulate or up-regulate cystatin C in rat’sbrain and cultured primary neurons to explore the underlying mechanism of theneuroprotective effect of HBO preconditioning.Experiment I: Study on the effect of cystatin C in the ischemic tolerance induced byHBO preconditioning in rat subjected to MCAOObjective: To investigate whether cystatin C is involved in the neuroprotection of HBOpreconditioning.Methods:1) To observe the expression of cystatin C in the ischemic penumbra of rat’sbrain who is preconditioned with HBO.24h after HBO preconditioning (2.5ATA,100%oxygen,1h/d×5d), male adult SD rats were subjected to middle cerebral atery occlusion(MCAO) for2h. We detected expressions of cystatin C protein in the ischemic penumbrabefore the onset of MCAO and at2,4,6,12,24h after reperfusion.2) To evaluate theeffects of cystatin C on the neuroprotective effect of HBO preconditioning. CystatinC-siRNA and cystatin C protein were administered respectively to down-regulate orup-regulate cystatin C level in rat’s brain after HBO.24h after reperfusion, we evaluatedneurological score and infarct volume.3) To justify the effects of HBO preconditioning onthe mTOR phosphorylation and autophagy. We detected expressions of p-mTOR, mTOR,LC3II/I, Beclin-1protein in the ischemic penumbra before the onset of MCAO and at2,4,6,12,24h after reperfusion. Furthermore, Immunofluorescent statining was used to detectthe expression level and localization of p-mTOR and Beclin-124h after reperfusion.Results: HBO preconditioning effectively improved rat’s neurological score and reducedinfarct volume24h after reperfusion. The expression of cystatin C was increased from4h to at least24h after reperfusion. HBO preconditioning significantly down-regulatedmTOR phosphorylation from4h to at least24h after reperfusion as compared with theMCAO group. The autophagy related protein LC3II/I and Beclin-1was increased from6hto at least24h after reperfusion. P-mTOR was primarily expressed in cytoplasm ofneurons, and Beclin-1was mainly in the nuclei of neurons in ischemic penumbra. CystatinC-siRNA could reverse the neuroprotective effect of HBO preconditioning. Additionaladministration of cystatin C could induce ischemia tolerance as HBO preconditioning.Conclusion: HBO preconditioning induces tolerance against transient focal cerebralischemia through upregulation of cystatin C. HBO preconditioning downregulates mTORphosphorylation and promotes autophagy.Experiment II: Study on the effect and mechanism of cystatin C in the ischemiatolerance induced by HBO preconditioning in cultured rat’s cortical neurons.Objective: To investigate whether cystatin C is involved in the neuroprotection inducedby HBO preconditioning in cortical neurons of rat, and to explore the underlyingmechanism.Methods:1) To observe the effects of HBO preconditioning on the expression of cystatinC after OGD/R. The Cortical primary neurons were obtained from brains of E18–E19SDrat embryos. HBO preconditioning (3.5ATA, oxygen>90%,3h) was performed at6d incluture. Neurons were subjected to a2h OGD injury at24h after HBO preconditioning.24h after OGD/R, we examined the cell viability, LDH leakage ratio and expression ofcystatin C.2) To evaluate the effects of cystatin C in the neuroprotection of HBOpreconditioning. Cystatin C-siRNA and cystatin C protein were used respectively todown-regulate or up-regulate cystatin C expression in primary neurons. Cystatin C-mRNAand cystatin C protein were detected by RT-PCR and Western Blot respectively afterinterference. Cells were subjected to a2h of OGD24h after interference.24h after OGD/R,we examined the cell viability and LDH leakage ratio.3) To examine the effects ofcystatin C on the mTOR phosphorylation and autophagy in primary neurons in HBOpreconditioning. Cystatin C-siRNA and cystatin C protein were used24h before OGD.And24h after OGD/R, we detected p-mTOR, mTOR, LC3II/I, Beclin-1protein. Results: Results of immunofluorescent statining assay indicates that the percentage ofβ-tublinⅢ positive neurons was more than95%. HBO preconditioning increased cellviability and decreased LDH leakage ratio of primary neurons after OGD/R injury. HBOpreconditioning could upregulate cystatin C expression in neurons after OGD/R injury.Adding of cystatin C and application of cystatin C-siRNA (10μM) could up-regulate ordown-regulate cystatin C expression effectively. Down-regulation of cystatin C attenuatedthe protective effect of HBO preconditioning; Up-regulation of cystatin C could protectprimary neurons from OGD/R injury as HBO preconditioning. Up-regulation of cystatin Ccould inhibit mTOR phosphorylation and promot autophagy, while mTORphosphorylation increased and autophagy decreased after down-regulated cystatin C.Conclusion: Cystatin C plays an important role in the protective effect on the primaryneurons induced by HBO preconditioning. HBO preconditioning induced toleranceagainst OGD/R injury through upregulating cystatin C expression, inhibiting thephosphorylation of its downstream protein mTOR, and promoting autophagic activity.Summary: Results from both the in vitro and in vivo studies indicates that Cystatin Cplays an important role in the protective effect of HBO preconditioning. HBOpreconditioning upregulates cystatin C expression, inhibits the phosphorylation of itsdownstream protein mTOR, and promots autophagic activity which then induced toleranceagainst OGD/R injury.