Role Of CPKCγ In HPC Protects N2a Cells Against OGD-induced Injury And Its Molecular Mechanism

Related domestic and foreign research data show that vascular diseases are theleading cause of death nationwide, and as a kind of high death and disabling disease,stroke have brought huge economic losses and spiritual burden to families and societyfrom all over the world. However, the clinical treatment for stroke is still lack ofeffective measures. Ischemic/hypoxic preconditioning (I/HPC) can improve thetissues and organs’ tolerance to subsequent serious ischemic/hypoxic injury,, so theunderstanding of the molecular mechanism underlying this endogenous protectionmay open new horizons for clinical treatment of stroke. In recent years, researchesshow that the participation of protein kinase C (PKC) was involved in many possiblemechanisms of HPC forming. In addition, our lab have found in earlier studies thatthe increases of the typical PKC (cPKC) II and γ, and novel PKC (nPKC) membrane translocation (activation) levels participated in the mouse HPCdevelopment, and at the same time, cPKCγ and its interacting proteins play animportant role in the process of HPC-induced neuroprotection against cerebralischemic injuries.. Accordingly, the pure low oxygen and oxygen-glucose deprivation(OGD) models of mouse neuroblastoma cells (N2a) were used to simulate the HPCand ischemic damage in vitro and explore the role of cPKCγ in HPC protects N2acells against OGD-induced injury and its molecular mechanism.In this experiments, mouse neuroblastoma cells (N2a) were cultured in DMEMcontaining10%fetal bovine serum (FBS) complete medium, and cultivated in ahumidity saturated chamber containing21%O2/5%CO2/74%N2mixed gas at37℃as normoxic condition, The medium was changed every24h and N2a cells were subcultured for2-3d. N2a cell HPC model: N2a cells were cultured in high glucoseDMEM containing10%FBS,1%O2/5%CO2/94%N2low oxygen gas at37℃for20min. N2a cell OGD model: N2a cells were cultured in serum-free DMEM (noglucose) medium with1%O2/5%CO2/94%N2low oxygen gas at37℃for2,3or4h OGD as ischemic treatment, and then the N2a cells were cultured in normoxiccondition for24h to restore glucose and oxygen to mimic reperfusion. For theinhibition of cPKCγ activation, Go6983was added before HPC treatment and its finalconcentration was maintained at6nmol/L in the cell culture medium. The techniquesof thiazolyl blue tetrazolium bromide (MTT) and lactate dehydrogenase (LDH)assays, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick endlabeling (TUNEL) staining and Western blot for LC3II/I were applied to detect thelevels of N2a cell death, apoptosis and autophagy, respectively. Statistical analysiswas conducted by using One-Way Analysis of Variance followed by all pairwisemultiple comparison procedures using Bonferroni test. Significance was regarded asp<0.05. The results were reported as follows:1. Effects of cPKCγ and HPC on OGD-induced ischemic injury in N2a cellsN2a cells were treated2or4h OGD, and then were restored to normoxiccondition as reoxygenation for24h. MTT results showed that OGD2and4h canmake N2a cells survival rate dropped to (79.0±2.1)%and (36.9±2.0)%respectively;and according to the results of LDH leakage rate, we found that the mortality rates ofOGD2and4h treated N2a cells increased obviously (P <0.05, n=6per group). Tofacilitate the observation of the effects of Go6983(cPKCγ inhibitor) and HPC onOGD-induced N2a cell apoptosis, autophagy and necrosis, the3h OGD and24h,reoxygenation (simply described as3h OGD) was adopted as ischemia/reperfusionstimulation for the following tests.The cell survival rate results showed that HPC (20min) could significantly reduce3h OGD-induced ischemic damage of N2a cells (P <0.05, n=16per group),and cPKCγ inhibitor Go6983(6nmol/L) could block the protective effects of HPC on3h OGD treated N2a cells (P <0.05, n=16per group).2. Effects of cPKCγ and HPC on3h OGD-induced N2a cell apoptosisTUNEL staining results showed that3h OGD could obviously increase theapoptosis cell number of N2a cells (P <0.05, n=6per group), but20min HPC andGo6983+HPC could not obviously affect the apoptosis level of3h OGD-treated N2acells, suggesting that cPKCγ activation and HPC-induced neuroprotections on3hOGD treated N2a cells probably were not affected by the process of cell apoptosis.3. Effects of cPKCγ and HPC on3h OGD-induced N2a cell autophagyThe levels of N2a cell autophagy was determined by using Western blot todetect the proportion of LC3II/I. The results showed that3h OGD could obviouslyincrease the N2a cell autophagy level when compared with that of Control group (P <0.01, n=6per group); and compared with3h OGD group, inhibition of cPKCγactivation (Go6983) and HPC+Go6983could significantly reduce3h OGD-inducedN2a cell autophagy levels (P <0.05, n=6per group), but20min HPC could notsignificantly decrease3h OGD-induced N2a cell autophagy levels. These resultssuggested that cPKCγ activation is involved in the process of3h OGD-induced N2acell autophagy, while the HPC protactive effect on3h OGD-treated N2a cells may notbe affected by the process of cell autophagy. However, why HPC+Go6983canreduce3h OGD-induced N2a cell autophagy levels more obviously than cPKCγinhibition, it needs further experimental analysis.4. Effects of cPKCγ and HPC on3h OGD-induced N2a cell necrosisDetection of LDH leakage rate was used to determine N2a cell necrosis level.Results showed that20min HPC could obviously improve the necrosis level of3hOGD-treated N2a cells (P <0.05, n=16per group), whereas cPKCγ inhibitor Go6983abolished the protective effect of HPC (P <0.05, n=16per group),suggesting that the protective effect of HPC on3h OGD-treated N2a cells is mainlymediated through reducing N2a cell necrosis and cPKCγ activation.In conclusion, our results further confirmed the important role of cPKCγ inHPC-induced neuroprotection against N2a cell ischemic injury in vitro. In addition,the HPC-induced neuroprotectin on OGD-treated N2a cells is mediated throughreducing N2a cell necrosis and cPKCγ activation. This achievement may enrich ourunderstanding of molecular mechanism underlying HPC-induced endogenousneuroprotection and provide a potential therapeutic target for ischemic stroke.