The Role And Mechanism Of Glycolysis In The Reactive Proliferation Of Astrocytes After Oxygen-glucose Deprivation Under Cdh1 Regulation

Background and ObjectiveIschemic brain injury is a common clinical problem and cardiac arrest, stroke, and cerebral ischemia induced by various factors during perioperative period are the common origin diseases. It is not only the leading causes of disability to death in the elderly; in recent years it has seriously jeopardizing the health of teenagers. To clarify the intrinsic mechanism of cerebral ischemia reperfusion injury is important for the treatment of ischemic brain injury.Astrocyte is a multifunctional cell, which is believed to be the cornerstone of the structure and function of the brain cells. It is widely existed in the central nervous system. Reactive astrocyte proliferation is an important cellular response to cerebral ischemia. Reactive astrocyte proliferation may play a beneficial and a detrimental role in recovery of neurological deficits. Initially, it may protect the neurons from exposure to toxic elements in the damaged area and secrete neurotrophic factors to provide a permissive substrate for neuron regeneration. However, at the later stage of cerebral ischemia, glia scars, formed from reactive astrocyte proliferation and subsequent accumulation, act as an astrocytic barrier that physically impedes axon regeneration and growth. Thus, to explore the mechanism of astrocytes reactive proliferation after cerebral ischemia and early and effective regulation of excessive astrocyte proliferation to inhibit glial scar formation may create a favorable environment for neuronal regeneration and thereby improve recovery from ischemic injury.Eukaryotic cell proliferation depends strictly on the E3 ubiquitin ligase activity of anaphase promoting complex/cyclosome (APC/C). Cdhl is an important co-activator of APC/C during late mitosis and G1. Cdhl forms complexes with APC/C and controls cell cycle transition through ubiquitination and degradation of multiple cyclins. The recent studies showed that, PFKFB3 contains a KEN box that targets it to APC/C-Cdh1-mediated ubiquitination and degradation. In previous studies, we found that Cdhl activity in hippocampus was significantly decreased after global cerebral ischemia in rats in vivo, and that the Cdhl protein expression was decreased in astrocytes after oxygen-glucose deprivation/reperfusion in vitro. In the physiological state, PFKFB3 protein is expressed in astrocytes in high level, while the activity of APC/C-Cdhl is low. Therefore, we further hypothesized that in the pathological process after cerebral ischemia reperfusion injury, APC/C-Cdhl can regulate the glycolysis in astrocytes; thereby inhibit astrocytes reactive proliferation through ubiquitination and degradation of PFKFB3.Oxygen-glucose deprivation/reperfusion (OGD/R) model is a kind of experimental methods, which mimics ischemic hypoxic brain injury in vitro. Studies have confirmed that this method can induce the reactivity of astrocyte proliferation. Thus, in this study, we will firstly establish the OGD/R model in vitro to mimic the astrocytes reactive proliferation after cerebral ischemia and observe the effect of OGD/R treatment on the glycolysis in astrocytes. Secondly, we will explore the relationship between glycolysis change and astrocytes proliferation through constructing PFK1 overexpression lentivirus and PFKFB3 siRNA to regulate the expression of glycolysis key enzymes. In addition, we will construct Cdhl overexpression lentivirus and Cdhl siRNA lentivirus to regulate Cdhl activity, and explore the effect of Cdhl change on glycolysis and astrocytes proliferation after OGD/R.The purpose of this study is to investigate the effect of OGD/R treatment on the glycolytic ability in astrocytes; to explore the relationship between glycolysis changes and astrocytes proliferation and its mechanisms after OGD/R and to explore the role of Cdhl activity change in regulation astrocytes proliferation from the view of glycolysis, which may provide a new target and experimental evidence of future therapy for cerebral ischemic injury.Methods and Results1. The construction and identification of the model of oxygen-glucose deprivation/reperfusion of cerebral astrocytes in rat.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group and oxygen-glucose deprivation/reperfusion group (group OGD/R). The culture medium was replaced with glucose-free DMEM perfused with an anaerobic gas mixture (5% CO2,95% N2) at 37℃ for 3 h to produce OGD. OGD was terminated by replacing the exposure medium with standard medium and then returning the cells to the incubator under normoxic conditions (37℃,5% CO2) for 24 h. Control cells were treated identically except that they were not exposed to OGD. The expression of GFAP, which is the marker protein of astrocytes, and PCNA was assayed by double-immunofluorescence methods. Red fluorescence represented PCNA and green fluorescence represented GFAP.Results:Compared with the control group, the number of PCNA-positive cells in the group OGD/R is significantly increased, which indicated that the OGD/R model is successfully constructed.2. To explore the effect of OGD/R on the expression of glycolysis key enzymes in the level of mRNA and protein of astrocytes in rat.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group and oxygen-glucose deprivation/reperfusion group (group OGD/R). After 3 h of oxygen-glucose deprivation and recovery for 24 h, the total mRNA and protein in astrocyte was extracted. Then the expression of PFKFB3 and PFK1 in the mRNA and protein level was detected by PCR, western blot and immunocytochemistry.Results:Immunocytochemistry showed that PFKFB3 and PFKl were present in nucleus of astrocytes in the control group. After OGD/R, PFKFB3 and PFK1 protein expression increased significantly. The result of PCR analysis showed that compared with the control group, the expression of PFKFB3 mRNA in astrocytes was increased in the group OGD/R (P<0.05), while the expression of PFK1 mRNA was not significantly different in group OGD/R (P>0.05). The results of western blot showed that the expression of PFKFB3 and PFK1 protein was upregulated significantly in group OGD/R compared with the control group (P<0.05).3. To explore the effect of PFK1 expression lentivirus intervention on the astrocytes proliferation after OGD/R.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group, group OGD/R, OGD/R and PFK1 lentivirus group (group OGD/R+PFK1-LV) and OGD/R and PFK1 control lentivirus group (group OGD/R+PFK1-CV). When the cell confluence was up to about 50%, the old cell medium was removed and PFKl expression lentivirus and control lentivirus were added to the astrocytes in the lentivirus intervention group according to the manufacturer’s instructions.3 days after infection, the cells were subjected to OGD/R. The proliferation of astrocytes in different group was detected by EdU assay and western blot.Results:The results of EdU showed that compared with group OGD/R+PFK1-CV, the number of EdU-positive cells was significantly increased in the group OGD/R+PFK1-LV (P<0.05). The results of western blot showed that the expression of PCNA protein was upregulated in the group OGD/R+PFK1-LV compared with the group OGD/R+PFK1-CV (P<0.05).4. PFKFB3 siRNA inhibited reactive astrocytes proliferation after OGD/R.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group, group OGD/R, OGD/R and PFKFB3 siRNA group (group OGD/R+siPFKFB3) and OGD/R and negative control siRNA group (group OGD/R+NC siRNA). When the cell confluence was up to about 50%, the old cell medium was removed and PFKFB3 siRNA or NC siRNA were added to the astrocytes in the siRNA intervention group according to the manufacturer’s instructions.3 days after transfection, the cells were subjected to OGD/R.Results:Compared with the control group, EdU(+) cells increased remarkably after OGD/R (P<0.05). While the number of EdU(+) cells in group OGD/R+siPFKFB3 was decreased compared with the group OGD/R+NC siRNA (P<0.05). The western blot analysis showed that PCNA protein was decreased by siPFKFB3 transfection after OGD/R (P<0.05).5. PFKFB3 siRNA decreased glycolysis activation of astrocytes in rat after OGD/R.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group, group OGD/R, OGD/R and group OGD/R+siPFKFB3 and group OGD/R+NC siRNA. When the cell confluence was up to about 50%, the old cell medium was removed and PFKFB3 siRNA or NC siRNA were added to the astrocytes in the siRNA intervention group according to the manufacturer’s instructions.3 days after transfection, the cells were subjected to OGD/R. The expression of PFKFB3 and PFK1 protein was detected by western blot and the lactate concentration in the culture medium were measured a spectrophotometer.Results:The results of western blot showed that the expression of PFKFB3 and PFK1 protein was decreased in the group OGD/R+siPFKFB3 compared with the group OGD/R+NC siRNA (P<0.05). Lactate concentrations in the medium were significantly decreased in the group OGD/R+siPFKFB3 compared with the group OGD/R+NC siRNA (P<0.05).6. To explore the effect of Cdhl expression lentivirus intervention on the glycolysis of astrocytes after OGD/R.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group, group OGD/R, OGD/R and Cdhl lentivirus group (group OGD/R+Cdhl-LV) and OGD/R and Cdhl control lentivirus group (group OGD/R+Cdh1-CV). When the cell confluence was up to about 50%, the old cell medium was removed and Cdhl expression lentivirus and control lentivirus were added to the astrocytes in the lentivirus intervention group according to the manufacturer’s instructions.3 days after infection, the cells were subjected to OGD/R. The expression of PFKFB3 and PFK1 protein was detected by western blot and the lactate concentration in the culture medium were measured a spectrophotometer.Results:The results of western blot showed that the expression of PFKFB3 and PFK1 protein was decreased in the group OGD/R+Cdhl-LV compared with the group OGD/R+Cdhl-CV (P<0.05). Lactate concentrations in the medium were significantly decreased in the group OGD/R+Cdhl-LV compared with the group OGD/R+ Cdh1-CV(P<0.05).7. To identify the efficiency of Cdhl siRNA lentivirus of the astrocytes.Methods:The astrocytes were respectively infected by MOI for 1,5,10 and 20 with Cdhl siRNA lentivirus or control lentivirus. To determine the best MOI by observing the GFP expression with inverted fluorescence microscope. The purified astrocytes were randomly assigned to control group, Cdhl siRNA lentivirus group (group siCdhl-LV) and control lentivirus group (group siCdhl-CV). The astrocytes were infected by the Cdhl siRNA lentivirus or control lentivirus by MOI for the best one in the lentivirus infection group. The expression of Cdhl protein was detected by the western blot.Results:The results of fluorescence showed that the GFP expression was observed when MOI for 1,5,10 and 20. When MOI=10, it could achieve more effective transfection effect, thus we selected MOI for 10 in follow-up experiments from the economic point of view. The results of western blot showed that the expression of Cdhl protein decreased significantly in the group siCdhl-LV compared with the group siCdhl-CV (P<0.05).8. To explore the effect of Cdhl siRNA lentivirus intervention on the expression of PFKFB3 protein of astrocytes after OGD/R.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group, group OGD/R, OGD/R and Cdhl siRNA lentivirus group (group OGD/R+siCdhl-LV) and OGD/R and control lentivirus group (group OGD/R+siCdhl-CV).3 days after infection, the cells were subjected to OGD/R. The expression of PFKFB3 protein was detected by western blot.Results:Compared with the group OGD/R+siCdhl-CV, the expression of PFKFB3 protein was increased in the group OGD/R+siCdh1-LV (P<0.05).9. To explore the effect of Cdhl siRNA lentivirus intervention on the expression of PFKFB3 protein of astrocytes after OGD/R.Methods:The cerebral cortex astrocytes of rats were purified and cultured in vitro, and randomly assigned to control group, group OGD/R, group OGD/R+ siCdh1-LV+siPFKFB3, group OGD/R+siCdhl-LV+NC siRNA and group OGD/R+ siCdh1-CV+siPFKFB3.8 h after lentivirus infection, the siRNA was added to the astrocytes in the combined intervention with lentivirus or siRNA group.3 days after transfection, the cells were subjected to OGD/R. The expression of PFKFB3 and PCNA protein was detected by western blot.Results:Compared with the group OGD/R+siCdhl-CV, the expression of PFKFB3 and PCNA protein were increased in the group OGD/R+siCdhl-LV (P<0.05). Down-regulation of Cdhl with Cdhl-siRNA lentivirus aggravated the increase in PFKFB3 and PCNA protein expression, while, this effect was reversed when in combination with PFKFB3 siRNA intervention.10. Statistical analysisAll data are presented and graphed as mean ± SEM. The statistical package for the social sciences 20 software was used for the statistical analyses. Student’s t-test, one-way ANOVA were used as appropriate for comparisons between different groups. Differences were deemed statistically significant if P value<0.05.1. It was found that glycolysis key enzymes PFK1 and PFKFB3 protein were highly expressed in rat cortical astrocytes in vitro. The expression of PFK1 and PFKFB3 protein in astrocytes were increased and glycolysis of astrocytes was upregulated after OGD/R.2. It was confirmed that the lentivirus-mediated over-expression of PFK1 could not induce the proliferation of astrocytes in normal condition, while it aggravated the astrocyte proliferation and glycolytic activation after OGD/R. On the contrary, PFKFB3 siRNA inhibits the astrocyte proliferation and glycolytic activation after OGD/R in vitro.3. Over-expression of Cdhl with Cdhl expression lentivirus decreased the expression of PFKFB3 and PFK1 protein, reduced the lactate release, and inhibited astrocytes proliferation in vitro. Down-regulation of Cdhl with Cdhl-siRNA lentivirus aggravated the increase in PFKFB3 protein expression, while, this effect was reversed when in combination with PFKFB3 siRNA intervention.OGD/R significantly increased the expression of PFKFB3 and PFK1 protein. Regulation of glycolysis key enzymes with PFK1 lentivirus or PFKFB3 siRNA regulated the reactive proliferation of astrocytes. PFKFB3 and PFK1 expression positively correlated with reactive proliferation and ischemic brain injury induced reactive astrocyte proliferation is accompanied by the activation of glycolysis. Using Cdhl lentivirus regulated Cdhl expression, an upstream regulator of PFKFB3, and subsequently regulated the ubiquitination and degradation of PFKFB3 and PFK1 expression, which then regulated glycolytic flux.