The Mechanism Of Soce In Hyperglycemia Induced Neuron Injury In Vitro AND In Vivo

Diabetes mellitus (DM) is a common metabolic disorder characterized by hyperglycemia. DM can lead to a variety of complications, affecting end-organ like kidney and eyes, which can result in a worse outcome. Much attention has been paid to diabetes induced peripheral neuropathy. However, accumulating evidence from recent studies has indicated that central nervous system may also be involved in diabetes, which mainly leads to diabetic cognitive dysfunction. Hippocampus belongs to limbic system that is essential to learning and memory formation in mammal animals. Diabetic cognitive dysfunction is closely associated with hyperglycemia-induced hippocampus injury. However, the specific mechanism of hyperglycemia-induced hippocampus neuron injury remains tobe known.Calcium ion (Ca2+) is a universal second messenger that is involved in various cellular process, such as proliferation, transcription, exocytosis, and apoptosis. Calcium influx is mediated by ligand-gated channels, voltage-gated channels and store-operated calcium channels (SOCs). Store-operated Ca2+entry (SOCE), as an essential Ca2+entry mechanism in both excitable and non-excitable cells, is mediated by SOCs which is activated by depletion of internal Ca2+stores. SOCs include two main mediators, stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (CRAR, also called Orail). STIM1 is a single-pass transmembrane protein located in the endoplasmic reticulum (ER) membrane and functions as an ER Ca2+sensor to sense ER luminal Ca2+ concentration. SOCE has close relationship with calcium homeostasis. When Ca2+ concentration in ER is decreased, STIM1 will translocate toward plasma membrane where it activates SOCE and mediate extracellular calcium influx.Over the past decade, a growing body of evidence has demonstrated that alterations of SOCE play a crucial role in neurodegenerative diseases such as Parkinson and Alzheimer. However, the association of SOCE with hyperglycemia-induced hippocampus injury remains obscure.Therefore, the present study was aimed to investigate the role of SOCE on neuron injury induced by high glucose application and the effectiveness of SOC inhibitor in alleviating high glucose induced apoptosis in cultured neurons and Sprague-Dawley rat hippocampus, which may provide an experimental basis for finding a new target for clinical treatment of diabetic cognitive dysfunction.PART I:THE ROLE OF CALCIUM IONS IN HIGH GLUCOSE-INDUCED NEURON INJURYObjectives:To evaluate primary cultured neuron injury and the calcium concentration in the cytoplasm after high glucose exposure. To evaluate the calcium concentration in the cytoplasm and primary cultured neuron injury after intracellular and extracellular calcium chelators application, in order to analysis the role of Ca2+in high glucose-induced neuron injury.Methods:1. Cell culture:Primary cultures of hippocampus neurons from day 18 embryos of Sprague-Dawley rats were prepared as follow. After hippocampi were dissected, digested and counted, neurons were placed in 6-well or 96-well culture plates in neurobasal medium, supplemented with 1xB27. Neurons were maintained at 37? in a humidified 5% CO2 incubator. Cultivating mediums were half changed every 3 days.2. Cell treatment groups:After 7-day culture, cells were randomly divided into the groups as follow:0,25mM,50mM,75mM, 100mM different concentrations of high glucose stimulation (48 hours) group; normal control group, high glucose stimulation group, high glucose added EDTA group and high glucose added BAPTA-AM group.3. Evaluation of cytosolic calcium concentration:The Ca2+in the cytoplasm of primary cultured neurons were labeled by Fluo-3/AM, which was tested by flow cytometry to evaluate the concentration of Ca2+in neurons.4. Assessment of primary cultured neuron injury:We calculated the survival rates of primary cultured neurons by MTT assay to evaluate neuron injury and select the appropriate concentration of glucose in subsequent research.Results:1. The impacts of high glucose on the injury of primary cultured neurons:The glucose of 0,25mM,50mM,75mM, 100mM concentration-dependently reduced neurons viability. The survival rate of primary cultured neurons after exposure to 50mM glucose was 54.29%±3.17%. Thus,50mM glucose was an appropriate concentration in the subsequent research.2. The effect of high glucose on cytosolic Ca2+concentration:The cytosolic Ca2+ concentration was concentration-dependently increased with glucose of 0,25mM, 50mM,75mM, 100mM application in primary cultured neurons.3.The effects of Ca2+chelators on cytosolic Ca2+concentration:The cytosolic Ca2+ concertration elevated by high glucose was decreased by extracellular calcium chelator EDTA and intracellular calcium chelator BAPTA-AM. There was no significant difference between two Ca2+chelators.4. The effect of Ca2+chelators on primary cultured neurons injury:Both EDTA and BAPTA-AM can elevate cell survival rate decreased by high glucose. There was no significant difference between two chelators.Conclusions:1. Glucose can concentration-dependently decrease survival rate of primary cultured neurons2. Ca2+may be involved in high glucose-induced neuron injury.PART II:THE MECHANISM OF SOCE IN HIGH GLUCOSE-INDUCED NEURON INJURYObjectives:To investigate the gene and protein expressions of SOC protein STIM1 after exposure to high glucose. To evaluate neuron injury by MTT; To evaluate cytosolic Ca2+concentration, apoptosis rate, and mitochondrial membrane potential by flow cytometry; To evaluate cytochrome C in neuron cytoplasm and mitochondria by Elisa; To evaluate variation of the apoptotic proteins by western blot to analysis the role and mechanism of SOCE in high glucose-induced neuron injury.Methods:1. Neuron transfection and efficiency of transfection:Design and synthesize siRNA sequences for STIM1. The synthesized siRNA STIM1 was transfected into primary cultured neurons by using Hpo2000. After 48 hours, RNA and proteins were extracted from transfected cells to evaluate the efficiency of transfection by qPCR and western blot.2. Cell treatment groups:PBS group, stimulation of 50mM glucose 24 hours group, 48 hours group,72 hours group; PBS group,50mM glucose stimulation group, glucose+La3+group, glucose+siRNA control interference group, glucose+siRNA STIM1 interference group.3. Investigation of the gene and protein expressions of SOCE associated protein STIM1:After 48-hour stimulation, RNA and protein were extracted from each group. Gene and protein expression of STIM1 were detected by qPCR and western blot.4. Detecting the cytoplasmic Ca2+concentration:Flow cytometry was used to detect the Ca2+in neuron cytoplasm labeled by Fluo-3/AM. And neuron cytoplasmic Ca2+ concentration were detected after blockade of SOCE by both inhibitor and siRNA interference.5. Evaluation of neuron injury:We evaluated the neuron survival rate by MTT assay after SOCE inhibitor and siRNA interference application.6. Evaluation of apoptosis rate and mitochondrial membrane potential from each group:After cell collection from each group, flow cytometry was used to evaluate apoptosis rate and mitochondrial membrane potential.7. Detection of the concentration of the cytosolic and mitochondrial CytC and the protein expression of different apoptotic proteins in each group:After cytosolic and mitochondrial portions were collected respectively from each group, CytC content were detected in cytoplasm and mitochondria of neurons; expression of apoptotic proteins were also evaluated by western blotResults:1. The efficient transfection of siRNA STIM1 into primary cultured neurons:The result showed that the expression of STIM1 was significantly inhibited by siRNA STIM1.2. High glucose upregulated the gene and protein expression of STIM1:Compared with normal control group,50mM glucose significantly upregulated the gene and protein expression of STIM1, and the effect lasts at least 72 hours.3. The variation of cytoplasmic Ca2+concentration after blockade of SOCE:La3+ and siRNA STIM1 could significantly downregulate the high glucose-increased cytoplasmic Ca2+concentration. There were no statistical differences between La3+ and siRNA STIM1.4. The effect of SOCE inhibitor and siRNA interference on primary cultured neuron injury:La3+and siRNA STIM1 could significantly increase the high glucose-decreased cell survival rate. There were no statistical differences between La3+and siRNA STIM1.5. Cell apoptosis and mitochondrial membrane potential in each group:Compared with normal control group, apoptosis rate of neurons was remarkably elevated after 50mM glucose stimulation. Concentration of Rh123 fluorescence was also reduced by 50mM glucose stimulation, meaning reduction of the mitochondrial membrane potential. Compared with 50mM glucose stimulation group, inhibitor and siRNA interference can downregulate the apoptosis rate of neurons and upregulate mitochondrial membrane potential.6. The change of CytC content in each group:Compared with normal control group, 50mM glucose can increase the CytC content in cytoplasm, while decrease the CytC content in mitochondria; Compared with high glucose stimulation group, the inhibition of SOCE can remarkably downregulate the CytC content in cytoplasm, while relatively upregulate the CytC content in mitochondria, indicating that blockade of SOCE can significantly limit the release of mitochondrial cytochrome C into the cytoplasm.7. The change of protein expression of STIM1 and various apoptotis proteins in each group:Compared with normal control group,50mM glucose can increase the protein expression of STIM1, upregulate Bax/Bcl-2 ratio and elevate caspase3 activation. Compared with high glucose stimulation group, inhibition of SOCE can reduce the protein expression of STIM1, downregulate Bax/Bcl-2 ratio and caspase3 activation.Conclusions:1. High glucose remarkably increased the mRNA and protein expression of SOC protein STIM1.2. The mechanism of SOCE in high glucose-induced neuron injury may be mediated by inducing mitochondrial dysfunction and activating mitochondrial apoptosis.PART ?:THE MECHANISM OF SOCE IN HYPERGLYCEMIA INDUCED HIPPOCAMPUS INJURY IN DIABETIC RATSObjective:To evaluate the protein expression of STIM1 in each group. To observe the improvement of cognitive function in diabetic rats after giving SOCE inhibitor corosolic acid. To investigate the cytochrome C contents in the mitochondria of hippocampus tissue by Elisa and the expression of apoptotic proteins by western blot. To analyze the role and mechanism of SOCE in hippocampus injury caused by hyperglycemia.Methods:1. Rat experiment groups:80 adult male Sprague-Dawley rats were randomly divided into four groups:normal control group, diabetes group, diabetes+corosolic acid (lmg/kg, dissolved in 2ml saline) group, diabetes+corosolic acid (2mg/kg, dissolved in 2ml saline) group. Diabetic rat model:diabetes was induced with a single intraperitoneal injection of streptozotocin(STZ,50mg/kg, dissolved in lOmM sodium citrate buffer, pH4.5). The confirmation of diabetes was made by collecting blood samples from the tail vein after 48h. Rats with a blood glucose level exceeding 250mg/dl were considered diabetic. Normal group was given same amount of saline for intraperitoneal injection. Diabetes+corosolic acid (1mg/kg) group:At the beginning of the 5th week, daily oral lmg/kg corosolic acid at the given time. diabetes+corosolic acid (2mg/kg) group:At the beginning of the 5th week, daily oral 2mg/kg corosolic acid at the given time. The treatment lasted 12 weeks. Morris water maze test were carried out after 12 weeks treatment. Rats were weighed before and after the experiments, and blood specimens were collected and brain tissues were stored in liquid nitrogen or 4% formaldehyde.2. Detection of the expression of SOC protein STIM1 in each group:The expression and of STIM1 was evaluated by western blot and immunohistochemistry.3. Observation of hippocampus tissue injury in each group:The pathological changes in hippocampus was observed by Nissl staining.4. Evaluation of cytochrome C in mitochondria in each group:The mitochondria portions of the hippocampus tissue were collected from each group. The contents of cytochrome C in mitochondria were detected by Elisa.5. Detection of the expression of apoptotic proteins in each group:Total proteins in each group were extracted from the hippocampus tissue, and the expressions of SOC protein STIM1 and apoptotic proteins were detected by western blot.Results:1. The change of cognitive function in diabetic rats. Analysis revealed that diabetic rats had significantly longer escape latencies compared with control rats. Diabetic rats spent less time in the target quadrant compared with control rats. There were no significant differences in swimming speed in each group.2. The pathological changes in hippocampus of diabetic rats. Compared with control group, Nissl staining showed that cells were sparsely arranged and the shapes of pyramidal cells were fuzzy. The number of nissl body was decreased.3. The expression of SOC protein STIM1 and apoptotic proteins in diabetic group: Compared with control group, the expression of STIM1 was significantly increased, the ratio of Bax/Bcl-2 was rising and caspase3 activity was elevated.4. The effect of inhibition of SOCE on cognitive function and pathological changes in hippocampus of diabetic rats. Compared with diabetes group, diabetic rats treated with corosolic acid displayed a significant decrease in escape latency and spent more time in the target quadrant. Nissl staining revealed that cells were arranged densely, the shape and structure of the cells were normal compared with diabetic group.5. The effect of inhibition of SOCE on cytochrome C:Compared with diabetic group, administration of corosolic acid increased mitochondrial cytochrome C, indicating that blockade of SOCE can inhibit the release of cytochrome C into cytoplasm.6. The effect of inhibition of SOCE on the expressions of STIM1 and apoptotic proteins:Compared with diabetic group, corosolic acid reduced the protein expressions of SOC protein STIM1, downregulated the ratio of Bax/Bcl-2 and decrease caspase3 activity.Conclusions:1. Alteration of SOCE led to hippocampus injury by inducing neuron apoptosis, resulting in cognitive dysfunction in diabetic rats.2. Blockade of SOCE alleviated hippocampus injury and improved cognitive function in diabetic rats.