Roles Of NF-кB And PI3K/Akt Signal Pathway In H₂O₂ Preconditioning-induced Adaptive Cytoprotection

BackgroundWith the progress of aging, neurodegenerative diseases became a menace to human's health. Therefore the pivot of medical research turned to how to prevent this kind of disease. Necrosis and apoptosis of neuron induced by oxidative stress is the main reason of neurodegenerative diseases. At present, prevention and treatment to this kind of diseases was limited to reducing the injury and apoptosis resulting from oxidative damages by using anti-oxidant, which belong to a passive process. How to resist oxidative injury actively will be a new direction for prevention of neurodegenerative diseases through enhancing ability of endogenous defensive system stimulated by extrinsic substance.Adaptive cytoprotection means treated cells with mild insults before strong insults can relieve cell injury induced by strong damages, and the mechanism of which is due to the sublethal stress (mild insults) triger endogenous defense system which make organism adapt to subsequent strong insults. Serve as a common mechanism on maintaining steady state, adaptive cytoprotection have a characteristic of enhancing ability of endogenous defensive system through extrinsic stimuli without changing quality of insults. Therefore adaptive cytoprotection provides possibility for prevention of neurodegenerative diseases actively.Cytoprotection model of H₂O₂ precondition had been established in PC12 cells in our laboratory, and previous study indicated that H₂O₂ precondition induced cytoprotection via reducing intracellular reactive oxygen species (ROS) level, preventing the reduction of mitochondrial membrane potential (MMP), and enhancing expression of Bcl-2 and Survivin. Moreover increasing expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and activation of JAK-STAT signal pathway participated in anti-apoptotic induced by H₂O₂ precondition. The results suggest that the mechanism of H₂O₂ precondition-induced cytoprotection is complicated, which may involve in activation of multi-pathway.NF-κB and PI3K/Akt signal pathway gathered more attentions because of its closed relation with cell fate (survival and death) under oxidative stress circumstance.1. NF-κB signal pathway and oxidative stressNF-κB was the first eukaryotic transcription factor reported to response to oxidative stress such as ROS and H₂O₂. A number of study demonstrated that NF-κB mediates cell survival and death. When Confronted with oxidative injury constitutive NF-kB activity was necessary for neuron survival, and inhibition of NF-κB reduce neuron survival under different oxidative stress. On contrary, some evidences indicated blocking of NF-κB transition into nuclei can reduce neuron death induced by oxidative stress.2. PI3K/Akt signal pathway and oxidative stressActivation of PI3K/Akt pathway is very important for cell survival to oxidative stress. Moreover activation of PI3K/Akt pathway played an anti-oxidant role in central and peripheral neuron and can protect neuron against apoptosis induced by oxidative toxicant. But different study showed that some apoptosis–induced stimuli down-regulate expression of Akt, which suggest PI3K/Akt pathway may not participate in cell survival under some stress.Therefore the effect of NF-κB and PI3K/Akt signal pathway on cell fate is complicated when confronted with oxidative injury. Whether NF-κB and PI3K/Akt signal pathway play a role in H₂O₂ precondition-induced cytoprotection is still not clarified. This study will investigate this question. Part one Role of NF-κB signal pathway in H₂O₂ preconditioning-induced adaptive cytoprotectionObjectiveTo discover whether NF-κB signal pathway participate in H₂O₂ precondition induced- cytoprotection.Methods1. Estimation of damages on cells: MTT was used to detect the viability of PC12 cell, and the cytotoxicity was quantitatively assessed by measuring the amount of lactate dehydrogenase (LDH) released from damaged cells into the cell culture medium.2. Assessment of cell apoptosis: morphological changes were determined by using Hoechst 33258 staining, and flow cytometry (FCM) analysis was used to measure the rate of apoptosis.3. Immunocytochemical staining was adopted to detect the nuclear translocation of NF-κB.4. Electrophoretic mobility shift assay (EMSA) was used to estimate the ability of NF-κB binding to DNA.5. Activity of caspase-3 was measured by colorimetry.6. Western blot analysis was used to measure the expression of protein.7. All data are expressed as mean + SD. The differences between groups were evaluated by one-way analyses of variance (ANOVA) using SPSS 14.0. Unless indicated, all experiments were performed at least three times with similar results. Differences were considered significant at P<0.05.Results1. H₂O₂ precondition causes expression of NF-κBAnalysis of western blot indicated oxidative stress and H₂O₂ precondition induced expression of NF-κB in PC 12 cells. Moreover inhibitor of NF-κB, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) reduced the expression of NF-κB induced by H₂O₂ precondition.2. H₂O₂ precondition results in nuclear translocation of NF-κBImmunocytochemical staining showed that H₂O₂ precondition significantly promote nuclear translocation of NF-κB.3. H₂O₂ precondition enhances NF-κB DNA binding activityAnalysis of EMSA showed that H₂O₂ precondition enhance the amount of nuclear protein and DNA complex, which indicated that H₂O₂ precondition caused increasing in NF-κB DNA binding activity in PC12 cells.4. NF-κB mediates adaptive cytoprotection induced by H₂O₂ preconditionâ‘ TPCK antagonize relieving in cell damages induced by H₂O₂ precondition: measurement of MTT showed that H₂O₂ precondition resist reducing in viability induced by high dose H₂O₂ in PC 12 cells, and TPCK administered before H₂O₂ precondition significantly decreased the viability of cell. Assessment of LDH in cell culture medium showed that H₂O₂ precondition decrease release of LDH induced by high dose H₂O₂ in PC 12 cells, and TPCK administered before H₂O₂ precondition significantly increased the release of LDH in PC 12 cell.â‘¡TPCK antagonize relieving in cell apoptosis induced by H₂O₂ precondition: Hoechst 33258 staining showed that H₂O₂ precondition reduce numbers of apoptic cell, but TPCK administered before H₂O₂ precondition significantly increase numbers of apoptic cell. Analysis of FCM indicated that H₂O₂ precondition reduce increasing in rate of apoptosis induced by high dose, TPCK administered before H₂O₂ precondition significantly increased rate of apoptosis in PC 12 cell.5. NF-κB affects apoptosis in caspase-dependant manner H₂O₂ precondition significantly decreased the activity of caspase-3 induced by high dose H₂O₂.TPCK administered before H₂O₂ precondition obviously increased the activity of caspase-3.6. H₂O₂ precondition induces expression of heat shock protein (HSP) 70 and 90 via NF-κB pathwayH₂O₂ precondition up-regulated the expression of HSP 70 and HSP 90 induced by 300μM H₂O₂, and TPCK administered before H₂O₂ precondition inhibited the expression of HSP 70 and HSP 90.Conclusion1. Oxidative stress and H₂O₂ precondition induced overexpression of NF-κB, and H₂O₂ precondition enhance nuclear translocation and transcriptional activity of NF-κB.2. Expression and activation of NF-κB participate in adaptive cytoprotection induced by H₂O₂ precondition. The mechanisms of anti-apoptosis of H₂O₂ precondition involved in caspase-dependant pathway.3. HSP 70 and 90 served as effective protein after NF-κB triger transcription, and may be one of mechanism in H₂O₂ precondition induced-cytoprotection.Part two Role of PI3K/Akt signal pathway in H₂O₂ preconditioning-induced adaptive cytoprotectionObjectiveTo discover whether PI3K/Akt signal pathway participate in H₂O₂ precondition induced-cytoprotection.Methods1. Estimation of damages on cells: MTT was used to detect the viability of PC12 cell, and the cytotoxicity was quantitatively assessed by measuring the amount of lactate dehydrogenase (LDH) released from damaged cells into the cell culture medium.2. Assessment of cell apoptosis: morphological changes were determined by using Hoechst 33258 staining, and flow cytometry (FCM) analysis was used to measure the rate of apoptosis.3. Activity of caspase-3 was measured by colorimetry.4. Western blot analysis was used to measure the expression of protein.5. All data are expressed as mean + SD. The differences between groups were evaluated by one-way analyses of variance (ANOVA) using SPSS 14.0. Unless indicated, all experiments were performed at least three times with similar results. Differences were considered significant at P<0.05.Results1. H₂O₂ precondition induces expression of p-akt in a PI3K-dependant manner Analysis of western blot indicated H₂O₂ precondition induced expression of p-akt in PC 12 cells. Inhibitor of PI3K, ly294002 reduced the expression of p-akt induced by H₂O₂ precondition.2. PI3K/Akt pathway mediates adaptive cytoprotection induced by H₂O₂ preconditionâ‘ Ly294002 antagonize relieving in cell damages induced by H₂O₂ precondition: measurement of MTT showed that H₂O₂ precondition resist decreasing in viability induced by high dose H₂O₂ in PC 12 cells, and ly294002 administered before H₂O₂ precondition significantly reduced the viability of cell. Assessment of LDH in cell culture medium showed that H₂O₂ precondition decrease release of LDH induced by high dose H₂O₂ in PC 12 cells, and ly294002 administered before H₂O₂ precondition significantly increased the release of LDH in PC 12 cell.â‘¡Ly294002 antagonize relieving in cell apoptosis induced by H₂O₂ precondition: Hoechst 33258 staining showed that H₂O₂ precondition reduce numbers of apoptic cell, but ly294002 administered before H₂O₂ precondition significantly increase numbers of apoptotic cell. Analysis of FCM indicated that H₂O₂ precondition reduce increasing in rate of apoptosis induced by high dose, ly294002 administered before H₂O₂ precondition significantly increased rate of apoptosis in PC 12 cell.3. PI3K/Akt pathway affects apoptosis in a caspase-dependant manner H₂O₂ precondition significantly decreased the activity of caspase-3 induced by high dose H₂O₂. Ly294002 administered before H₂O₂ precondition obviously increased the activity of caspase-3.4. H₂O₂ precondition induces expression of heat shock protein (HSP) 70 and 90 via PI3K/Akt pathway H₂O₂ precondition up-regulated the expression of HSP 70 and HSP 90 induced by 300μM H₂O₂, ly294002 administered before H₂O₂ precondition inhibited the expression of HSP 70 and HSP 90.5. H₂O₂ precondition activates NF-кB pathway via PI3K/Akt pathway It is showed that ly294002, inhibtor of PI3K block the activation of NF-кB induced by H₂O₂, which indicated that PI3K/Akt pathway lies in upstream of NF-кB in H₂O₂ precondition-induced adaptive cytoprotection.Conclusion 1. H₂O₂ precondition induced activation of Akt via PI3K-dependant pathway.2. Activation of PI3K/Akt pathway participated in adaptive cytoprotection induced by H₂O₂ precondition. The mechanisms of anti-apoptosis of H₂O₂ precondition involved in caspase-dependant pathway.3. HSP 70 and 90 served as effective protein after activation of PI3K/Akt triger transcription, and may be one of mechanisms in H₂O₂ precondition induced-cytoprotection.4. PI3K/Akt pathway lies in upstream of NF-кB in H₂O₂ precondition-induced adaptive cytoprotection.Our study demonstrated that both PI3K/Ak and NF-κB signal pathway mediate H₂O₂ precondition-induced adaptive cytoprotection. H₂O₂ precondition activates Akt in PI3K-dependant manner, and activation of Akt induce activate of NF-κB signal pathway, which antagonize apoptosis via inhibition caspase-3 activity. Simultaneously NF-κB signal pathway promote expression of some gene related with stress and synthesize protective protein HSP 90 and HSP 70, which confer cell ability to resist subsequent serous injury.