The Mechanism Research: The Role Of Protein XIAP In Periodontal Ligament Cells Apoptosis Induced By Hydrogen Peroxide

The research background and purpose:Cells apoptosis can be induced by many factors. The attack for cells from oxygen isone of the important. Hydrogen peroxide is a strong oxidizer, which can induce cells toproduce large amounts of reactive oxygen species(ROS). ROS can take the initiative toattack the double-stranded DNA, leading to abnormal variation. Hydrogen peroxide is thecommon use of the clinical treatment of oral drugs, commonly used in dental bleachingand periodontal washing through direct effects on gingival and periodontal tissue. But infact it is also a double-edged sword. In addition to its a therapeutic effect, it may alsoinduce tissue oxidative stress reaction, leading to cell death. In addition, hydrogenperoxide as the key metabolites of oxidation, plays an important role in the diseasemediated by oxidative stress. Hydrogen peroxide can be produced in almost all oxidativestress reaction including periodontitis, which is the main cause for teeth loose and fallingoff. More and more evidence indicate that the hydrogen peroxide can regulate the functionof cells and induce cell apoptosis and cell death. Hydrogen peroxide can penetrate cellmembrane and have an impact on sensitive factors in cells, playing a role as the second messager in the signal transdunction pathways. Resaerches show that the manners of deathinduced by hydrogen peroxide have a relationship with cell type, hydrogen peroxideconcentration and the way of stimulation. XIAP protein is the strongest apoptosisinhibiting factor in the antiapoptotic proteins family. It can restrains caspases directly,shows a powerful role to protect cells from apoptosis stimulation, and regulates cellapoptosis via many ways.At present, researches about the potencial toxicity of hdrogen peroxide for theperiodontal membrane is less, which can be produced by oxidative stress or used in oraltreatment. Additionally, as an important inhibitor of apoptosis, the role of XIAP protein inthe signal pathway, which exists in the course of periodontal ligament cells’ apoptosisinduced by oxidative damage, is unknown. So the research on the molecular mechanism ofthe periodontal ligament fibroblasts’ apoptosis induced by hydrogen peroxide will provideimportant theory basis for the prevention and treatment of periodontitis, and thereconstruction of the periodontal tissue.The reseach contents: this experiment includes three parts. First part: observing thebiological react of periodontal ligament cells under hydrogen peroxide, including cellmorphology change, cell proliferation activity, cell cycle and apoptosis rate. Second part:after overexpression XIAP protein gene and gene silencing of XIPA protein, observing thechange of periodontal ligament cells’ activity. Third part: research on the regulationmechanism in the antiapoptotic kinase signaling pathways.MethodsUsing HE staining and vimentin staining techniques to identify and establish humanperiodontal ligament cells line, cell viability was detected by MTT, and cell cyclequantified by flow cytometry, TUNEL method and flow cytometry PI-FITC doublestaining. With the helps of optical microscopy, fluorescence microscopy, theestablishment of the apoptosis model over the periodontal ligament cell induced byhydrogen peroxide was created, and then via western blotting and immunofluorescenceconfocal laser microscope, XIAP protein expression was checked. Using molecular cloning techniques to overexpress XIAP or gene silencing it, simultaneously, detectionof different signaling pathways including AKT, JNK2, and GSK3β. Over-expression ofXIAP protein and gene silencing, respectively, using hydrogen peroxide treating cells for72h with250μM of H2O2, then, with the helps of ELISA and western blotting, weexplored the molecular mechanism of the cell apoptosis and cell revival. Finally, detectedCasepase-3, Casepase-8, PTEN protein expression by western blotting.Results1. Using different concentrations of hydrogen peroxide to treat periodontal ligamentcells,MTT showed after24hours, for the hydrogen peroxide concentration higher than125μM, the cell viability decreased significantly, which also confirmed by TUNEL assayand flow cytometry. Treating cells with250μM hydrogen oxidation after72hours wewere able to observe the apoptotic rate was36.5%, which means hydrogenperoxide-induced periodontal ligament cells apoptosis model successfully established.2.72hours treatment with250μM of hydrogen oxidation, western blotting showedthat XIAP protein levels decreased (p <0.05), while JNK2, AKT, PAKT and GSK3βprotein levels compared with the control group also had significantly decreased. Theresults also showed confocal results of AKT, PAKT protein levels significantly decreased.Using250μM hydrogen peroxide-treated cells with increasing duration, for24h,48h and72h, respectively, western blotting detected intracellular Caspase-3, Caspase-8, Ptenprotein levels increased significantly compared with the control group(p <0.05).3. Overexpressing XIAP, we observed JNK2, AKT, PAKT and intracellularexpression of GSK3β protein levels did not change significantly. However,72hours250μM of hydrogen peroxide treating XIAP overexpression cells or normal cells, we foundthat overexpressing cells for XIAP, the protein levels of JNK2, AKT, PAKT, and GSK3βwere significant higher than normal cells (p <0.01). While with the over-expression group,which treated by hydrogen peroxide treatment, we detected a significant increase in cellviability by MTT and cell cycle, in addition with cell apoptosis assay, meanwhile, lasercopolymer light microscopy revealed after over-expression of XIAP,72h of250μM hydrogen peroxide treatment induced the c-jun, and AP-1protein significantly increased.4. In the role of gene silencing, MTT detected significantly decreased cell viabilitycompared with the control group, while compared to the control group, JNK2, AKT,PAKT, and GSK3β protein levels decreased significantly.Conclusion1. The inhibition of H2O2for periodontal ligament cells can be enhanced by the higherconcentration of H2O2and longer action time.2. The optimum model of the induction periodontal ligament cell apoptosis: H2O2concentration250μM and72h action time.3. The expression level of XIAP protein was decreased by H2O2induction. So thecombination of XIAP and Caspase-3was restrained, which leading the apoptosis ofperiodontal ligament cells. The expression level of XIAP protein had an impact on thecell activity.4. Protein AKT、JNK2、GSK3β had a great role in the mechanism during the apoptosiscourse of periodontal ligament cell induced by H2O2.5. The transcription factors of C-jun and AP-1was to be activated by XIAP. Themechanism might be that JNK2would be positive regulated by XIAP through C-junphosphorylation.6. The repair of AKT by XIAP might be implemented through the inhibition of PTEN.