Pi3k/akt Signaling Pathway In Cyclical Tensile Stress Mediated The Role Of The Periodontal Ligament Fibroblast Apoptosis

ObjectivePeriodontitis is a complex etiology, which is caused by the interaction of many factors of destructive periodontal disease, and regulated by the body defense mechanisms and a variety of systemic and local factors. Bite force, as one of the local factors, plays a very important role in the whole development of periodontitis.And apoptosis have the same role PI3K/Akt signaling pathway play essential roles in apoptosis、migration and proliferation。However, little was known on whether PI3K/Akt pathway will be involved in the mechanical strain-induced apoptosis of hPDLFs. This study first constructed a hPDLFs model of mechanical stimulation in vitro, and applied cyclic strain with different loading duration to investigate the apoptosis of hPDLFs by Hochest、RT-PCR、Flow Cytometry.To study the expression and activity change of Akt in fibroblast under the cyclic tensile stress by RT-PCR、Identify the relationship among PI3K/Akt、cyclic tensile stress and apoptosis of fibroblast. It could be helpful to understand the pathogenic mechanism of occlusal trauma and periodontitisMethodsIn vitro culture-tensile stimulate models of hPDLFs were established by using a multi-passage load adding system. Cyclic stretch was applied on the fibroblasts for1,6,12and24hours, respectively; The loading was set for15%surface elongation, with frequency0.5Hz. each cycle including the3-s stretch/3-s relaxation Meanwhile, the normal control group and0h+LY294002was established by static group. The cell apoptosis was determined by Hoechst33258staining; flow cytometry analysis of cell cycle. The expression of mRNA was detected on Bcl-2、Bax and Akt by RT-PCR technology. This time makes the hPDLFs prior to impose cell-specific inhibitor LY294002, and then imposed on the same tensile stress, and then we detected the changes of cell apoptosis.The experimental data were analyzed used SPSS17.0Statistical Package.Results1. The results show that the nucleus for the group without unloading disperseduniformly round or oval-shaped fluorescent, while the loading groups can be seen within the nucleus or cytoplasm, dense stain particles, crescent or ring fluorescence, nuclear fast shrinkage, some slices of nuclear fragmentation can be seen2. Flow cytometry analysis showed that hPDLFs cycle has changed as the loading time prolonged.And12h have most remarkable chang.3. There were significant differences in the Bcl-2、Akt and Bax mRNA among the six groups The Bcl-2、Akt mRNA expression decreased in loaded hPDLFs group compared with that in unloaded hPDLFs.(p<0.05), and the lowest at12hour following loading, and then enhanced gradually. And Bax mRNA expression increased(p<0.05).4. Compared with the control group,the Bcl-2and Akt levels of hPDLFs began to decrease after loading, and LY294002group had more remarkable decrease. By contraries, the Bax mRNA levels increased in LY294002group.Conclusions1. Cyclical tensile stretch of15%.0.5Hz may induce the apoptosis of hPDLFs2. the apoptosis of hPDLFs has positively correlation with the time of cyclical tensile stress in a certain range3. PI3K/Akt signaling pathway may participate in the process of apoptosis of hPDLFs induced by cyclic stretch.,while the apoptosis process was participated by a series of enzyme and controlled by genes.