Preliminary Study On The Function And Mechanism Of Apoptosis Protein Inhibitor 5 In Periodontitis

Objective: This study was to compare the differences in cell activity and cell apoptosis related gene in human gingival fibroblasts(HGFs) from healthy volunteers and patients with chronic periodontitis, to analysis the change of apoptosis inhibiting factor 5(API5) and cell apoptosis related factors in gingival tissue from healthy volunteers and patients with chronic periodontitis, to detect the influence of API5 on HGFs cell activity and apoptosis through si RNA technology, and to explore its possible regulating mechanism, so as to provide experimental information for elucidating the pathogenesis of periodontitis.Methods: 1. After informed consent, healthy gingival tissues from healthy volunteers and patients with chronic periodontitis were collected during extracting the third molar, immediately washed in PBS solution repeatedly, cut into small pieces(about 1 mm3). HGFs were obtained from tissue explants in vitro, observed cell morphology and growth by using inverted phase contrast microscope.2. Cell activity of HGFs from healthy volunteers and patients with chronic periodontitis was detected in different time point in vitro. At 2d and 4d, real-time polymerase chain reaction(PCR) was applied to detect the expression of apoptosis related factors B cell lymphoma/lewkmia-2(Bcl-2), Bcl-2-associated X protein(Bax) and the ratio of both was analyzed.3. After informed consent, gingival tissues were collected from healthy volunteers and patients with chronic periodontitis, immediately put into RNAlater preservation solution. Total RNAs were extracted from gingival tissues, and then real-time PCR was applied to detect m RNA expression levels of cell apoptosis related factors API5, Bcl-2, Bax, B-cell lymphoma-extra large(Bcl-xl) and cyclin E1(CCNE1).4. After si RNA API5 transfection in HGFs, API5 protein level was detected by Western blotting, and then the si RNA inhibitory efficiency was detected.5. After siRNA API5 transfection in HGFs, cell activity and apoptosis were detected by MTT and flow cytometry, respectively. Blank control group, negative control group, si RNA API5 group were set up.6. After si RNA API5 transfection in HGFs, the expressions of apoptosis related genes were detected using reverse transcription-polymerase chain reaction(RT-PCR). Blank control group, negative control group, si RNA API5 group were set up.Results: 1. The primary cells were migrated from the edge of tissue, tended to be consistent, long spindle and thrived. Cell activity of HGFs from patients with chronic periodontitis was lower than that of HGFs from the healthy group at day 4, 6 and 8(P<0.05).2. At day 2 and 4, Bax m RNA levels in HGFs from chronic periodontitis group was significantly higher than these in HGFs from healthy group(P<0.05). Meanwhile, the ratio of Bcl-2/Bax reduced in chronic periodontitis group at day 2 and 4, although Bcl-2 m RNA levels were significantly elevated at day 4(P<0.05).3. The mRNA expressions of API5, Bcl-2 and Bcl-xl in chronic periodontitis group were lower than these in healthy group, however, Bax and CCNE1 m RNA expressions were higher(P<0.05).4. siRNA API5 was successfully transfected into HGFs and the inhibitory efficiency is 49.54%. Knock-down of API5 in HGFs reduced cell activity significantly, increased cell apoptosis(P<0.05). At day 0, 1and 3 after transfection, API5, Bcl-2 and CCNE1 expressions were significantly lower than healthy group. The expression of Bcl-xl showed significant decrease at day 3(P<0.05), but not at day 0 and 1(P>0.05). However, the expression of Bax was significantly higher and the ratio of the Bcl-2/Bax was significantly lower than these in healthy group(P<0.05).Conclusion:1. Compared with healthy volunteers, cell activity of HGFs from patients with chronic periodontitis obviously decreased, and cell apoptosis increased, which suggests that patients with chronic periodontitis have poor ability of repair and increase susceptibility of periodontitis.2. Knock-down of API5 in HGFs reduced cell activity and promoted cell apoptosis, meanwhile, API5 expression level decreased in gingival tissue from chronic periodontitis, which suggests that API5 is involved in cell apoptosis in chronic periodontitis.3. Knock-down of API5 reduced Bcl-2, Bcl-xl and CCNE1, increased the expression of Bax, lowered the ratio of Bcl-2/Bax, which indicates that API5 plays an important role in the process of chronic periodontitis through regulating the expressions of cell apoptosis related genes.