Study for periodontal pathogenesis and apoptosis in periodontal tissues induced by volatile sulfur compounds

硫化氢(H_2S)是引起口臭和牙周病的主要原因之一。H_2S可引起主动脉平滑肌细胞等组织细胞的凋亡性死亡，细胞凋亡在牙周炎的发生发展中起重要作用。因此，我们研究了H_2S是否会引起人牙龈成纤维细胞(HGF)的凋亡。材料和方法：用乳酸脱氢酶测定法测定坏死细胞。通过检测组蛋白复合体DNA片段和流式细胞术确定细胞的凋亡情况。Caspase3是细胞凋亡信号转导的关键酶。同时检测细胞内活性氧(ROS)含量，证实H_2S对超氧化物歧化酶(SOD)的抑制作用。用单细胞凝胶电泳法(SCGE)检测DNA损伤。结果：100 ng/mlH_2S孵育72小时，HGF的坏死率低于10%，而胞浆DNA片段化明显增加(p&lt；0.05)。流式细胞仪检测显示肝细胞生长因子细胞的凋亡率也有所增加。Caspase3活性在孵育72h时显著升高(p&lt；0.01)。H_2S作用48小时和72小时后，HGF的尾长、尾长和尾矩DNA百分率均增加。与H_2S孵育48小时和72小时后，ROS显著升高(p&lt；0.005和p&lt；0.001)，而肝细胞生长因子的超氧化物歧化酶受到强烈抑制。结论：H_2S可诱导HGF细胞发生凋亡，H_2S对超氧化物歧化酶的抑制作用可增加细胞内ROS水平，进而诱导细胞凋亡和DNA断裂。

Hydrogen sulfide (H_2S) is one of the main reasons of halitosis and periodontally pathogenic. H_2S causes apoptotic cell death in aorta smooth muscle cells and other tissues, and apoptosis plays an important role in the onset and progress of periodontitis. Therefore, we investigated if H_2S causes apoptosis in human gingival fibroblasts (HGF). Materials and Methods : Necrotic cell were determined using lactate dehydrogenase assay. Apoptosis was ascertained with detecting histone-complexed DNA fragments and a flow cytometry. Caspase 3, a key enzyme in apoptotic signaling, was assessed. Reactive oxygen species (ROS) were also detected and inhibition of superoxide dismutase (SOD) by H_2S was verified. DNA damage was examined with single-cell gel electrophoresis (SCGE). Results : Necrosis was found less than 10% of HGF during 72 hours incubation with 100 ng/ml H_2S, whereas significant increment of DNA fragmentation in cytoplasm was found (p < 0.05). Apoptotic HGF cells were also increased in a flow cytometry. Caspase 3 activity was significantly increased at 72 hours incubation (p < 0.01). Tail length, %DNA in tail and Tail moment obtained by SCGE increased in HGF exposed to H_2S for 48 and 72 hours. Moreover, ROS was significantly increased at 48 and 72 hour incubation with H_2S (p < 0.005 and p < 0.001, respectively), and SOD of HGF was strongly inhibited. Conclusion : H_2S caused apoptosis in HGF, and an inhibition of SOD by H_2S increased ROS, then induced apoptosis and DNA strand breaks.