Liu Kai-Lei, Yu Shao-Jie, Fu Yun
Department of Periodontology, Guanghua School and Hospital of Stomatology & Institute of Stomatological Research, Sun Yat-sen University, Guangzhou 510055, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2011 Nov;46(11):673-7.
To investigate the effect of synthesized advanced glycation end products (AGE) on apoptosis of human gingival fibroblasts (HGF) and the possible role of caspase-dependent pathway in the process of AGE-induced apoptosis.
HGF were incubated with AGE-human serum albumin (AGE-HSA). The activity of caspase-8, caspase-9 and caspase-3 were detected by microplate reader after 12 and 24 hours. HGF were incubated with caspase inhibitors for 1 hour, and then incubated with AGE-HSA for 24 hours, HGF was first stained by Hoechst33258 and observed under inverted microscope, and then double stained by annexin V and propidine iodide (PI) and observed by flow cytometry (FCM). The activity of caspase-3 was determined by caspase-3 assay kit and observed by microplate reader.
Caspases activity of caspase-8, -9, -3 was 0.1097 ± 0.0051, 0.0965 ± 0.0051 and 0.1280 ± 0.0103 after 12 h of incubation with AGE-HSA and HGF, respectively, and 0.1558 ± 0.0053, 0.1308 ± 0.0035 and 0.1954 ± 0.0051 after 24 h of incubation with AGE-HSA and HGF, respectively (P < 0.05). Positive cells number was 247.7 ± 32.4, 200.1 ± 14.6, 154.1 ± 14.4 and 131.3 ± 14.6 in caspase inhibitor groups by Hoechst33258 staining, respectively. Apoptotic rate was (25.57 ± 2.20)%, (38.87 ± 3.31)%, (17.17 ± 2.24)% and (14.73 ± 2.48)% in caspase inhibitor groups by annexin V-PI staining, respectively. The difference between different groups was significant (P < 0.05). Caspase-3 activity was reduced to 0.1274 ± 0.0076, 0.1465 ± 0.0062, 0.1044 ± 0.0051 in caspase inhibitor groups, respectively. The difference between different groups was significant (P < 0.05).
Apoptosis of HGF induced by AGE-HSA may be mainly through activation of caspase-dependant pathway in which cytoplasmic pathway may play a predominant role.
