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Effect of caspase-dependent pathway on apoptosis of human gingival fibroblasts induced by advanced glycation end products

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Author:
No author available
Journal Title:
Chinese Journal of Stomatology
Issue:
11
DOI:
10.3760/cma.j.issn.1002-0098.2011.11.008
Key Word:
成纤维细胞;糖基化终产物,高级;半胱氨酸天冬氨酸蛋白酶;细胞凋亡;Fibroblasts;Glycosylation end products,advanced;Caspases;Apoptosis

Abstract: Objective 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.Methods 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.Results 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 ).Conclusions Apoptosis of HGF induced by AGE-HISA may be mainly through activation of caspase-dependant pathway in which cytoplasmic pathway may play a predominant role.

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