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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (8): 888-893.doi: 10.3736/jcim20110811

• Original Experimental Research • Previous Articles     Next Articles

Active ingredients of Plastrum Testudinis inhibit epidermal stem cell apoptosis in serum-deprived culture

Lan Chen1, Hui Li1(), Chun Li2, Jia-hui Cao1, Yi-ling Wu1, Yang Liu1, Jian-hong Zhou1, Dong-feng Chen1, Yi-wei Li2   

  1. 1. Teaching and Research Section of Anatomy, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
    2. Teaching and Research Section of Nursing, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Received:2011-03-20 Accepted:2011-04-12 Online:2011-08-20 Published:2011-08-15

Objective: To investigate the effects of active ingredients of Plastrum Testudinis (PT) on serum deprivation-induced apoptosis of epidermal stem cells (ESCs).
Methods: ESCs were isolated from the back skin of fetal Sprague-Dawley rats with 2 weeks of gestational age and were divided into normal group (10% fetal bovine serum), control group (serum-deprived culture) and groups treated with serum deprivation plus active ingredients of PT, including ethyl acetate extract (2B), stearic acid ethyl ester (S6), tetradecanoic acid sterol ester (S8) and (+)-4-cholesten-3-one (S9). The vitality of ESCs after 24, 48 and 72 h of culture was measured with MTT method; apoptotic ESCs double-stained with Annexin V-FITC and propidium iodine were detected by flow cytometry (FCM); Bcl-2 and caspase-3 expressions were measured by Western blotting.
Results: MTT results indicated that the vitality of ESCs in the active ingredients of PT groups at 48 h was increased compared with the control group and 2B had better effects than the others. FCM results indicated that 2B had the most significant anti-apoptotic effect compared with the control as well as S6, S8 and S9. Western blot results indicated that 2B, S6, S8 and S9 up-regulated the expression of Bcl-2 protein and down-regulated the expression of caspase-3 protein compared with the control.
Conclusion: Ethyl acetate extract of Plastrum Testudinis inhibits epidermal stem cell apoptosis in serum-deprived culture by regulating the expressions of Bcl-2 and caspase-3 proteins and has a stronger anti-apoptotic effect than its constituents S6, S8 and S9.

Key words: Plastrum Testudinis, epidermal stem cells, apoptosis, Bcl-2, caspase-3

Figure 1

Activity of ESCs with serum-free DMEM after 24-, 48- and 72-hour culture tested by MTT method △△P<0.01, vs control group; ▲P<0.05, ▲▲P<0.01, vs 2B group; ■P<0.05, vs S8 group. DMEM: Dulbecco’s modified Eagle’s medium; ESC: epidermal stem cell; 2B: extracts from Plastrum Testndinis; S6: stearic acid ethyl ester; S8: tetradecanoic acid sterol ester; S9: (+)-4-cholesten-3-one."

Figure 2

Apoptosis of ESCs after 48 h serum-free culture A: Graph of flow cytometry; B: Quantitative results of apoptosis ratios of ESCs. △P<0.05, △△P<0.01, vs control group; ▲▲P<0.01, vs 2B group; □P<0.05, vs S6 group; ■P<0.05, vs S8 group. ESC: epidermal stem cell; 2B: extracts from Plastrum Testndinis; S6: stearic acid ethyl ester; S8: tetradecanoic acid sterol ester; S9: (+)-4-cholesten-3-one."

Figure 3

Expressions of Bcl-2 and caspase-3 proteins after 48 h serum-free culture A: Original drawing of Western blot; B: Statistical results of Western blot. **P<0.01, vs normal group; △△P<0.01, vs control group; ▲P<0.05, ▲▲P<0.01, vs 2B group; □□P<0.01, vs S6 group; ■■P<0.01, vs S8 group. ESC: epidermal stem cell; 2B: extracts from Plastrum Testndinis; S6: stearic acid ethyl ester; S8: tetradecanoic acid sterol ester; S9: (+)-4-cholesten-3-one."

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