title: Eucommia ulmoides Oliv. antagonizes H2O2-induced rat osteoblastic MC3T3-E1 apoptosis by inhibiting expressions of caspases 3, 6, 7, and 9.
abstract: Eucommia ulmoides Oliv. (EuO), also known as Duzhong, native to China, has been reported to have antioxidative function, but its cellular mechanism is not fully examined yet. We investigated inhibitory effects of EuO leaf ethanol extracts on H(2)O(2)-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms. Locally-grown Duzhong leaves were extracted with ethanol. MC3T3-E1 cells were treated with EuO (6.25, 12.5, 25, 50, and 100 µg/ml) for 24 h, and then H(2)O(2) (800 µmol/L) for an additional 24 h. Cell survival rate, percentage of apoptosis, and expressions of caspases 3, 6, 7, and 9 were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, microscopic analysis, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR). The final EuO leaf ethanol extract powder was detected to contain caffeotannic acid at 58 mg/g and geniposide at 3.45 mg/g by high performance liquid chromatography (HPLC). EuO remarkably restrained cell oxidative damage and increased cell survival rate in a dose-dependent manner: 0 µg/ml, 0.21; 6.25 µg/ml, 0. 28; 12.5 µg/ml, 0.31; 25 µg/ml, 0.48; 50 µg/ml, 0.54; and 100 µg/ml, 0.66 (P<0.05), with the half-effective concentration being around 25 µg/ml. MTT results were confirmed by microscopic analysis. Western blotting and RT-PCR analyses showed that the expressions of caspases 3, 6, 7, and 9 were significantly decreased in the EuO-treated cells compared with the control (EuO- and H(2)O(2)-free) (P<0.05), with the half-effective concentration of EuO ranging from 12.5 to 25 µg/ml. We conclude that the ethanol-extracted EuO leaf extracts promoted the growth of MC3T3-E1 cells, and suppressed the H(2)O(2)-induced apoptosis in a rat MC3T3-E1 osteogenic cell model, likely due to the inhibition of caspases' activities. The results indicate that EuO is a potent antioxidant, which may contribute to its many cellular protective functions, including the promotion of bone growth.
authors.affiliation: Department of Stomatology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Lin
authors.forename: Yi-jing
authors.initials: YJ
authors.lastname: Fan
authors.forename: Christian
authors.initials: C
authors.lastname: Mehl
authors.forename: Jia-jun
authors.initials: JJ
authors.lastname: Zhu
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Ling-yan
authors.initials: LY
authors.lastname: Jin
authors.forename: Jing-hong
authors.initials: JH
authors.lastname: Xu
authors.forename: Hui-ming
authors.initials: HM
authors.lastname: Wang
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D031283:Eucommiaceae; D015870:Gene Expression / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000633:toxicity*; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology*; D010936:Plant Extracts / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats
chemical_list: D000975:Antioxidants; D061945:Caspase Inhibitors; D017931:DNA Primers; D010936:Plant Extracts; D012333:RNA, Messenger; D006861:Hydrogen Peroxide; D020169:Caspases