Objective: To observe the protective effects of Zuoguiwan (ZGW) on tertiary butyl hydroperoxide-induced MC3T3-E1 cell apoptosis

in order to study the possible mechanisms of ZGW via mitochondria-dependent pathways. Method: ZGW drug-contained serum was prepared. With murine osteoblastic MC3T3 cells as the research object

the oxidative stress model was established by tertiary butyl hydroperoxide (t-BHP)

and then divided into 3 groups

blank control group

t-BHP group

ZGW+t-BHP group and Estradiol Valerate+t-BHP group. After incubation for 24

48

72 hours

the effects of ZGW on viability and apoptosis of t-BHP-induced MC3T3-E1 cells were investigated by MTT assay. After incubation for 48 hours

cell apoptosis was measured by acidine orange/ethidium bromide (AO/EB) staining. The changes of nucleus were observed by Hoechst 33342 staining. The changes of mitochondrial membrane potential were analyzed with rhodamine 123 (Rh123) staining. Western blot analysis was used to evaluate expressions of Bcl-2

Bax and Caspase-3 protein expressions. Result: Compared with blank model group

the proliferation of MC3T3-E1 cell was significantly inhibited in t-BHP group (P < 0.05

P < 0.01)

cell apoptosis increased

mitochondrial membrane potential decreased

and the protein expression of Bcl-2 was significantly down-regulated (P<0.01). Compared with t-BHP group

the survival of MC3T3-E1 cells exposed to the oxidative damage induced by t-BHP was promoted in ZGW+t-BHP group (P<0.01)

particularly at 48 h

cell apoptosis reduced

mitochondrial membrane potential significantly improved

and the protein expression of Bcl-2 was increased (P<0.05)

while Bax and caspase-3 expression were decreased (P<0.01). Conclusion: Drug-contained serum of ZGW can inhibite t-BHP-induced MC3T3-E1 cell apoptosis. The possible mechanism may be related to mitochondria-dependent pathways.