| Objective:To investigate the anticancer effects and mechanisms of tanshinone IIA (Tan IIA) microemulsion (ME) in H22 murine hepatoma cells both in vitro and in vivo.Methods:TanⅡA was encapsulated into a microemulsion as a drug delivery system, TanⅡA ME was prepared to a targeting preparation. H22 murine hepatoma cells were treated with 0.5μM, 2μM TanⅡA ME for 48h, control groups were 0.5μM empty ME and 2μM TanⅡA solution. H22 tumor bearing mice were treated with 2, 4, 8mg/kg TanⅡA ME by iv daily for a total of 7 consecutive days, control groups were 4mg/kg empty ME and 8mg/kg TanⅡA solution, positive group is 25mg/kg 5-FU. In vitro, Bcl-2 expression was performed by flow cytometry, Bcl-2, Bax and Caspase-3 mRNA levels were performed by reverse transcription-PCR assay. In vivo, body weight, spleen index and inhibition rate were investigated, Bcl-2, Bax and Caspase-3 mRNA levels in H22-transplanted tumor were performed by reverse transcription-PCR assay, the distribution of Bcl-2, Bax and Caspase-3 in H22-transplanted tumor were performed by double immunofluorescence staining.Results:1.In vitro, FCM analysis showed that Bcl-2 protein level of H22 murine hepatoma cells were 28.68(Empty ME), 27.17(TanⅡA solution), 27.36(0.5μM TanⅡA ME), 2.65(2μM TanⅡA ME).2.PCR assay showed that the H22 murine hepatoma cell mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in TanⅡA solution group were 0.10, 1.55, 0.75; the H22 murine hepatoma cell mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in Empty ME group were 0.09, 0.85, 0.40; the H22 murine hepatoma cell mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in 0.5μM TanⅡA ME group were 0.16, 0.70, 0.85; the H22 murine hepatoma cell mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in 2μM TanⅡA ME group were 0.33, 0.44, 1.24.3.In vivo, 4, 8mg/kg TanⅡA ME and 25mg/kg 5-FU had significant inhibitory effects on the growth of inoculated H22 cells in mice, and the inhibition rates were 41.74%, 52.89%, 67.06%, respectively. The spleen index of the 5-FU group was smaller than other groups, and the body weight of 5-FU group was lower than other groups, *p<0.05.4.PCR assay showed that the mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in H22 tumor bearing mice treated with TanⅡA solution were 0.65, 1.23, 0.12; the mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in H22 tumor bearing mice treated with Empty ME were 0.26, 1.34, 0.09; the mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in H22 tumor bearing mice treated with 5-FU were 1.43, 0.68, 0.30; the mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in H22 tumor bearing mice treated with 2mg/kg TanⅡAME were 0.87, 0.97, 0.09; the mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in H22 tumor bearing mice treated with 4mg/kg TanⅡAME were 1.06, 0.94, 0.18; the mRNA levels of Bax/β-actin, Bcl-2/β-actin, Caspase-3/β-actin in H22 tumor bearing mice treated with 8mg/kg TanⅡAME were 1.36, 0.59, 0.31.5.Double immunofluorescence staining showed that up-regulated protein levels of Bax and Caspase-3 and a down-regulated protein level of Bcl-2 induced by 4, 8mg/kg TanⅡA ME and 25mg/kg 5-FU.Conclusions:1.Compared with TanⅡA solution, TanⅡA ME induced apoptosis in H22 murine hepatoma cells with a dose-dependent manner. 2. TanⅡA ME had significant inhibition rate for H22 tumor bearing mice, compared with 5-FU group, TanⅡA ME has no major side effects to the mice.3. The anti-cancer mechanism of TanⅡA ME might be related to the regulation of the expression of Bcl-2, Bax, and Caspase-3. |
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