Study On Action Mechanism Of FuFangDanShen In Protecting Oxidative Stressed Trophoblast Cells

Chapter I Effects of FuFangDanShen on Oxidative Stressed Trophoblast CellsObjective:To decide the protective concentrations of FuFangDanShen (FFDS) on oxidative stressed trophoblast cells.Methods:JEG-3 and JAR trophoblast cells were pre-incubated with different concentrations (0 mg/ml,1.25 mg/ml,2.50 mg/ml,6.25 mg/ml and12.5 mg/ml)of FFDS. Then 5 mM H2O2 was added to induced oxidative stress. For evaluation of cell viability and selection of the best protective concentration of FFDS, MTT staining method was used. The morphological characteristics of cells were observed by the inverted microscope and transmission electron microscope(TEM).Results:As compared to cells without pre-incubation with FFDS, Cells pre-incubated with low levels (1.25 mg/ml,2.50 mg/ml and 6.25 mg/ml) of FFDS showed a significantly higher survival rate, but cells preincubated with high level of FFDS(12.5 mg/ml), compared with other groups, showed a significantly reduced value of cell survival rate。observed by the inverted microscope, cells without pre-incubation with FFDS, were severely injured. The intercellular spaces were wide and debris increased. FFDS pre-incubated cells were slightly injured. Observed by TEM:pre-incubated without FFDS, cells showed a condensed chromatin close to nuclear membrane, cytoplasm were vacuolated, nuclear membrane disappeared and apoptotic body formed. However, morphologically, cells in FFDS group were similar with the normal cells.Conclusion:FFDS show a protective effect on oxidative trophoblast. The protective levels of FFDS include 1.25 mg/ml,2.50 mg/ml and 6.25 mg/ml, and the best one is 6.25 mg/ml. But hight levels of FFDS may cause an injured effect.Chapter II Protective action mechanism of FuFangDanShen on Oxidative Stressed Trophoblast CellsObjective:To explore the protective action mechanism of Fu Fang Dan Shen(FFDS) on oxidative stressed trophoblast cells.Methods:JEG-3 and JAR trophoblast cells were pre-incubated with different concentrations (0 mg/ml,1.25 mg/ml,2.50 mg/ml and 6.25 mg/ml) of FFDS. Then 5 mM H2O2 was added to induced oxidative stress. For evaluation of cell oxidative stress and analysis of MDA, TPA in supernatant of curltured medium, DCFH-DA, colorimetry and ELISA were used, respectively. For analysis of SOD-activity, SOD mRNA and protein expression, colorimetry, real time fluorescent quantitative RT-PCR and Western Blot were respectively used. Dysferlin expression was tested by immunofluorescent staining technique.Results:As compared to cells without pre-incubation with FFDS, Cells pre-incubated with of FFDS showed a significantly reduced contents of ROS, MDA and TPA. So did the expression of dysferlin. And the more concentration of FFDS, the lower value of index above. As well as a significant increase of SOD-activity, SOD-mRNA and protein expression. And the more concentration of FFDS, the higher value of index above.Conclusion:(1) FFDS show a protective effect on oxidative stressed trophoblast. This effect may be related to the increased SOD-activity, upregulated expression of SOD mRNA and the reduction of ROS, MDA and TPA production. (2) Oxidative stressed trophoblast JEG-3 and JAR show an expression of dysferlin. The increasing of expression in severe injured cells may be a compensatory response. Chapter III Effects of FuFangDanShen on Expression of NF-κB, ICAM-1 and sFlt-1 in Trophoblast Cells.Objective:To explore the effects of FuFangDanShen (FFDS) on expression of NF-κBp65, ICAM-1 and sFlt-1 in trophoblast cells.Methods:JEG-3 and JAR trophoblast cells were pre-incubated with different concentrations (0 mg/ml,1.25 mg/ml,2.50 mg/ml and 6.25 mg/ml) of FFDS. Then 5 mM H2O2 was added to induced oxidative stress. For evaluation of the expression of NF-KBp65 and ICAM-1, immunofluorescent staining technique was used. The level of sFlt-1 and VEGF-A in supernatant of curltured medium were tested by ELISA. The mRNA and protein expression of sFlt-land VEGFA were tested by real time fluorescent quantitative RT-PCR and Western blot techniques.Results:As compared to cells without pre-incubation with FFDS, cells pre-incubated with of FFDS showed a significantly reduced expression of NF-KBp65, ICAM-1, and the higher concentration of FFDS, the lower value of index above, and a significantly reduced level of protein and mRNA of sFlt-Iand VEGF-A.Conclusion:FFDS show an anti-inflammatory effect. This may be related to inhibition of the expression of NF-κBp65, ICAM-1 and sFlt-1/VEGF in oxidative stressed trophoblast.Chapter IV Effects of FuFangDanShen on apoptosis induced by preeclampsia serum in trophoblast cellsObjective:To explore the effects of FuFangDanShen on apoptosis induced by preeclampsia serum in trophoblast cells.Methods:Primary placental trophoblast (PT) cells were isolated by digestion of normal human term placental villous tissue with trypsin/DNase and purified by Percoll density gradient centrifugation. To identify the PT cells, immunocytochemistry fluorescent staining technique were used to observe the expression of cytokemtin-7 and vimentin in trophoblast cells. JEG-3 and JAR and PT cells were divided into 5 groups:normal control NC (cultured with 10%fetal bovine serum, FBS), control NP (cultured with 10%normal pregnancy serum), preeclampsia PE (mPE and sPE, mild and severe PE,cultured with 10%preeclampsia serum), and sPE+DS(cells Pre-incubated with 6.25mg/ml FFDS for 24h, then 10%sPE serum added). Cells in NC groups were cultured in medium with 10%FBS. Other cells were pre-cultured 24h in medium containing 0.1%fetal bovine albumin(without FBS), at the same time, FFDS was added into medium of sPE+DS groups, then incubated 24h with pregnancy serum. The apoptosis was tested by flow cytometry annexin V/PI and TUNEL staining.Results:Flow cytometrically, the apoptosis rate in NC was significantly lower than other groups. in sPE groups, the apoptosis rate was significantly higher than other groups, But there was no difference in NP, mPE and sPE+DS groups. PT cells showed significantly higher apoptosis rates than JEG-3 and JAR cells (P<0.05). The cells in sPE+DS had significantly lower apoptosis rates than sPE groups (P<0.05) There was no difference of apoptosis rate in the same group of JEG-3 and JAR cells. TUNEL showed:the numbers of positive stained cells in sPE were significantly higher than NC and sPE+DS groups. The apoptotic cell in NC groups was lowest (P <0.05). PT cells had higher apoptosis rate than JEG-3 and JAR cells, but no difference between JEG-3 and JAR cells.Conclusion:The sPE serum significantly promotes the apoptosis of trophoblast cells. FFDS can inhibit the apoptosis induced by sPE serum in trophoblast cells. It may be may be related to FFDS antioxidant effect.