Preventive Effect Of Schisandrin B Against Apoptosis Damage Of Extravillous Trophoblast Cell Line Caused By BaP

PAHs are common air pollutants,Benzo(a) pyrene(Ba P) is the most representative of PAHs pollutants that mainly from coal combustion, tobacco smoke, cook lampblack and automobile exhaust,easily in soil, water and crop residue accumulation [1]. studies have shown that benzo(a) pyrene is associated with the occurrence of adverse pregnancy outcomes, such as abortion, fetal growth restriction, eformity, also related with fertility decline and infertility [2-5], which may affect the metabolism of mechanism include oxidative damage, DNA damage,apoptosis, cytochrome P450 enzymes and anti-estrogenic effects, etc. [6-11]Schisandra chinensis are magnoliaceae plants,which is good for heart, lung,kidney and so on. Its main ingredients Schisandrin B not only has antioxidant and antiapoptotic functions[12-15], but also can the bacteriostasis in vitro[16], anti-tumor[17-18], DNA damage resistance[19], etc. However, the preventive effect of Schisandrin B on embryo damage caused by air pollutants had not be researched till now.We used human chorionic trophoblast cells in this study to explore the probable effect of Schisandrin B on inhibiting apoptosis injury caused by benzopyrene.Objective: To explore the preventive mechanism of Schisandrin B against apoptosis damage caused by Ba P,based on HTR8-SVneo cells,which provides the theoretical basis and the experimental basis for the development of protective agent of reproductive toxicity in pollutants environmental.Methods:1 The study found 1 ~ 20μmol / L Ba P at 24 hours a decrease in cell survival, and cell survival with increasing concentrations of benzopyrene decreases. According to cell survival and the exposure intensity, we selected 20μmol / L 24 hours as exposure intensity to establish the injury model of Ba P. 1 ~ 2μmol / L Schisandrin B at 24 hours make a increase in cell survival,but 10 ~ 200μmol / L Schisandrin B at 24 hours make a decrease in cell survival. Experimental groups: control group, Ba P( 20μmol/L) exposure group, Schisandrin B(0.5, 1, 2μmol/L)exposure to Ba P(20μmol/L) group. Using MTS to observe the cell viability of each groups.2 Respectively using Hochest33342, flow cytometry to observe apoptotic morphological and apoptosis rate in each cells group,using mitochondrial membrane potential kit measure the change of mitochondrial membrane potential in each cells group.3 Respectively using ELISA measure the concentration of bax,bcl-2 and Cyt-c protein in each cells groups. using Western blot analysed the expression of caspase-9, caspase-3 in each cells groups.Results:1 Preventive effects of Schisandrin B on the the damage of HTR8-SVneo cell lines induced by Ba P.MTS method found that OD value of blank control group was 1.568±0.012, Ba P infected group was 1.187 ± 0.015, 0.5μmol/L Schisandrin B was 1.483 ± 0.022, 1 μmol/L Schisandrin B was 1.489±0.048, 2μmol/L Schisandrin B was 1.531± 0.240, compared with Ba P infected group, the difference was statistically significant(P<0.05).2 Hochest33342 observed the morphological changes of apoptosis and flow cytometry determined the apoptosis rateThe result showed that Schisandrin B can prevent the apoptosis damage of extravillous trophoblast cell line caused by Ba P. Hochest33342 experiment shown that HTR8-SVneo cells exposed to Ba P had significantly morphological changes of apoptosis and flow cytometry determined the apoptosis rate shown that Schisandrin B significantly decreased the apoptosis rate of HTR8-SVneo cells induced by Ba P. the apoptosis rate of Blank control group was 0.031±0.001, Ba P infected group was 0.123±0.04, 0.5μmol/L Schisandrin B was 0.064±0.008, 1 μmol/L Schisandrin B was 0.052±0.020, 2μmol/L Schisandrin B was 0.036±0.014, compared with Ba P infected group, the difference was statistically significant(P<0.05).3 The change of mitochondrial membrane potentialThe mitochondrial membrane potential test shown that Schisandrin B significantly decreased the depolarization of HTR8-SVneo cells induced by Ba P. Red/green fluorescence intensity of blank control group was10.20±2.37, Ba P infected group was 3.35±0.85, 0.5μmol/L Schisandrin B was 6.99±0. 53, 1 μmol/L Schisandrin B was 7.15±1.22, 2μmol/L Schisandrin B was 8.73±1.05, compared with Ba P infected group, the difference was statistically significant(P<0.05).4 The change of bax, bcl-2 and Cyt-c protein concentrationELISA test shown that compared with the Ba P group, 0.5, 1, 2μmol/L Schisandrin B significantly decreased the concentration of bax, Cyt-c protein and increased concentration of bcl-2 in HTR8-SVneo cells induced by Ba P(P<0.05).5 The expression of Caspase-9 and caspase-3Western blot test shown that compared with the Ba P group, 0.5, 1, 2μmol/L Schisandrin B significantly decreased the expression of Caspase-9 and caspase-3 in HTR8-SVneo cells induced by Ba P(P<0.05).Conclusions:This study from the aspects of mitochondrial apoptosis signaling pathways, explore part of the mechanism of Schisandrin B prevent the apoptosis damage of HTR8-SVneo cells induced by Ba P in vitro. The mechanism may be related to reduce the concentration of bax, enhance the concentration of bcl-2, decrease the depolarization of mitochondria to block Cyt-c from mitochondria release into cytosol and then inhibit the expression of downstream Caspase-9 and caspase-3to play a role of antiapoptotic.These results indicated that Schisandrin B can prevent the apoptosis damage of HTR8-SVneo cells induced by Ba P in vitro.