| Objectives: As one of polycyclic aromatic hydrocarbons(PAHs), Benzo(a)pyrene(Ba P) has been recognized for its teratogenic, carcinogenic and mutagenic potential. Yet, evidences that Ba P can induce reprotoxicity have emerged in recent years. Based on previous studies, we speculate that Bap can induce damage in trophoblast cells and therefore detect the effect of Ba P on human trophoblast HTR-8/SVneo(HTR) cells in vitro. Traditional Chinese drug Five-Seed Procreating Pill has been found to improve fertility in humans. The principal component of the drug, Schizandrin B(Sch B) has been proven to activate the Nrf2-ARE signal pathway to perform anti-oxidative stress, anti-inflammatory, anti-apoptosis as well as against cells injury. Studies have agreed that activated Nrf2-ARE can not only accelerate the Ba P metabolism but also attenuate the oxidative stress damage in cells induced by Ba P. However, no study has identified that Sch B can inhibit Ba P in HTR cells up to date. We speculate that Sch B most likely attenuate damage of Ba P exposure in HTR cells via activating the Nrf2-ARE signal pathway. In present study, we aimed to detect whether Ba P can induce toxicity in HTR cells or not, and if so, whether Sch B can play a protective role on inhibiting toxicity of Ba P by activating the Nrf2-ARE signal pathway.Methods:1 MTS cell proliferation assay was performed to detect the effect of Ba P-alone, Sch B-alone and Sch B combining with Ba P treatment on HTR cell proliferation.2 Nrf2 Si RNA transfection was performed to interfere the Nrf2 m RNA expression and MTS cell proliferation assay was performed to detect the effect of Sch B and Ba P treatment on HTR cell proliferation.3 Real-time polymerase chain reaction(real-time PCR) was performed to detect the Nrf2, HO1, NQO, SOD and KEAP1 m RNA expression before Nrf2 Si RNA transfection and after Nrf2 Si RNA transfection.4 Western blot was used to detect the Nrf2, HO1 and NQO1 protein expression before Nrf2 Si RNA transfection and after Nrf2 Si RNA transfection.5 Enzyme-linked immnosorbent assay(ELISA) was performed to detect the HO1 and NQO1 protein expression in the cell supernatant before Nrf2 Si RNA transfection and after Nrf2 Si RNA transfection.6 SOD activity assay was performed to detect the total SOD(T-SOD) activity in the cell supernatant before Nrf2 Si RNA transfection and after Nrf2 Si RNA transfection.Results:1 MTS cell proliferation assay expressed message that all concentrations of 1, 2.5, 5, 10, 20, 40 μM Ba P-only group inhibited HTR cells proliferation(P<0.01) compared with the control group, while 20 μM Ba P led a 28.1% HTR cells death. It indicated that Ba P induced toxicity in HTR cells. And all concentrations of 0.25, 0.5, 1, 2, 5 μM Sch B did not induce toxicity in HTR cells except for 10 μM Sch B, even 0.5, 1, 2μM Sch B performed a slight proliferation promotion(P<0.01). In addition, 0.25, 0.5, 1, 2, 5 μM Sch B combining with Ba P groups attenuated the damage induced by Ba P-only group in HTR cells(P<0.01).2 Both real-time PCR and western bolt analysis agreed with that 1 μg Nrf2 Si RNA transfection made almost 75% Nrf2 m RNA silent. After Nrf2 Si RNA transfection in MTS cell proliferation assay, compared with Ba P-only group, the celluar protection rate of 0.5, 1 and 2 μM Sch B combining with Ba P groups were decreased to 4.5%, 9.8% and 15.6% in HTR cells from 10.4%, 19.1% and 24.0% before Nrf2 Si RNA transfection, respectively.3 In real-time PCR analyses, Nrf2 m RNA expression in Ba P-only group, 0.5, 1 and 2 μM Sch B combining with Ba P groups were respectively 1.45, 2.05, 3.48 and 4.83 times as much as the control group before Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups), while these four groups were respectively 1.27, 1.67, 2.05 and 2.96 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups). HO1 m RNA expression in the four groups were respectively 1.62, 2.27, 3.30 and 4.20 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while they were respectively decresed to 1.39, 1.77, 2.29 and 2.67 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups). NQO1 m RNA expression in the four groups were respectively 1.41, 2.49, 3.80 and 5.20 times as much as the control group before Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups), while they were respectively decresed to 1.30, 1.79, 2.82 and 3.59 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups). SOD m RNA expression in the four groups were respectively 1.63, 2.84, 4.66 and 6.40 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while they were respectively decresed to 1.43, 2.10, 3.04 and 3.98 times as much as the control group after Nrf2 Si RNA transfection(P<0.01). In addition, both before and after Nrf2 Si RNA transfection, Ba P-only group significantly downregulated the KEAP1 m RNA expression compared with the control group(P<0.01) while no statistical difference was observed between 0.5, 1 and 2 μM Sch B combining with Ba P groups and the control group(P>0.05).4 In western blot analyses, Nrf2 expression in nucleoprotein in Ba P-only group, 0.5, 1 and 2 μM Sch B combining with Ba P groups were respectively 1.55, 1.84, 2.61 and 3.51 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while these four groups were respectively decreased to 1.32, 1.56, 2.05 and 2.66 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups). Nrf2 expression in total protein in the four groups were respectively 1.36, 1.69, 1.93 and 2.33 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while they were respectively decresed to 1.18, 1.43, 1.54 and 1.76 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups). HO1 expression in total protein in the four groups were respectively 1.15, 1.42, 1.86 and 2.28 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while they were respectively decresed to 1.09, 1.23, 1.50 and 1.72 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups). NQO1 expression in total protein in the four groups were respectively 1.14, 1.42, 1.77 and 2.23 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while they were respectively decresed to 1.08, 1.23, 1.52 and 1.80 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups).5 In ELISA analyses, HO1 protein expression in cell supernatant in Ba P-only group, 0.5, 1 and 2 μM Sch B combining with Ba P groups were respectively 1.08, 1.16, 1.27 and 1.40 times as much as the control group before Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups), while these four groups were respectively decreased to 1.05, 1.09, 1.16 and 1.25 times as much as the control group after Nrf2 Si RNA transfection(P>0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups). NQO1 protein expression in cell supernatant in the four groups were respectively 1.13, 1.37, 1.56 and 1.67 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while they were respectively decresed to 1.07, 1.15, 1.26 and 1.35 times as much as the control group after Nrf2 Si RNA transfection(P<0.01).6 In SOD activity assay, T-SOD activity in cell supernatant in Ba P-only group, 0.5, 1 and 2 μM Sch B combining with Ba P groups were respectively 1.08, 1.23, 1.36 and 1.48 times as much as the control group before Nrf2 Si RNA transfection(P<0.01), while these four groups were respectively decreased to 1.05, 1.16, 1.24 and 1.33 times as much as the control group after Nrf2 Si RNA transfection(P<0.05 for Ba P-only group, P<0.01 for 0.5, 1 and 2 μM Sch B combining with Ba P groups).Conclusions:1 Ba P can directly induce toxicity in HTR cells.2 Moderate concentration of Sch B(no more than 5μM) has no hazards and even can attenuate the injury of Ba P exposure in HTR cells.3 Sch B can inhibit the injury induced by Ba P exposure in HTR cells by activating the Nrf2-ARE signal pathway. |
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