| Objective: 1.Through constructed the oxidized stress damage of human umbilical vein endothelial cell induced by PO to preliminary explore the role of Notch1 pathway plays in this cell model. 2. To investigate an protective effect of phytoestrogens Gen on oxidized stress damage induced by PO and the relationship between the Notch1 signaling pathways.Methods: 1.The establishment of the OSI model by PO: HUVECs cells were divided into seven groups, which were treated with 0, 300, 600, 900, 1200, 2400 ?M PO and PO solvent(0.1% DMSO) for 24 h respectively. Different kits were used to detect the cell viability as well as the concentration of Lactate dehydrogenase(LDH), Superoxide dismutase(SOD)and Malondialdehyde(MDA) of each group. Western blot was used to detect the protein expression levels of Notch1, Hes1, Bcl-2, Bax and Caspase3 of each group. Then, observe the dose-related effects of PO on oxidative damage of HUVECs. Next, select the optimal concentration of PO(1200 ?M) to treat HUVECs for 0, 6, 12, 24 h respectively. Test biomedical index and protein exprssion levels of 1200 ?M PO-treated cells as described above to evaluate the time- response relationship between PO and oxidative damage of HUVECs. 2.The observation of dose-related and time-response effects of Gen on ameliorating oxidative stress damage of HUVECs: pretreat HUVECs with different concentration of Gen(0, 0.05, 0.1, 0.5, 1 ?M) for 10 h respectively, and then treat all HUVECs with 1200 ?M PO for 24 h. Test related biomedical index and protein exprssion levels of each group of HUVECs to evaluate the dose-response effect of Gen on ameliorating the oxidative damage of HUVECs. Then, pretreat HUVECs with optimal concentration of Gen(0.5 ?M) for 0, 6, 10, 14 h and then treated with 1200 ?M PO for 24 h. Next, time-effect relationship between Gen and the amelioration of oxidative damage of HUVECs was assesed. 3.Exploration of relationship between Gen's amelioration of oxidative damage induced by PO and Notch1 signaling pathways. HUVECs was divided into six groups: control group, PO-stimulation group, PO+DAPT group, DAPT group, Gen+PO+DAPT group and PO+Gen group. Pretreat each group of HUVECs with 0.5 ?M Gen for 10 h and then add 10?M DAPT(specific inhibitor of Notch1) and /or 1200 ?M PO for co-culture for 24 h. Next, detect the cell vitality, the concentration of LDH, SOD and MDA, and test related protein expression levels.Results: 1. HUVECs were treated with different concentrations of PO for 24 h. Under microscope observation, changes did not happen in the 300 ?M PO-treated and 600 ?M PO-treated groups, while the cavity and fragmentation were increased, and the intercellular arrangement was sparsed in the 1200 ?M PO-treated and 2400 ?M PO-treated groups. The control group and DMSO group were in good condition. In CCK8 analysis, cell viability was dose-dependently decreased in the PO(900, 1200, 2400 ?M) treated groups compared to control group(P<0.01). Besides, the cell viability in the DMSO group did not decrease significantly compared with the control group(P>0.05). Enzyme assay showed that the content of LDH and MDA was significantly increased in PO(600, 900, 1200, 2400 ?M) groups compared with control group, while the content of SOD decreased significantly(P<0.01). The results of Western Blot showed that the expression of Notch1, Hes1, Bax, Caspase3 were increased in PO(1200 ?M) group, while the expression of Bcl-2 and the Bcl-2/Bax ratio were decreased compared with control one(P<0.05). 2. HUVECs were treated with PO(1200 ?M) for different hours. Treatment of 1200 ?M PO significantly decreased the cell viability and the SOD level, while the LDH and MDA levels were significantly increased. Besides, 24 h of PO treatment increased the expression of Notch1, Hes1, Bax and Caspase3, while the expression of Bcl-2 and the Bcl-2/Bax ratio were decreased. 3. The cell viability and SOD level were significantly increased in Gen(0.5, 1 ?M)-pretreated HUVECs damaged by PO, while, the content of LDH and MDA were significantly decreased, and such effects were most obvious under pretreatment of 0.5 ?M Gen. The results of Western Blot showed that pretreatment of 0.5 ?M Gen significantly decreased the expression of Notch1, Hes1, Bax and Caspase3(P<0.05), while, the expression of Bcl-2 and the Bcl-2/Bax ratio were increased compared with PO(1200 ?M) group(P<0.05). 4. HUVECs were Pretreated with Gen(0.5 ?M) for different hours. With the increase of pretreat time, cell viability and SOD levels were significantly increased, while the LDH and MDA levels were decreased. The results of Western Blot showed that the expression of Notch1, Hes1, Bax and Caspase3 were decreased in Gen(10, 14 h) group, while the expression of Bcl-2 and the Bcl-2/Bax ratio were increased in Gen(10, 14 h) group compared with Gen 0 h group in time-dependent pattern. 5.Under the microscope observation, the cells in P + D + G group were significantly damaged, more cells were detached and the cell interval was increased compared to PO + Gen group. In CCK8 analysis, cell viability did not change apparently in DAPT group compared with control group(P>0.05). In groups that was added with PO, the cell viability was significantly decreased(P<0.05). The cell viability was decreased in the PO + DAPT + Gen group compared with PO + Gen group(P<0.05). In enzyme assay, the content of LDH and MDA were significantly increased, while the content of SOD was significantly decreased in groups adding PO compared with control group(P<0.05). The content of LDH and MDA were significantly increased, while the content of SOD was significantly decreased in the Gen + PO + DAPT group compared with Gen + PO group(P<0.05). The results of Western Blot showed that the expression of Notch1, Hes1, Bax and Caspase3 were increased(P<0.05), yet, the expression of Bcl-2 and Bcl/Bax ratio were decreased(P<0.05)in group treated with PO(1200 ?M) for 24 h. Compared with Gen+PO group, the PO + DAPT + Gen group had significantly increased expression of Notch1, Hes1, Bax and Caspase3(P<0.05) and decreased expression of Bcl-2 and the Bcl-2/Bax ratio. These results implied that Gen may ameliorate the oxidative damage induced by PO through regulation of Notch1 signaling pathway.Conclusions: 1.The damage effects of PO in HUVECs cells in time and dose-dependent manner, Gen plays a protective role in PO-induced oxidative stress damage in HUVECs. 2.The damage effects of PO in HUVECs though the upregulation of the expressions of Notch1, Hes1, Caspase3, Bax, downregulation of Bcl-2, Bcl-2/Bax expressions. Gen reverse the PO-induced Notch1, Hes1, Caspase3, Bax downregulation of Bcl-2, Bcl-2/Bax expressions contributing to the plays a protective role in PO-induced oxidative stress damage in HUVECs. |
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