| Objectives Exploring the effects ofhuman umbilical cord mesenchymal stem cellsconditioned medium(h UC-MSCs-CM)and N-acetylcysteine(NAC)on radiationrelated oxidative stress and Hippocampal neuron proliferation and apoptosis.Methods HT-22 cells were treated with irradiation of different X-rays doses(0,5,10,15Gy),and the optimal radiation dose was selected.Enzyme digestion was used to extract human umbilical cord mesenchymal stem cells(h UC-MSCs).After culturing and passage,the morphology was observed under a microscope.Flow cytometry was used to identify immunophenotype.The third generation cells were cultured with serum-free medium for 48 h,and the supernatant was collected for subsequent experiments.Experimental groups were set as follows: control(Control)group,radiation treatment(RT)group,radiation treatment+MSCs-CM(RT+MSCs-CM)group,MSCs-CM(MSCs-CM)group,radiation treatment+NAC(RT+NAC)group,radiation treatment+MSCs-CM+inhibitor of PI3K(LY294002)group.Each group was treated with 10 Gy X-ray radiation except control group and MSCs-CM group.After 24 hours of radiation,we assessed a panel of basic cellular indexes regarding radiation response including the number and morphology,cell proliferation,apoptosis,intracellular reactive oxygen species(ROS),and glutathione(GSH),malondialdehyde(MDA)content and superoxide dismutase(SOD)activities,and mitochondrial membrane potential(MMP).Furthermore,?-H2 AX,PI3K-AKT,P53,Cleaved caspase-3,Bax and BCl-2 proteins were analyzed by Western blot.Results 1 After treatment of HT-22 cells with 0,5,10,15 Gy doses,the cell viability decreased in a dose-dependent manner.HT-22 cells apoptosis rate is proportional to X-rays doses(P<0.01).2 Under the inverted phase contrast microscope,the cell morphology of the third generation h UC-MSCs was arranged in a fusiform and swirling arrangement.3 Flow cytometry detection h UC-MSCs conform to the immunophenotype of MSCs.4 In the treated groups including MSCs-CM(P<0.01)and NAC group(P<0.01)were significantly reversed radiation-induced proliferation inhibition,intracellular MMP,ROS and MDA content ascend,and GSH and SOD inactivity in HT-22 cells increased.5 MSCs-CM and NAC suppressed ?-H2 AX,P53,Bax,Cleaved-caspase-3 but raised anti-apoptotic protein Bcl-2(P<0.01).6 Westernblot results showed that compared with the control group,the levels of p-PI3 K protein in HT-22 cells in the RT+MSCs-CM group and MSCs-CM group were significantly increased.PI3 K inhibitor(LY294002)can antagonize the up-regulation of p-PI3 K in HT-22 cells by MSCs-CM.NAC has no significant effect on regulating the expression of p-PI3 K protein.Conclusions 1 Both h UC-MSCs-CM and NAC can improve the proliferation inhibition induced by radiation,reduceapoptosis.2 The protective mechanisms of h UC-MSCs-CM and NAC on HT-22 cells after radiation exposure include reducing oxidative stress,then reducing DNA double-strand breaks,and upregulating mitochondrial membrane potential.3 h UC-MSCs-CM exerts neuron protection through activating the PI3K-AKT pathway,thereby reducing radiation-induced neuronal cell apoptosis.4 The protective effect of h UC-MSCs-CM on HT-22 cells exposured radiation was better than that of NAC.Figure 13;Table 17;Reference 147... |
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