| The effect of H2O2on spleen of MT-Ⅰ/Ⅱ KO miceObjectivesIn the early stage of cerebral ischemia-reperfusion, there is a large number of inflammatory cytokines expression in spleen. These inflammatory cytokines lead to apoptosis in spleen after cerebral ischemia-reperfusion. We use hydrogen peroxide (H2O2) induced wild type (WT) mice and Metallothionein-Ⅰ/Ⅱ Knockout (MT-Ⅰ/Ⅱ KO) mice spleen cells and mitochondria, and investigate the N-Acetyl-L-Cysteine (NAC) and Diethyldithiocarlamate (DETC) for its intervention effect. We aim to investigate the effect of exogenous H2O2on the spleen cell viability and the effect of NAC and DETC on H2O2induced oxidative stress in the spleen of MT-Ⅰ/Ⅱ KO mice.Methods1. Preparation of the WT mice and MT-Ⅰ/Ⅱ KO mice spleen cell suspension, were divided into various concentrations of H2O2(0,0.1,0.2,0.5,1,2mM H2O2) group, the cell viability was detected by MTT colorimetric method after2hours;2. The spleen cells with different concentrations of H2O2(0,0.1,0.2,0.5,1,2mM H2O2) were stained by fluorescent probes MitoSOX in WT mice and MT-Ⅰ/Ⅱ KO mice after2hours, observed under light and fluorescence microscope;3. On the cells level, treatment of NAC and DETC on0.5mM H2O2-induced spleen cells, the cell viability was detected by MTT colorimetric method after2hours;4. NAC, DETC treated the spleen cell of WT mice and MT-Ⅰ/Ⅱ KO mice with0.5mM H2O2, lactate dehydrogenate (LDH) activity in culture supernatant were assayed by microplate reader;5. The spleen cells treated with NAC and DETC were stained by fluorescent probes MitoSOX in MT-Ⅰ/Ⅱ KO mice after2hours, observed under light and fluorescence microscope;6. Mitochondria were extracted from WT mice and MT-Ⅰ/Ⅱ KO mice spleen, on the mitochondria level, the effect of NAC and DETC on0.5mM H2O2induced oxidative stress in the mitochondria of spleen was evaluated by mitochondrial swelling.Results1. MTT detection results from treatment of various concentrations of H2O2on the spleen cells of WT mice and MT-Ⅰ/Ⅱ KO mice showed that both in WT mice or MT-Ⅰ/Ⅱ KO mice, compared with control group, with the increase of the concentration of H2O2, spleen cell viability was significantly decreased (p<0.05), but compare with WT mice, MT-Ⅰ/Ⅱ KO mice spleen cell viability decreased more pronounced extent in the same treatment (p<0.05);2. Observed under light microscope and fluorescence microscope showed that compared with the control group, with the increase of the concentration of H2O2, mean fluorescence intensity (MFI) of MitoSOX increased gradually; but compare with WT mice, MT-Ⅰ/Ⅱ KO mice spleen cell MitoSOX MFI increased significantly in the same treatment (p<0.05);3. Results from treatment of NAC on0.5mM H2O2induced spleen cells showed that compared with control group, spleen cell viability decreased, LDH viability increased and MitoSOX MFI enhanced in0.5mM H2O2group; compared with0.5mM H2O2group, NAC+0.5mM H2O2group spleen cell viability increased (p<0.05) and the LDH release reduced (p<0.05), and MitoSOX MFI decreased; compared with WT mice, MT-Ⅰ/Ⅱ KO mice spleen cell viability decreased (p<0.05), the LDH release increased (p<0.05) and MitoSOX MFI increased in0.5mM H2O2group and NAC+0.5mM H2O2group;4. Results from treatment of NAC on0.5mM H2O2induced spleen cells mitochondrial showed that compared with control group, mitochondrial absorbance values decreased in0.5mM H2O2group (p<0.05); compared with0.5mM H2O2group, mitochondrial absorbance values increased in NAC+0.5mM H2O2group; compared with WT mice, MT-Ⅰ/Ⅱ KO mice mitochondrial absorbance values no statistical difference in0.5mM H2O2group and NAC+0.5mM H2O2group (p>0.05);5. Results from treatment of DETC on0.5mM H2O2induced spleen cells showed that compared with control group, spleen cell viability decreased (p<0.05) and LDH activity increased (p<0.05), MitoSOX MFI enhanced in0.5mM H2O2group and DETC group; compared with0.5mM H2O2group, spleen cell viability decreased (p<0.05) and LDH activity increased (p<0.05), MitoSOX MFI enhanced in DETC+0.5mM H2O2group; compared with WT mice, spleen cell viability decreased (p<0.05) and LDH activity increased (p<0.05), MitoSOX MFI enhanced in0.5mM H2O2group and DETC+0.5mM H2O2group and DETC group of MT-Ⅰ/ⅡKO mice (p<0.05);6. Results from treatment of DETC on0.5mM H2O2induced spleen cells mitochondrial showed that compared with control group, mitochondrial absorbance values was significantly decreased in0.5mM H2O2group (p<0.05), and no statistical difference with DETC group (p>0.05); compared with0.5mM H2O2group, no statistical difference with DETC+0.5mM H2O2group in mitochondrial absorbance values (p>0.05); compared with WT mice, MT-Ⅰ/Ⅱ KO mice mitochondrial absorbance values no statistical difference in0.5mM H2O2group, DETC+0.5mM H2O2group and DETC group (p>0.05).Gonelusion1. MT-Ⅰ/Ⅱ may have protective effect.2. NAC can improve the H2O2induced oxidative damage in mice spleen cells.3. DETC increased H2O2induced oxidative damage in mice spleen cells. The effect of excessive iodide on spleen of MT-Ⅰ/ⅡKO miceObjectivesIn this study, we aimed to observe the effect of excessive iodide intake on peripheral immune organs spleen in MT-Ⅰ/Ⅱ KO mice, and explore the relationship between the immune system and MT-Ⅰ/Ⅱ.MethodsRandomly selected the mature health MT-Ⅰ/Ⅱ KO mice and WT mice were divided into two groups:the control group and100KI group respectively, each group of10,14days after feeding used in the experiment. Weighing the body weight of mice, then all the mice were sacrificed before dissection of spleen. Weighing the weight of the spleen, observe the spleen size and calculate the organ-to-body ratio; Frozen sections of the spleen for HE staining to observe the changes in the spleen structure.ResultsCompared with the control group, spleen relative weight and size are decreased (p<0.05), may appear the humoral immune response in spleen of100KI group; Compared with WT mice, spleen relative weight and size is decreased significantly (p<0.05), and also may appeared the humoral immune response with visible void damage in the spleen in MT-Ⅰ/Ⅱ KO mice.ConclusionExcessive dietary iodide reduced spleen size, and may appear the humoral immune response. Spleen size decrease is more significantly in MT-Ⅰ/Ⅱ KO mice, and also may appear the humoral immune response but visible void damage in the spleen of MT-Ⅰ/Ⅱ KO mice, these showed that MT-Ⅰ/Ⅱ can protect the spleen from damage caused by excessive iodide intake. |
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