The Study Of High Concentrations Of Iodide Oxidative In The Thyroid Of Mt-â… /â…¡ KO Mice

ObjectivesTo investigate the effect of high concentrations of iodide on mitochondrial oxidative stress-antioxidant defense in the thyroid of metallothionein Ⅰ/Ⅱ knockout (MT Ⅰ/Ⅱ KO) mice. Study the mechanism of oxidative stress-antioxidant defense in the thyroid with or without MT Ⅰ/Ⅱ. We use the competitive inhibitor of iodine transport KC1O4, thyrotropin, TPO inhibitor PTU, to study the effect of high concentrations of iodide on mitochondrial oxidative stress-antioxidant defense in the thyroid of MT Ⅰ/Ⅱ KO mice.MethodsCellular level1.Thyroid cell suspension of MT-Ⅰ/Ⅱ KO mice and WT mice were prepared, the cells were exposed to high concentrations (100μM,1mM,10mM) of KI, or1mM H2O2for2hours respectively.①ell viability was evaluated with the MTT (methyl thiazolyl tetrazolium, MTT) assay.②A mitochondrial superoxide indicator (MitoSOX Red) was used to measure intramitochondrial superoxide production by flow cytometry.③Lactate dehydrogenase (LDH) leakage rate was measured using a cytotoxicity detection kit.④Western blot was used to detect the protein level of Prx3.2.After the exposure of100μM KI with or without30μM KC1O4for2hours respectively, we detect the intramitochondrial superoxide production, cell viability, LDH leakage rate of thyroid cells of MT-Ⅰ/Ⅱ KO mice and WT mice.3.After the exposure of100μM KI with or without300μM PTU for2hours respectively, we detect the intramitochondrial superoxide production, cell viability, LDH leakage rate of thyroid cells of MT-Ⅰ/Ⅱ KO mice and WT mice.4.After the exposure of100μM KI with or without10U/L TSH for2hours respectively, we detect Cell viability, LDH activity of thyroid cells of MT-Ⅰ/Ⅱ KO mice and WT mice.Animal levelUse MT-Ⅰ/Ⅱ KO mice and WT mice to set up short-term iodine excess modle, they were divided randomly to three groups,①normal iodine intake group (NT),②10times high iodine intake group (10HI),③100times high iodine intake group (100HI), they all have normal iodine chow for14days.1.Use ammonium persulfate digestion arsenic cerium catalytic spectrophotometry to measure urinary iodine content.2.Calculate thyroid organ/body weight ratio.3.Use HE staining to observe thyroid morphology.4.Measure thyroid hormone levels.5.Cell viability was evaluated with the MTT assay.6.LDH leakage rate was measured using a cytotoxicity detection kit.7.MitoSOX Red was used to measure intramitochondrial superoxide production by flow cytometry.8.Western blot was used to detect the protein level.ResultCellular level1.In vitro study, following100μM,1mM,10mM of KI, or1mM H2O2exposure, the mitochondrial superoxide production exhibited a significantly increased MitoSOX Red fluorescence intensity (P<0.01), with increased LDH leakage rate (P<0.01) and decreased cell viability (P<0.01) in both MT-Ⅰ/Ⅱ KO and WT group, more significant increase of mitochondrial superoxide production can be detected in MT-Ⅰ/Ⅱ KO group compared to WT group (P<0.01). With the increase of KI concentration, the Prx3level of thyroid incresed (P<0.01), and there was difference between WT and MT-Ⅰ/Ⅱ KO mice (P<0.05).2.PTU, KClO4and TSH supressed the increase of mitochondrial superoxide production,the increase of LDH activity and decrease of cell viability induced by the iodine excess.①100μM KI decreased cell viability of thyroid in both MT-Ⅰ/Ⅱ KO and WT mice.But after disposing with300μM PTU,30μM KClO4or10U/L TSH seperately, cell viability increased compared with the100μM KI group (P<0.01). Compared with WT mice, the cell viability of MT-Ⅰ/Ⅱ KO mice decreasd more significantly (P<0.01).②300μM PTU,30μM KClO4and10U/L TSH depressed the decrease of mitochondrial superoxide production induced by100μM KI in both MT-Ⅰ/Ⅱ KO and WT mice (P<0.01). Compared with WT mice, the decrease of mitochondrial superoxide production of MT-Ⅰ/Ⅱ KO mice was increased more significantly (P<0.05).③100μM KI increased LDH leakage rate of thyroid in both MT-Ⅰ/Ⅱ KO and WT mice.But after disposing with300μM PTU,30μM KC1O4or10U/L TSH seperately, LDH leakage rate decreased compared with the100μM KI group (P<0.01). Compared with WT mice, the LDH leakage rate of MT-Ⅰ/Ⅱ KO mice increasd more significantly (P<0.01).Animal level1.After MT-Ⅰ/Ⅱ KO mice and WT mice had normal iodine chow for14days, urinary iodine content of the three groups double increased accompany with the incresed iodine intake (p<0.05). Compared with WT mice, the urinary iodine content of MT-Ⅰ/Ⅱ KO mice increasd more significantly (P<0.05).2After MT-Ⅰ/Ⅱ KO mice and WT mice had normal iodine chow for14days, thyroid organ/body weight ratio had difference between NI group and100HI group (p<0.05), and no difference between NI group and10HI group.3.After MT-Ⅰ/Ⅱ KO mice and WT mice had normal iodine chow for14days, compared with the NI group, serum thyroid hormone level of T3、T4、FT3、FT4had no difference in10HI group and100HI group (P>0.05). Compared with the WT mice, serum level of FT4of100HI group increased in MT-Ⅰ/Ⅱ KO mice.4.Under the microscopic observation, in MT-Ⅰ/Ⅱ KO mice and WT mice, there were medium large follicles in NI group and10HI group, compared with the NI group, part of the follicles were significantly larger, part were next to normal, having a slight hyperplasia, while part of them had smaller follicles and follicular cavity, those changes manifested more obvious in WT mice.5.Compared with the NI group, cell viability of100HI group decreased in both MT-Ⅰ/Ⅱ KO and WT group (P<0.01), more significant decrease of cell viability can be detected in MT-Ⅰ/Ⅱ KO group compared to WT group (P<0.01). And there was no difference between NI group and10HI group.6.Compared with the NI group, cell damage of100HI group increased in both MT-Ⅰ/Ⅱ KO and WT group (P<0.01), more significant increase of cell damage can be detected in MT-Ⅰ/Ⅱ KO group compared to WT group (P<0.01). And there was no difference between NI group and10HI group.7.Compared with the NI group, mitochondrial superoxide production of100HI group increased in both MT-Ⅰ/Ⅱ KO and WT group (P<0.01), more significant increase of mitochondrial superoxide production can be detected in MT-Ⅰ/Ⅱ KO group compared to WT group (P<0.05). And there was no difference between NI group and10HI group.8.Compared with the NI group, Prx3expression level of100HI group increased in both MT-Ⅰ/Ⅱ KO and WT group (P<0.05), more significant increase of Prx3expression level can be detected in MT-Ⅰ/Ⅱ KO group compared to WT group (P<0.05). And there was no difference between NI group and10HI group.Conclusion1.MT Ⅰ/Ⅱ showed its antioxidative effect in short-term iodine excess induced oxidative stress in the thyroid.2.Iodine excess may increase the mitochondrial superoxide production in thyroid of both MT-Ⅰ/Ⅱ KO and WT mice.3.300μM PTU may relieve the oxidative stress induced by iodine excess in both MT-Ⅰ/Ⅱ KO and WT mice.4.30μM KClO4may relieve the oxidative stress induced by iodine excess in both MT-Ⅰ/Ⅱ KO and WT mice.5.10U/L TSH may relieve the oxidative stress induced by iodine excess in both MT-Ⅰ/Ⅱ KO and WT mice.6.Iodine excess may induce hypothyroidism, and change as the change of iodine intake.