Mechanism Investigation Of Nickel Sulfate Induced Autophagy In Thyroid Tissue And Cells

Objective To clarify the effect and significance of autophagy on thyroid metabolism of rats exposed to nickel by detecting the expression levels of thyroid autophagy related indexes.To evaluate the toxic effect of nickel on metabolic injury of rats by the index changes of thyroid function and histomorphology in rats treated with nickel sulfate(NiSO₄).To speculate the dose-effect of thyroid injury caused by NiSO₄by measuring the effects of different concentrations of NiSO₄ on the proliferation of human thyroid follicular epithelial cells(Nthy-ori 3-1)in vitro.The effect of NiSO₄ on thyroid metabolism and the possible mechanism were investigated by detecting autophagy marker protein and related molecules of nuclear factor kappa B(NF-?B)signaling pathway,which would provide theoretical basis for the prevention of thyroid metabolic injury caused by environmental nickel exposure.Methods1.32 SPF healthy male Wistar rats were randomly divided into 4 groups according to weight stratification:normal control group(5 m L/kg saline),low dose group(2.5mg/kg NiSO₄),medium dose group(5.0 mg/kg NiSO₄),and high dose group(10.0mg/kg NiSO₄),8 rats in each group and intraperitoneal injection was given once a day for 40 consecutive days.The expression levels of autophagy related markers LC3B,Beclin1 and p62 in thyroid tissues were detected using immunohistochemistry.The m RNA expression of autophagy related genes ATG5,ATG7,ATG12,LC3B,p62 and Beclin1 were detected by Real-time fluorescence quantitative polymerase chain reaction(RT-PCR),and thyroid function and thyroid tissue morphology changes in rats after different doses of NiSO₄ treating were detected.2.In human Nthy-ori 3-1 cells,effects of different concentrations of NiSO₄ on cell growth were detected by CCK-8 assay,and the effects of NiSO₄ on cell cycle were detected by flow cytometers to determine the appropriate damage concentration of NiSO₄.The NiSO₄ injuried human thyroid cell model was constructed.3.The morphological changes of cells after NiSO₄ intervention were observed by using an inverted phase contrast microscope,and the ultra-microstructure changes of cells were further observed with a transmission electron microscope.Meanwhile,the occurrence of autophagy was identified by fluorescence microscope after MDC staining.Western blot was used to detect the expression levels of autophagy related molecular markers LC3,p62 and Beclin1.4.NiSO₄-induced autophagy were regulated by using the autophagy inhibitor 3-MA and autophagy inducer rapamycin.PI staining was used to detect the effect on NiSO₄-induced Nthy-ori 3-1 cells mortality,and Annexin V-FITC/PI double staining was used to detect their effect on cell apoptosis rate.Western blot was used to detect the expression levels of apoptosis-related proteins Bax,Bcl-2 and Caspase-3,further elucidate the relationship between autophagy and apoptosis induced by NiSO₄.5.To investigate the possible regulatory mechanism of NiSO₄ induced autophagy in Nthy-ori 3-1 cells,NF-?B p65 transcription factor assay kit was used to detect the activity of NF-?B signaling pathway and Western blot was used to detect the pathway related proteins p65,I?B?,p-I?B?,IKK?and p-IKK?.Results1.Comparing with control group,m RNA expression of ATG5 increased significantly in thyroid tissues treated with various concentrations of NiSO₄(P<0.001).However,m RNA expression of p62 decreased significantly as the dose of NiSO₄increased(P<0.001).In addition,the level of m RNA expression in ATG7,ATG12,LC3B and Beclin1 was elevated only in the medium dose and high dose NiSO₄ exposed groups(P<0.05).LC3B protein expression in low dose NiSO₄ treated group showed no significant change when comparing with the control group,while the expression increased in the medium and high dose treated groups.The expression level of Beclin1protein in rats thyroid tissues was consistent with its m RNA expression.p62 protein in each treated group showed lower expression than in the control group and the expression level decreased with the increase of NiSO₄ concentration.2.Comparing with the control group,concentrations of serum FT₄ in each NiSO₄treated rat decreased significantly(P<0.05).However,there was no significant difference between the treated groups(P>0.05).Normal thyroid structure was observed in the control and low dose group rats,while in the medium dose group,the hyperplasia of thyroid follicular epithelium was cubic or low columnar,interstitial hyperemia and hyperplasia of fibrous tissue was observed.In the high dose group,some follicular epithelial hyperplasia was columnar,some papillary hyperplasia,and colloid were sparse in some follicular lumen,epithelial absorption vacuoles of varying sizes appear in the colloid surrounding the follicles,and interstitium was scattered in red blood cells.3.NiSO₄ dose-and time-dependently inhibits the growth of Nthy-ori 3-1 cells,and with the increase of NiSO₄ concentration and the extension of exposure time,the decrease of cell vitality became more obviously.In addition,with the increase of NiSO₄concentrations,the cells become smaller and rounder,and could not be walled and suspended in culture solution.Cell cycle results showed that high dose NiSO₄could arrest the cell cycle of Nthy-ori 3-1 cells in S and G2/M phases.4.Observing using a fluorescence microscope,the NiSO₄treatment group displayed stronger fluorescent signals and more MDC-labeled positive cells than the control group.When the ultrastructure of NiSO₄ intervention of Nthy-ori 3-1 cells were detected using transmission electron microscopy,the NiSO₄ treated groups showed a number of various sizes of autophagy vesicles.When treated with different NiSO₄concentrations,the expression of LC3-II increased with the increase of NiSO₄concentration.When comparing with the control group,the level of Beclin 1 protein expression in NiSO₄ treatment group increased,and the difference in high NiSO₄concentration group was statistically significant(P<0.01),p62 protein expression decreased significantly,and the difference was statistically significant(P<0.05).5.Autophagy and apoptosis in Nthy-ori 3-1 cells were activated after NiSO₄intervention.Inhibition of autophagy by 3-MA in Nthy-ori 3-1 cells significantly increased the NiSO₄-induced mortality,inhibited the NiSO₄-induced growth and increased the apoptosis rate.When using an autophagy inhibitor 3-MA,Caspase-3protein expression induced by NiSO₄ increased further with no significant change in Bax protein,however the ratio of Bcl-2/Bax decreased significantly(P<0.05).Combined with the autophagy inducer rapamycin,Nthy-ori 3-1 cell mortality was reduced with the vitality increased.Rapamycin alleviated the apoptosis rate of Nthy-ori3-1 cells.The expression of NiSO₄-induced Caspase-3 protein and Bax protein were decreased with the ratio of Bcl-2/Bax increased by treatment of rapamycin,and the difference was statistically significant(P<0.05).6.After NiSO₄ intervention,expression of nuclear p65 protein,p-I?B?and p-IKK?protein were increased.The autophagy inhibitor 3-MA significantly reduced the expression of NiSO₄-induced nuclear p65 protein and the expression of phosphorylation of IKK?and I?B?.At the same time,3-MA significantly reduced NiSO₄-induced NF-?B transcription activity with statistical significance(P<0.001).The NF-?B inhibitor QNZ decreased the expression of NiSO₄-induced LC3-II,while the expression of p62 protein was increased.Conclusions1.Intraperitoneal injection of NiSO₄can induce autophagy in thyroid tissues of rats,leading to morphological changes of thyroid tissue and thyroid dysfunction in Wistar rats.2.NiSO₄ significantly inhibited the growth of Nthy-ori 3-1 cells in a dose-and time-dependent manner and induced autophagy in Nthy-ori 3-1 cells.3.Both autophagy and apoptosis were activated during the NiSO₄-induced death of Nthy-ori 3-1 cells,NiSO₄-induced autophagy is an adaptive response and protects Nthy-ori 3-1 cells from apoptosis.4.Activation of NF-?B signaling pathway may participate in autophagy in Nthy-ori 3-1 cells induced by NiSO₄.