Mechanism Of Apoptosis Of Thyroid And Pancreatic Tissues In Rats And Cells Induced By Environmental Endocrine Disruptor-Nickel Sulfate

Background Nickel exposure through the water,air and soil pollution has become a serious threat to the ecological environment and human health,because of its interference effect on the body’s endocrine metabolic state,it had been considered as one of the environmental endocrine disrupting chemicals（EEDs）.Endocrine glands,especially the thyroid and pancreas,are the core organs of energy metabolism regulation,they balance the metabolism of carbohydrate,fat and protein through strict feedback mechanism.The interference of Ni on the regulation of metabolism is closely related to the apoptosis and proliferation of pancreas and thyroid.Objective In this study,a series of cell models with different doses of nickel sulfate（NiSO₄ ）were designed to investigate the dose-and time-response relationship of Ni induced injury of human thyroid follicular epithelial cells（Nthy-ori 3-1,Nthy）and pancreatic ductal epithelial cells（h TERT-HPNE,HPNE）,Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis rate and mitochondrial membrane potential,to explore the relationship between Ni induced apoptosis of human Nthy cells and human HPNE cells and mitochondrial apoptosis pathway.Then,the animal models of rats with different doses of NiSO₄ were established.The toxic effect of Ni on metabolism injury by detecting the changes of function and histomorphology of thyroid and pancreas tissues in rats exposed to different doses of NiSO₄ ,and to simultaneously detect the expression of apoptosis related indexes in mRNA and protein levels of thyroid and pancreas tissues in rats exposed to Ni in order to elucidate the development of metabolism injury induced by Ni exposure.The possible mechanism can provide scientific basis for finding effective prevention and intervention of Ni metabolic damage.Methods In this study,human Nthy cell line and HPNE cell line were used to detect the effect of different doses of NiSO₄ on the activity of human Nthy and HPNE cell line by CCK-8 method.The appropriate injury concentration and injury time were selected to construct the cell Ni injury model.Hoechst 33258 fluorescent staining and Annexin V-FITC/PI double staining flow cytometry were used to observe the effect of Ni on the proliferation and apoptosis of human Nthy cells and HPNE cells.Flow cytometry and laser confocal microscopy were used to detect the changes of mitochondrial membrane potential before and after the intervention,and to observe the relationship between Ni damage to human Nthy and HPNE cells and mitochondrial apoptosis.Thirty two healthy adult male Wistar rats were randomly divided into four groups:control group（N）、low dose group（L）、medium dose group（M）and high dose group（H）.Eight rats in each group were intraperitoneally injected with equal volume（5 ml/kg）once a day for 40 days.The control group was intraperitoneally injected with 5 ml/kg normal saline.The dose of NiSO₄ was calculated as 2.5,5.0 and 10.0 mg/kg respectively.The general condition,body weight,thyroid function,islet function,thyroid and pancreatic histomorphology of rats were observed.The expression of apoptosis related genes（Caspase-8、Caspase-9、Caspase-3、Bax、Bcl-2、Fas）mRNA and protein was detected by RT-PCR and immunohistochemistry.Results1.NiSO₄ has obvious time effect and concentration effect on the activity of human Nthy cells and HPNE cells.With the increase of NiSO₄ exposure dose or the prolongation of exposure time,the cell viability decreased significantly,the cell morphology became round,the number of adherent cells decreased,cell apoptosis and death occurred.2.Different concentrations of NiSO₄ were used to treat the human Nthy cells and human HPNE cells for 24h and 48h,24h-640μM、48h-480μM and 48h-640μM could increase the apoptosis of human Nthy cells,48h-640μM could increase the apoptosis of HPNE cells,and all the concentration groups could reduce the mitochondrial membrane potential level,and affect the energy metabolism of human Nthy cells and HPNE cells.3.After 40 days of continuous intraperitoneal injection of NiSO₄ ,the pathological changes of thyroid and pancreas in high dose group were obvious,and NiSO₄ causes thyroid and islet dysfunction in rats.Compared with the control group,the levels of free T₄ （FT₄ ）and thyroid stimulating hormone（TSH）in each group were significantly decreased（P<0.05）,but there was no significant difference in free T₃（FT₃）among the groups（P>0.05）.In the aspect of islet function,after middle and high doses of NiSO₄ ,the random blood glucose increased significantly（P<0.05）,while the serum insulin and C-peptide levels decreased significantly（P<0.05）.4.The expression of Caspase-3 mRNA in thyroid tissue of rats exposed to NiSO₄ was significantly increased than that of control group（P<0.05）,the mRNA expression levels of Caspase-8 and caspase-9 in high-dose NiSO₄ group were significantly higher than those in other groups（P<0.01）.The expression of Bcl-2mRNA in high-dose NiSO₄ group was significantly lower than that in other groups（P<0.05）.There was no significant difference in Bax gene expression between the control group and NiSO₄ exposed groups（P>0.05）.With the increase of NiSO₄ dose,the ratio of Bcl-2/Bax mRNA decreased significantly（P<0.05）.Compared with the control group,the expression of Fas mRNA in high-dose NiSO₄ group was significantly higher（P<0.01）,it was significantly higher than that in other dose groups（P<0.01）.The expression levels of Caspase-3、Fas and Bax protein were consistent with mRNA expression.The expression of Bcl-2 protein in NiSO₄ exposed groups was significantly lower than that in the control group（P<0.05）,but there was no significant difference between NiSO₄ groups（P>0.05）.5.The expression levels of caspase-3 mRNA and protein in pancreatic tissue of middle and high-dose NiSO₄ groups were significantly higher than those of control group（P<0.01）,especially in the high-dose group.The mRNA expression levels of Caspase-8 and caspase-9 in high-dose NiSO₄ group were significantly higher than those in other groups（P<0.01）.Compared with the control group,the mRNA expression level of Fas in NiSO₄ exposed groups was significantly increased（P<0.01）,while the mRNA expression of Bcl-2 in middle and high-dose NiSO₄ groups was significantly lower than that in other dose groups（P<0.01）.The expression of Fas protein in the middle and high-dose NiSO₄ groups were significantly higher than that in the control group（P<0.01）,there was no significant difference in the expression of Bcl-2 among the groups（P>0.05）.There was no significant difference in the expression of Bax gene and protein among the groups（P>0.05）.The mRNA ratio of Bcl-2/Bax decreased with the increase of exposure dose（P<0.01）,the protein ratio of Bcl-2/Bax was significantly lower in the high-dose group than in the control group（P<0.05）.Conclusion1.NiSO₄ can induce apoptosis of human Nthy cells and HPNE cells by reducing the mitochondrial membrane potential level,and the apoptotic effect is dose-dependent,thus affecting the energy metabolism of human Nthy cells and HPNE cells.2.NiSO₄ as an endocrine disruptor,can induce thyroid and pancreatic injury through apoptosis,resulting in thyroid and islet dysfunction.3.NiSO₄ may induce apoptosis of thyroid and pancreatic tissues through Caspase-dependent mitochondrial apoptosis pathway and Fas signaling pathway.