Depleted Uranium Induces Thyroid Damage Through Activation Of ER Stress Via The Thrombospondin 1/PERK Pathway

Background and ObjectivesDepleted uranium（DU）is a byproduct of the enrichment process of natural uranium.Its²³⁵U content is less than 0.711%.DU is similar to natural uranium in chemical properties,but is of only 60%radioactivity of natural uranium.It is of strong toughness,high density and good penetration,and is often used in military equipment,such as DU armor-piercing shells and DU protective armor.It is also used in civil activities,such as balance hammer of aircraft and radiation protective clothing.While DU is widely used in daily life,it also brings damage to human health.When DU enters the environment,it contaminates water,soil and air,and then enters our bodies through a variety of routes,such as respiratory tract,digestive tract and damaged skin,and accumulates and causes damage in multiple organs,including the kidneys,bones,and lungs.Studies have shown that DU aerosols entering the lungs caused a severe inflammatory reaction and formation of nodules,in some cases,lung abscess,pulmonary hemorrhage.The DU-induced damage to the human body is most prominent in the kidney.Nearly 90%of ingested DU will be excreted in urine within 24 hours.Absorbed DU will pass through the glomeruli and then to the proximal tubules,causing injury or necrosis of proximal tubule cells,even glomerular injury in case of high intake of DU,eventually leading to renal dysfunction and even renal failure.Studies have found that DU can cause damage to genetic material,which may lead to the occurrence and development of some tumors,and may also cause the occurrence of some genetic diseases.In terms of the damage induced by DU,the current researches are more focused on its effects on the kidney.However,it is still lacking for studies on the thyroid damage caused by DU and its mechanism.The thyroid is an important endocrine organ in human body.It secretes thyroid hormones,regulating various physiological functions.In particular,the thyroid plays an important role in the regulation of metabolic balance and nerve growth and development.For example,a lack of thyroid hormone during growth may lead to paralysis.While the thyroid is an organ that is particularly sensitive to environmental factors.These environmental factors include heavy metal and radioactive substances.Several heavy metals,such as cadmium,mercury,chromium and lead,have been shown to have damaging effects on the thyroid,and exposure to radioactive ¹³¹I may lead to thyroid disease and even thyroid cancer.Therefore,it should be possible for DU to have an damage effect on the thyroid.In fact,there have been several studies that have attempted to explore the relationship between DU and thyroid diseases.Studies have shown that uranium can cause thyroid hormones disruption in zebrafish.Other studies have investigated the relationship between uranium levels in the environment and thyroid disease and found that exposure to uranium may be associated with the development of thyroid disease.However,none of these studies have any in-depth analysis of how DU causes thyroid damage or any exploration of the mechanism.This study preliminarily explored the effects of DU on the thyroid and its mechanism by establishing models of DU-induced thyroid damage,trying exploring possible therapeutic targets for the prevention and treatment of DU-induced body damage.Methods1.The damage model of rat thyroid and Fischer rat thyroid cell line-5（FRTL-5）cells induced by DU was established,and the content of DU in thyroid was detected by inductively coupled plasma mass spectrometer（ICP-MS）,the damage of thyroid or FRTL-5 cells was evaluated by HE staining,electron microscopy,TUNEL staining,chemiluminescent particle immunoassay（CMIA）and CCK-8 assay.2.Gene chip technology was used to screen the sensitive differential genes of DU induced thyroid damage.Immunohistochemistry,immunofluorescence,Western blot and q RT-PCR were used to detect the expression of the sensitive differential gene thromboprotein1（TSP-1）.TSP-1 knockout mice were constructed,si RNA and TSP-1 overexpression plasmid were used to regulate the expression of TSP-1 in FRTL-5 cells,the exogenous TSP-1recombinant protein was used to increase the content of TSP-1 in FRTL-5 cells,establishing models of TSP-1 knockout mice and TSP-1 regulated FRTL-5 cells exposed to DU.HE staining,TUNEL staining,CMIA,CCK-8 assay and flow cytometry were used to observe the effects of DU,evaluating the role of TSP-1 in DU-induced damage of rat thyroid and FRTL-5cells.3.The effects of DU on endoplasmic reticulum（ER）stress were observed by immunohistochemistry,immunofluorescence,Western blot,q RT-PCR,etc.FRTL-5 cells and rat models of DU exposure after ER stress inhibited by 4-PBA were established,and the role of ER stress in DU-induced damage was observed by CCK-8 assay and CMIA.To establish the DU-induced damage model of TSP-1 knockout mice and TSP-1 regulated FRTL-5 cells,evaluating the effect of TSP-1 on ER stress.The expression of PERK in FRTL-5 cells was down-regulated by si RNA,to detecting the effect of DU exposure on the expression of CHOP and Caspase-3,evaluating the role of PERK in DU-induced FRTL-5 cells damage.Results1.DU accumulated in the rat thyroid after exposure,leading to thyroid tissue damage and apoptosis,resulting in the decrease of serum T4 and FT4.DU decreased the activity of FRTL-5 cells,increased the apoptosis of FRTL-5 cells,increased the activity of Caspase-3and ROS levels,and decreased the mitochondrial membrane potential.2.TSP-1 is the sensitive differential gene in DU-induced thyroid damage.DU exposure resulted in decreased expression of TSP-1 in rat thyroid and FRTL-5 cells.Down-regulation of TSP-1 aggravated the thyroid tissue damage,cell apoptosis,and decreased serum FT4 and T4 contents in mice after DU exposure,and aggravated the apoptosis of FRTL-5 cells and the increased activity of Caspase-3 after DU exposure.Up-regulation of TSP-1 alleviated the decreased activity of FRTL-5 cells induced by DU.3.DU increased the expression of PERK,CHOP and p-e IF2αin rat thyroid and FRTL-5cells.Inhibition of ER stress alleviated DU-induced decline in FRTL-5 cell activity and serum FT4 and T4 in rats.Up-regulation of TSP-1 alleviated DU-induced increased expression of PERK and activity of Caspase-3.Knocking out TSP-1 aggravated the increase expression of PERK in mice thyroid after DU exposure.Down-regulation of PERK reduced the increased expression of CHOP and Caspase-3 in FRTL-5 cells after DU exposure.ConclusionDU can accumulate in the thyroid after exposure,resulting in thyroid tissue structure disorder and cell damage,leading to thyroid insufficiency.TSP-1 is the sensitive differential gene of DU-induced thyroid damage.DU decreases the expression of TSP-1 in thyroid and may causes thyroid damage through activation of ER stress via the TSP-1/PERK pathway.TSP-1 may be a new therapeutic target for DU-induced body damage.