Effects Of DEHP On The Hypothalamus-Pituitary-Thyroid Axis In Adolescent Rats

Objective:Analysis the effects of DEHP exposure to thyroid hormones in rat,research the relationship between DEHP exposure and expression of gene and protein which regulate the secretion of thyroid hormones by hypothalamic pituitary thyroid axis,inquire into the rat endocrine system toxicity and the mechanism by DEHP,and provide some scientific evidence for the prevention and treatment of human thyroid-related diseases.Methods:80 healthy rats were selected,clean grade,21 days old,50 ~ 60 g body weight.They were randomly divided into 4 groups: control group(corn oil),low dose group(5 mg/kg/d,1/6000 LD50),middle dose group(50 mg/kg/d,1/600 LD50),high dose group(500 mg/kg/d,1/60 LD50).Each group was 20 rats,half males and half females.After 7 days conventional raised,the rats were intragastric administration by DEHP for the first time in the morning,then exposed for 4 weeks.Daily weighing,and recording the amount of food and water.HE staining was used to observe the hypothalamus,pituitary and thyroid organization form.Determination of serum FT3,FT4,T3,T4 and TSH levels by radioimmunoassay.The level of TRH in hypothalamus was measured by ELISA.The levels of FT3,FT4,T3,T4 and TSH were measured by radioimmunoassay.Real–time quantitative determination of hypothalamic-pituitary-thyroid axis-related gene m RNA expression.The localization and expression of hypothalamic-pituitary-thyroid axis-related proteins were observed by immunohistochemistry and Western Blot.Results:1.General toxicity by DEHP exposure in adolescent rats(1)After DEHP exposure,the weight of rats in low,middle and high dose group was significantly higher than control group(P<0.05).The weight of rats in middle and high dose group was significantly higher than low dose group(P<0.05).(2)The food and water intake of rats in treatment group was significantly higher than control group(P<0.05).2.Effects of DEHP on hypothalamic-pituitary-thyroid axis in adolescent rats(1)After DEHP exposure,there was no significant difference in the organs coefficients of pituitary and thyroid between each groups(P>0.05).(2)In each group,the structures of glial cells,neurons and nerve fibers were normal.Neurons were polygonal,large round nucleus,nucleolus centered,big and deep staining,with deep purple patches or granular Nissl bodies in the cytoplasm;glial cell nucleus was large;also visible oligodendrocyte cell bodies,small,round or oval.There was no significant difference between groups.(3)Each group of rats were observed under microscope pituitary eosinophils and basophils,chromophobe cells and capillaries.There was no significant change in each group.(4)The thyroid tissue in each group for a large number of thyroid follicular cavity,jelly stained red.There is a small amount of connective tissue and abundant capillaries.There was no significant difference between the low and middle dose groups of thyroid tissue,but in the high dose group,the thyroid follicular shape was regular but the volume was small,the density of the colloid in the cavity was decreased,and the morphology of the follicular epithelium was not abnormal.3.Effects of DEHP on thyroid hormones in adolescent rats(1)After DEHP exposure,the level of TRH in hypothalamus of rats in high dose group was significantly higher than that in control group and low dose group(P<0.05).(2)The content of TSH in serum of rats increased with the increase of dose,but there was no significant difference between the two groups(P>0.05).(3)The levels of T4,T3,FT4 and FT3 in the serum of rats were decreased with the increase of the dose,and the serum hormone level in the high dose group was significantly lower than the blank control group(P<0.05).4.The m RNA expression of hypothalamus-pituitary-thyroid axis in adolescent rats(1)After DEHP exposure,the expression level of TRH in hypothalamus of rats in high dose group was significantly higher than control group and low dose group(P<0.05).(2)The expression of TRHR in pituitary tissue of rats in high dose group was significantly higher than control group(P<0.05).The expression of TRHR m RNA in the middle dose group was significantly higher than control group(P<0.05).(3)There was no significant difference in the expression of TSH m RNA in different groups of rats(P<0.05).(4)The expression of TSHR in thyroid tissue of high dose group was significantly lower than control group and low dose group(P<0.05)5.The proteins expression of hypothalamic-pituitary-thyroid axis in adolescent rats(1)The level of in the hypothalamus of the rats exposed to DEHP was increased,and the MOD in the high dose group was significantly higher than control group and the middle dose group(P<0.05).Western Blot results showed that the high dose group of rat hypothalamus TRH protein expression was significantly higher than control group and middle dose group(P<0.05),low dose group rat protein expression was significantly higher than control group and middle dose group(P<0.05).(2)In the DEHP group,the level of immunohistochemistry in the rat pituitary tissue was deeper than that in the control group and the low dose group(P<0.05),which was significantly higher in the high dose group than in the MOD group.The results of Western Blot showed that the expression of TRHR in hypothalamus of rats in high dose group was significantly higher than that in control group and middle dose group(P<0.05).(3)The results of Western Blot showed that there was no significant difference in the protein expression of pituitary TSH in each group.(4)In the DEHP exposure group,the immunohistochemical staining of the pituitary tissue was relatively shallow,and the MOD in the high dose group was significantly lower than the control group and the low-dose group(P<0.05).The results of Western Blot showed that the expression of TSHR protein was significantly lower in the high dose group than in the control group and low dose group(P<0.05).The expression of TSHR protein in the middle dose group was significantly lower than control group and low dose group(P<0.05).Conclusions:1.With the dose of DEHP increased,the intakes of food and water were increased,as well as the body weight of adolescent rats.Those indicated that DEHP could cause the growth and development in adolescent rats.2.After DEHP intragastric administration,there was no difference in the organ coefficient of pituitary and thyroid tissue.But DEHP can cause toxicity effect on the thyroid gland in adolescent rats.3.DEHP exposure could disturb the balance of endocrine regulation by hypothalamic-pituitary-thyroid axis in adolescent rats.With increasing DEHP dose,the level of TRH in hypothalamus were elevated.And levels of T4,T3,FT4 and FT3 in serum were decreased.Dose-dependent relationship existed between DEHP and hormones levels.4.DEHP exposure may affect the expression of TRH,TRHR,TSH and TSHR on the hypothalamus-pituitary-thyroid axis of rats in different degrees,and destroy the homeostasis of adolescent rats.5.The regulation mechanism of hypothalamic-pituitary-thyroid axis were interferenced by DEHP endocrine toxicity in adolescent rat.The synthesis and secretion of thyroid related hormones and their receptor were effect by DEHP,and cause thyroid organ toxicity.