| Di（2-ethylhexyl）phthalate（DEHP）is currently the most consumed plasticizer,which is used in plastic products such as children’s toys,food packaging and medical equipment.DEHP is non-covalently bound to the plastic body,so it’s easy to leave the body and enter the environment,and then enter the body through diet,respiration.It is decomposed into mono-（2-ethylhexyl）phthalate phthalate（MEHP）within a few hours.Studies have confirmed that DEHP is an environmental endocrine disruptor,which can produce reproductive toxicity,developmental toxicity,liver toxicity and other toxicicity.In recent years,studies have shown that DEHP can be toxic to the thyroid and affect the normal function of the thyroid,but the mechanisam is still unclear.Estrogen receptors（ERs）play an important role in the occurrence and development of thyroid diseases.As an environmental estrogen,DEHP can affect the expression levels of ERs and can activate endoplasmic reticulum stress.It is speculated that ERs activating endoplasmic reticulum stress may play a role in the thyroid toxicity induced by DEHP.In this study,human thyroid follicular epithelial cells（Nthy-ori 3-1 cells）were exposed to MEHP to clarify the thyroid toxicity,the expression of ERs,and the occurrence of endoplasmic reticulum stress.induced by MEHP on Nthy-ori 3-1 cells;then inhibiting endoplasmic reticulum stress,detecting the changes in cytotoxicity,to clarify the role of endoplasmic reticulum stress in thyroid toxicity induced by MEHP;then inhibiting the expression of ERs gene,detecting the changes of endoplasmic reticulum stress and toxicity induced by MEHP,to clarify the regulatory effect of ERs on endoplasmic reticulum stress and its mechanism of thyroid toxicity induced by MEHP,and to provide a scientific basis for the prevention and treatment of DEHP-induced thyroid diseases.PartⅠToxicity and ERs expression levels of Nthy-ori 3-1 cells induced by MEHP Objective:To clarify the damage and the effect on the secretion of thyroid hormones of MEHP on Nthy-ori 3-1 cells,and to explore the effect of MEHP on the expression of ERs.Methods:Nthy-ori 3-1 cells were cultured in RPMI 1640 medium containing 10%FBS,and exposed to different concentrations of MEHP for 24 h.The cell viability was detected by CCK-8 method,and the dose of MEHP was determined.The experimental groups were divided into control group（Con）,solvent control group（VCon）,and 5μM,25μM and 125μM MEHP exposure groups.PI staining was used to detect the cell cycle,Annexin V-FITC/PI staining was used to detect the level of apoptosis,DCFH-DA probe staining was used to detect the level of reactive oxygen species（ROS）,and JC-1 probe staining was used to detect the level of mitochondrial membrane potential（MMP）.2m IU/L thyrotropin was added to the medium,and free triiodothyronine（FT3）and free thyroxine（FT4）levels were detected by ELISA,and the mRNA and protein expression levels of thyroid hormone synthesis-related genes and ERs were detected by Real-Time PCR and Western Blot.The results of each test index were expressed as mean±standard deviation（（?）±s）,and IBM SPSS 24.0 software was used for statistical analysis.One-way analysis of variance was used to compare the differences of test index results between groups,and LSD test was used for pairwise comparison between groups,the test level isα=0.05.Results:1.With the increase of MEHP exposure level,the shape of cells gradually changed from slender to round,and the lumen-like structure between cells decreased.2.The level of apoptosis and ROS level were significantly increased with the increase of MEHP dose（P<0.05）,and the level of MMP was significantly decreased with the increase of MEHP dose（P<0.05）.3.The levels of FT3 and FT4 in the supernatant of the cell medium of MEHP exposure group were significantly lower than those of the Con group and VCon group（P<0.05）.The levels of FT3 and FT4 in 125μM MEHP exposure group were significantly lower than those in the 5μM MEHP exposure group（P<0.05）.4.The mRNA expression levels of TG,TPO,TSHR,NIS,PAX8 and TTF1 genes in each MEHP exposure groups were significantly lower than those in the Con group and VCon group（P<0.05）.The mRNA expression levels of TPO,TSHR,NIS and PAX8genes in 125μM MEHP exposure group was significantly lower than that in 5μM MEHP exposure group（P<0.05）.The protein expression levels of TG,TPO,NIS,PAX8 and TTF1 in MEHP exposure groups were significantly lower than those in the Con group and VCon group（P<0.05）,and the protein expression levels of TG and TSHR in the125μM MEHP exposure group were significantly lower than those in the 5μM MEHP exposure group（P<0.05）.5.The mRNA and protein expression levels of ERαand GPR30 in each MEHP exposure group were significantly higher than those in the Con and VCon groups（P<0.05）,and the expression level in the 125μM MEHP exposure group was significantly higher than that in other groups（P<0.05）.Conclusions:1.MEHP exposure can increase the level of apoptosis and ROS in Nthy-ori 3-1cells,and decrease the level of MMP,resulting in damage to cells.2.MEHP exposure affects the expression of thyroid hormone synthesis-related proteins and transcription factors,reduces the levels of FT3 and FT4 and influence the normal function of cells.3.MEHP exposure can significantly up-regulate the expression levels of estrogen receptors ERαand GPR30 in Nthy-ori 3-1 cells.Part Ⅱ Effects of MEHP on endoplasmic reticulum stress of Nthy-ori 3-1 cells and its role in cytotoxicityObjective:To clarify the effect of MEHP exposure on the activation of endoplasmic reticulum stress in cells;and explore the effect of endoplasmic reticulum stress on Nthy-ori 3-1cell damage and abnormal cell function induced by MEHP.Methods:The experimental group of the effect of MEHP on endoplasmic reticulum stress is the same as the partⅠ.Fluo-4 AM fluorescent probe was used to detect the level of cellular Ca2+,and Real-Time PCR and Western Blot were used to detect the mRNA and protein expression levels of endoplasmic reticulum stress-related signaling pathways.4-PBA was used to inhibit the activation of endoplasmic reticulum stress,the dose of4-PBA was determined by CCK-8 method,and the action time of 4-PBA was determined according to preliminary experiments.Western Blot was used to detect the expression levels of endoplasmic reticulum stress marker proteins GRP78,p-PERK and p-IRE1αto determine the inhibition efficiency of 4-PBA,and the experimental groups were divided into control group（Con）,solvent control group（VCon）,ER stress inhibition group（4-PBA）,125μM MEHP exposure group（MEHP）,and MEHP exposure+ER stress inhibition group（MEHP+4-PBA）.The detection methods of apoptosis,ROS level,MMP level,FT3 and FT4 levels in the culture supernatant,and thyroid hormone synthesis-related protein expression levels were the same as those in the partⅠ.Results:1.With the increase of MEHP exposure dose,the level of cellular Ca2+increased.2.The expression level of endoplasmic reticulum chaperone protein GRP78 in25μM MEHP exposure group and 125μM MEHP exposure group was significantly higher than that in Con group,VCon group and 5μM MEHP exposure group（P<0.05）.3.MEHP exposure can activate the cellular PERK-e IF2α-ATF4-CHOP signaling pathway.The protein expression levels of PERK,p-PERK and p-e IF2αin MEHP exposure group were significantly higher than those in Con group and VCon group（P<0.05）,and the protein expression levels of PERK,p-PERK,p-e IF2α,ATF4 and CHOP in 125μM MEHP exposure group were the highest.4.MEHP exposure can activate cellular IRE1αsignaling pathway and ATF6signaling pathway.After exposure to MEHP,the protein expression levels of IRE1α,p-IRE1α,p-JNK,ATF6 p50 and mRNA expression level of XBP1s/XBP1u in 125μM MEHP exposure group were significantly higher than that in Con group and VCon group（P<0.05）.5.The cells were pretreated with 2m M 4-PBA for 6 hours and then acted on the cells together with MEHP for 24 hours.The expression levels of GRP78,p-PERK and p-IRE1αin the MEHP+4-PBA group were significantly lower than those in the MEHP exposure group（P<0.05）.6.After the endoplasmic reticulum stress was inhibited,the levels of apoptosis and ROS in the 4-PBA group were significantly lower than those in the VCon group（P<0.05）,the MMP level of cells in the 4-PBA group was significantly higher than that in the Con group and VCon group（P<0.05）.And the levels of apoptosis and ROS in the MEHP+4-PBA group were significantly lower than those in MEHP exposure group（P<0.05）.and the MMP level in the MEHP+4-PBA group was significantly higher than that in the MEHP exposure group（P<0.05）.7.The levels of FT3 and FT4 in the supernatant of cell culture medium in MEHP+4-PBA group were significantly higher than those in MEHP exposure group（P<0.05）.The expression levels of thyroid hormone synthesis-related proteins TG,TPO,NIS and PAX8 in MEHP+4-PBA group were significantly higher than those in MEHP exposure group（P<0.05）.Conclusions:1.MEHP exposure can increase the level of intracellular Ca2+,lead to the imbalance of Ca2+homeostasis,cause endoplasmic reticulum stress,increase the expression of endoplasmic reticulum chaperone GRP78,and activate the unfolded protein response signaling pathway PERK,IRE1α,ATF6 pathway.2.MEHP can damage cells by reducing the level of MMP,increasing the level of cellular ROS and apoptosis in Nthy-ori 3-1 cells after activating endoplasmic reticulum stress.3.After MEHP activates endoplasmic reticulum stress,it can reduce the expression levels of thyroid hormone synthesis-related proteins TG,TPO,NIS and PAX8,resulting in the reduction of FT3 and FT4 levels,which affects the normal function of Nthy-ori3-1 cells.Part Ⅲ The role of ERs-activated endoplasmic reticulum stress in Nthy-ori 3-1 toxicity induced by MEHPObjective:To explore the role of ERαand GPR30 in MEHP-activated endoplasmic reticulum stress,and to clarify the role of ERαand GPR30 in totoxicity.Methods:siRNA was used to inhibit the expression of ERαand GPR30 in cells,Real-Time PCR and Western Blot were used to detect the inhibition efficiency of siRNA,and the experimental groups were determined as Control group（Con）,negative control group（si NC）,gene inhibition group（si ERαor si GPR30）,125μM MEHP exposure group（MEHP）,and MEHP exposure+gene inhibition group（MEHP+si ERαor MEHP+si GPR30）.The detection methods of endoplasmic reticulum stress-related indicators and cytotoxicity-related indicators are the same as Part I and PartⅡ.Results:1.The mRNA and protein expression levels of ERαgene in cells in si ERαgroup were significantly decreased（P<0.05）,the mRNA inhibition efficiency was 83%;and the mRNA and protein expression levels of GPR30 gene in cells in si GPR30 group were significantly decreased（P<0.05）,the mRNA inhibition efficiency was 65%.2.The level of Ca2+and GRP78 protein expression in MEHP+si ERαgroup and in MEHP+si GPR30 group were significantly lower than those in MEHP exposure group（P<0.05）.The protein expression levels of p-PERK,p-e IF2α,ATF4,CHOP and ATF6p50 in MEHP+si ERαgroup were significantly lower than those in MEHP exposure group（P<0.05）.The expression levels of IRE1αand p-IRE1αin MEHP+si ERαgroup had no significant change compared with MEHP exposure group（P>0.05）.The protein expression levels of p-PERK,p-e IF2α,IRE1α,p-IRE1αand p-JNK in MEHP+si GPR30group were significantly lower than those in MEHP exposure group（P<0.05）.The expression level of ATF6 p50 protein in MEHP+si GPR30 group had no significant change compared with MEHP exposure group（P>0.05）.3.The apoptosis level of MEHP+si ERαgroup was significantly lower than that of MEHP exposure group（P<0.05）.The ROS level of cells in the MEHP+si GPR30 group was significantly lower than that in the MEHP exposure group（P<0.05）;and the MMP level in the MEHP+si GPR30 group was significantly higher than that in the MEHP exposure group（P<0.05）.4.The level of FT3 in the supernatant of cell culture medium in MEHP+si ERαgroup and in MEHP+si GPR30 group were significantly higher than those in MEHP exposure group（P<0.05）.The protein expression levels of TG,TPO,TSHR,NIS,PAX8and TTF1 in MEHP+si ERαgroup were significantly higher than those in MEHP exposure group（P<0.05）.The protein expression levels of TG,NIS and PAX8 in MEHP+si GPR30 group were significantly higher than those in MEHP exposure group（P<0.05）.Conclusions:1.MEHP can activate endoplasmic reticulum stress by up-regulating ERαexpression,and can activate the unfolded protein response mediated by PERK signaling pathway and ATF6 signaling pathway.2.MEHP can activate endoplasmic reticulum stress by up-regulating the expression of GPR30,and can activate the unfolded protein response mediated by PERK signaling pathway and IRE1αsignaling pathway.3.MEHP can induce apoptosis by up-regulating the expression of ERα,and can cause cell dysfunction by down-regulating the expression of thyroid hormone synthesis-related proteins TG,TPO,TSHR,NIS,PAX8 and TTF1 by ERα.4.MEHP can affect cellular ROS level and MMP level by up-regulating the expression of GPR30 and cause cell damage,and down-regulate the expression of thyroid hormone synthesis-related proteins TG,NIS and PAX8 by GPR30,causing cellular FT3 secretion disorder.5.ERs play an important role in Nthy-ori 3-1 cytotoxicity induced by MEHP by activating endoplasmic reticulum stress. |
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