Endoplasmic Reticulum Stress-inhibited Thyroglobulin Turnover Contribute To Apoptosis Induced By Microcystin-LR In Female

Background:In recent years,the outbreak of harmful algae blooms and the associated cyanotoxins have drawn global attention.After the occurrence of cyanobacteria bloom,numerous cyanobacteria toxins caused by the mortality of cyanobacteria have been detected in the water,and the most common and harmful cyanotoxin was microcystins（MCs）.There are more than 200 MCs isomers,microcystins-LR（MCLR）is the most toxic.A large number of studies have found that MCLR could damage the endocrine system and parental exposure to MCLR could cause serious growth retardation and thyroid endocrine disruption in F1 offspring,but its mechanism is not clear.Objective:In this experiment,we explored the effect of MCLR on the thyroid function of maternal and offspring zebrafish in vivo and the role of unfolded protein response（UPR）in MCLR-induced thyroid toxicity in vitro models.This study emphasized the potential impact of thyroid endocrine disruption on aquatic organism and human beings.Methods:Part 1 In vivo1.The embryos at 10 hpf were randomly divided into three groups.Three months later,the females were selected and mated with normal males to achieve the F1 generation.2.The hatching rate of F1 embryos and the survival rate of F1 zebrafish at 7 dpf were calculated,and the behavioral data of F1 zebrafish were measured.3.RT-PCR and Western blot were used to detect the expressions of genes and proteins related to the HPT axis in F0 and F1 zebrafish.4.The changes of hormones in F0 and F1 zebrafish were detected by ELISA.Part 2 In vitro5.ROS detection kit was uesd to detect the changes of ROS in FRTL-5 cells.6.The TUNEL assay was performed to calculate the rate of apoptosis.7.RT-PCR and Western blot were used to detect the expressions of genes and proteins related to the HPT axis in FRTL-5 cells.8.The co-localization of GRP78 and TG was detected by immunofluorescence and the co-localization rate was calculated.9.RT-PCR,Westernblot and immunofluorescence assay were used to verify whether endoplasmic reticulum stress was involved in thyroid toxicity induced by MCLR.10.The role of PERK and IRE1 pathway in thyroid toxicity induced by MCLR were verified by RT-PCR,Westernblot and immunofluorescence assay.Results Part 1 Maternal exposure to MCLR clearly perturbed THs in both F0 and F1 generations and affected the growth of offspring.（1）MCLR can perturb THs levels in F0 and F1 zebrafish:Compared to the control group,the levels of THs were elevated in the zebrafish testes treated with MCLR.（2）MCLR can affect the expressions of genes related to the HPT axis in F0 and F1 zebrafish: The results showed MCLR treatment affected the expressions of HPT genes in F0 and F1 zebrafish.（3）MCLR can induce serious growth retardation in the offspring: the hatching percentage at 3 dpf and the heart rates at 7 dpf of the F1 generation decreased significantly in the 5 μg/L and 25 μg/L MCLR group.And the survival rates were also significantly decreased in the 25 μg/L MCLR group.Importantly,the swimming speed of juveniles at7 dpf was decreased in the light and dark in the 5 μg/L and 25 μg/L MCLR group.Part 2 Excessive production of ROS caused by MCLR exposure induced endoplasmic reticulum stress and apoptosis.（1）MCLR can induce the level of the ROS in a dose-dependent manner.Pretreatment of the FRTL-5 cells with NAC or 4PBA significantly decreased the level of the ROS.（2）MCLR can stimulate the transcription and increased expression of the GRP78 protein.This result indicated that MCLR induced ER stress in the FRTL-5 cells.However,the levels of ER stress marker GRP78 were decreased in the 4PBA+10 μg/m L MCLR group when compared with cells treated with 10 μg/m L MCLR alone.Similarly,compared with 10 μg/m L MCLR alone,pretreatment of the FRTL-5 cells with NAC alleviated the ER stress.（3）MCLR can induce apoptosis of the FRTL-5 cells in a dose-dependent manner.Pretreatment of the FRTL-5 cells with NAC or 4PBA significantly decreased the rates of apoptosis.Part 3 The role of UPR pathway in thyroid toxicity induced by MCLR（1）The expression of TG was measured by Western blotting: The expression of TG in the 10 μg/m L MCLR group was significantly higher than that in control.The colocalization of GRP78 and TG by immunofluorescence staining showed that MCLR treatment stimulated the activity of GRP78 and TG.Further detection showed that the number of co-located cells in NAC+10 μg/m L MCLR group and 4PBA+10 μg/m L MCLR group were significantly lower than that in 10 μg/m L MCLR group.（2）To investigate the role of perk and ern1,perk-si RNA and ern1-si RNA were used in FRTL-5 cells,then the expression of TG was measured by Western blotting: The expression of TG in perk-si RNA+10 μg/m L MCLR group and ern1-si RNA+10 μg/m L MCLR group were significantly lower than that in 10 μg/m L MCLR group.Therefore,these results showed that both PERK pathway and IRE1 pathway were involved in this process.（3）The expressions of genes related to HPT axis were measured by PCR.Compared to the 10 μg/m L MCLR group,perk-si RNA and ern1-si RNA pretreatment could significantly reverse the expression of genes related to HPT axis.However,the gene expressions of nkx2.1,pax8 and hhex showed no change while ern1-si RNA pretreatment stimulated the gene expression of nkx2.1 and pax8,which indicates that only IRE1 pathway is involved in the process.ConclusionFemale zebrafish exposed to MCLR could affect thyroid hormones in F0 and F1 zebrafish.To explore the molecular mechanism of MCLR,our research showed that excessive production of ROS caused by MCLR induced endoplasmic reticulum stress and apoptosis in zebrafish and FRTL-5 cells.Endoplasmic reticulum stress hampered thyroglobulin turnover through IRE1 and PERK pathways and then affected the synthesis of thyroid hormones.Moreover,MCLR also affect thyroid development through the IRE1 pathway.Herein,MCLR damaged thyroglobulin and the development of thyroid in F0 and F1 zebrafish through endoplasmic reticulum stress.In summary,maternal MCLR exposure affected thyroid endocrine disruption through endoplasmic reticulum stress and the disrupting effects could be remarkably transmitted to its F1 offspring.