| Objective:1.To study whether low-dose 2,3',4,4',5-pentachlorobiphenyl(PCB118)could induce inflammatory responses in rat thyroid tissue and FRTL-5 cells.2.To investigate the influence of low-dose PCB118 on the thyroid functional gene of NIS.3.To explore whether low-dose PCB118 could induce inflammatory disorders and dysfunction in the thyroid through the AhR and JNK-mediated pathway.Methods:1.Forty aldult Wistar rats were assigned randomly into four groups,and admininstered with different concentrations of PCB118(0?10?100?1000 ?g/kg/d)by intraperitoneal injection,5 days a week for thirteen weeks.At the study end point,rats were euthanized with excess chloral hydrate by ip injection,and the thyroid gland was stored at-80? or fixed in 4%paraformaldehyde.2.Thyroids were placed in 4%paraformaldehyde for 48 h,stained with hematoxylin and eosin(HE)and Masson's trichrome for pathological observation of structural change and interstitial fibrosis.Real-time qRT-PCR and immunohistochemical staining was applied to detect the expression of proinflammatory factors,including interleukin-6(IL-6),tumor necrosis factor-a(TNF-a),and intercellular adhesion molecule-1(ICAM-1)in thyroid tissue.3.FRTL-5 rat thyroid cells were stimulated with various concentrations of PCB118(0?0.25?2.5?25 nM/L)for 24h or 48h,or stimulated with 25 nM/L PCB118 for different time(0?6h?12h?24h?48h),and then qRT-qPCR ELISA?Western-blot were used to detect the mRNA and protein expression expression of IL-6?TNF-? and ICAM-1.Western-blot was applied to detect the NIS protein expression.4.FRTL-5 cells were stimulated with the aboved concentrations of PCB118(0-25 nM/L)for 48h,and qRT-PCR was applied to detect the AhR and CYP1A1 expression level;cells were pre-treated with AhR antagonist a-NF?and then stimulated with DMSO or 25 nM/L PCB118 for 24h or 48h to detect the mRNA and protein expression of IL-6?TNF-a?ICAM-1 and NIS,respectively.5.FRTL-5 cells were stimulated with the above-mentioned concentrations of PCB118(0-25nM/L)for48h.and the expression levels ofJNK?p-JNK?P38?p-P38?ERK?p-ERK?c-jun?p-c-jun in thyrocytes were detected by Western-blot;Western-blot and Immunohistochemical staining was conducted for detection of of JNK and p-JNK expression in thyroid tissues.Small interefering RNA was conducted to silence JNK expression in FRTL-5 cells,afterwards,cells were stiulated with DMSO or 25 nM/L PCB118 for 24h or 48h,and the expression of IL-6?TNF-?.ICAM-1 and NIS were detected by qRT-PCR and Western-blot.6.Cells were pre-treated with 5 ?M a-NF or DMSO for 1 h,prior to DMSO or 25 nM/L PCB118 stimulation for 48 h,and then Western-blot was used to detect p-JNK expression.Results:1.No clinical symptoms of toxicity occurred in animals.There were no significant changes of weight gain among different groups(P>0.05),FRTL-5 cell viability and apoptosis were unaffected.HE staining showed that,PCB118 exposure could induce inflammatory infiltration,such as lymphocyte infiltration in the thyroid,follicular cell hyperplasia and expansion,and follicle collapse.Masson's trichrome staining showed that PCB118 caused thyroid mesenchymal fibrosis in a dose-dependent manner.In the two higher-dose groups,statistic difference occcured(P<0.05).PCB118 exposure upregulated the expression levels of IL-6?TNF-a and ICAM-1,with statistical significance.2.At the cell level,qRT-PCR,ELSIA and Western-blot were used to detect the mRN A and protein expression of proinflammatory factors,and results were that PCB 118 could induce both mRNA and protein levels of IL-6?TNF-a and ICAM-1 in time and concentration-dependent way(P<0.05).Meanwhile,PCB118 downregulated the NIS protein expression level significantly(P<0.05).3.FRTL-5 cells were stimulated with different concentraions of PCB118,compared with control,PCB118 did not alter AhR expression(P>0.05),but significantly upregulated CYP1A1 mRNA expression(P<0.05).After blocking AhR expression,the mRNA expression of AhR and CYP1A1 were inhibited(P<0.01).PCB118-induced IL-6 and ICAM-1 mRNA expression were significantly reduced,and NIS protein was restored(P<0.05);However,a-NF had no effect on PCB118-induced TNF-a expression(P>0.05).4.In the in vitro level,PCB118 significantly increased the p-JNK and p-c-jun protein expression(P<0.05),but no differences in P38 and ERK phosphorylation or total levels of JNK,P38 and ERK between the PCB118 and control groups were observed.At the aminal level,PCB118 promoted p-JNK protein expression in a concentration-dependent manner(P<0.05).Pre-treatment with JNK small interfering RNA(siJNK)significantly inhibited IL-6,TNF-a and ICAM-1 mRNA production and restored NIS protein expression.However,siJNK had no effect on PCB118-induced TNF-a expression.5.FRTL-5 cells were pretreated with a-NF for 1h,afterwards they were stimulated with PCB118 for 48h.Compared to the PCB118 control group,pretreatment with a-NF prior to PCB118 did not inhibit p-JNK induction significantly(P>0.05).Conclusions:1.Low concentrations of PCB118 could induce IL-6,TNF-a and ICAM-1 expression in rat thyroid tissue and cells,and promote inflammatory reactions in the thyroid.2.Low concentrations of PCB 118 could inhibit NIS expression in rat thyroid cells.3.Low concentrations of PCB118 could induce inflammatory responses and dysfunction in the thyroid through AhR and JNK-mediated pathway. |
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