腹腔注射多氯联苯118的雄性大鼠甲状腺功能变化观察及其机制探讨

Effect of intraperitoneal injection of PCB118 on thyroid function in male rats and its mechanism

目的观察多氯联苯118(polychlorinated biphenyl 118,PCB118)腹腔注射的雄性大鼠甲状腺功能变化及组织衰老程度,并探讨其可能作用机制。方法24只雄性Wistar大鼠随机分为A、B、C及对照组4组,每组6只。A、B及C组大鼠分别用10、100、1000μg/(kg·d)的PCB118腹腔注射,对照组用等容积玉米油腹腔注射,各组均注射1天/次,一周5次,共注射13周。造模结束时处死各组大鼠,心脏取血,ELISA法检测大鼠血清游离甲状腺素(FT4)、游离三碘甲状腺原氨酸(FT3)及促甲状腺激素(TSH)。取甲状腺组织,采用衰老相关β-半乳糖苷酶(SA-β-gal)染色评估甲状腺组织衰老情况;实时荧光定量PCR法检测各组大鼠甲状腺组织衰老基因(p16、p21、p53)、衰老相关分泌表型(SASP)中的部分因子[白细胞介素-1α(IL-1α)、肿瘤坏死因子α(TNFα)、白细胞介素-6(IL-6)、基质金属肽酶13(MMP13)]mRNA以及线粒体动力学相关蛋白[线粒体自噬调节因子PTEN诱导激酶1(PINK1)、线粒体动力相关蛋白1(DNM1L)、线粒体融合蛋白1(MFN1)、线粒体融合蛋白2(MFN2)]mRNA。结果A、B及C组大鼠血清FT3和FT4水平呈PCB118剂量依赖性降低,TSH水平呈剂量依赖性升高;与对照组比较,B、C组大鼠血清FT3和FT4水平显著下降,TSH水平显著升高(P均<0.05)。与对照组比较,A、B、C组大鼠甲状腺组织SA-β-gal染色阳性率高(P均<0.05);与A、B组比较,C组大鼠甲状腺组织SA-β-gal染色阳性率进一步增高(P均<0.05)。与对照组比较,B、C组大鼠甲状腺组织p16、p21基因相对表达量升高(P均<0.05)。与对照组比较,A、B、C组大鼠甲状腺组织IL-6 mRNA及MMP13 mRNA相对表达量升高,B组大鼠甲状腺组织TNFαmRNA相对表达量升高(P均<0.05);与B组比较,C组大鼠甲状腺组织TNFαmRNA相对表达量下降(P<0.05)。与对照组比较,A组甲状腺组织DNM1L mRNA、MFN1 mRNA相对表达量升高(P均<0.05),B组甲状腺组织PINK1 mRNA、DNM1L mRNA、MFN1 mRNA相对表达量升高(P均<0.05),C组甲状腺组织DNM1L mRNA及MFN2 mRNA相对表达量降低(P均<0.05)。结论PCB118腹腔注射的大鼠甲状腺功能减退、甲状腺组织衰老程度高,甲状腺组织衰老基因p16、p21表达升高,衰老相关分泌表型标志物IL-6 mRNA、TNFαmRNA及MMP13 mRNA表达升高,线粒体线粒体动力学相关蛋白PINK1 mRNA、DNM1L mRNA、MFN1 mRNA表达升高。PCB118可能通过诱导大鼠甲状腺组织线粒体动力学紊乱、上调衰老基因表达、促进衰老衰老相关分泌表型标志物表达,促进甲状腺组织衰老,导致大鼠甲状腺功能减退,诱导大鼠甲状腺衰老。

Objective To observe the secretory function and aging degree in thyroid tissues of rats injected with polychlorinated biphenyl 118(PCB118)intraperitoneally,and to explore the underlying mechanism.Methods Twenty-four male Wistar rats were randomly divided into four groups:groups A,B,C,and control group.Rats of groups A,B,C were intraperitoneally injected with 10,100,and 1000μg/(kg·d)PCB118,respectively;rats in the control group were intraperitoneally injected with corn oil,five days a week,for 13 weeks.At the end of modeling,the rats were euthanized and sacrificed,the blood was collected from the heart.Rats’serum levels of free thyroxine(FT4),free triiodothyronine(FT3)and thyroid-stimulating hormone(TSH)were measured by enzyme-linked immunosorbent assay(ELISA).Thyroid tissues of rats were taken out and their senescence was assessed by aging-relatedβ-galactosidase(SA-β-gal)staining.The qRT-PCR was used to detect mRNA expression levels of senescence genes(p16,p21,p53),senescence-associated secretory phenotype(SASP)factors[interleukin-1α(IL-1α),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and matrix metallopeptidase 13(MMP13)],and mitochondria dynamics regulators[PTEN induced putative kinase 1(PINK1),dynamin1-like(DNML1),mitofusin1(MFN1),and mitofusin2(MFN2)].Results FT3 and FT4 serum concentrations of rats decreased in the groups A,B and C in a PCB118 dose-dependent manner,while TSH increased in a PCB118 dose-dependent manner.Compared with the control group,FT3 and FT4 significantly decreased,and TSH significantly increased in groups B and C(all P<0.05).SA-β-Gal staining positive rate of rats’thyroid tissues was significantly higher in the groups A,B and C than in the control group(all P<0.05);compared with groups A and B,SA-β-Gal staining positive rate increased meaningfully in the group C(P<0.05).Compared with the control group,the mRNA expression levels of p16 and p21 in the rats’thyroid tissues significantly increased in groups B and C(all P<0.05).Compared with the control group,the relative expression levels of IL-6 mRNA and MMP13 mRNA in the thyroid tissues of rats increased in the groups A,B,and C,and the relative expression of TNFαmRNA in the thyroid tissues of rats increased in the group B(all P<0.05).Compared with group B,the relative expression of TNFαmRNA in the thyroid tissues of rats decreased in the group C(P<0.05).Compared with the control group,the relative expression levels of DNM1L mRNA and MFN1 mRNA in the thyroid tissues increased in the group A(all P<0.05),and the relative expression levels of PINK1 mRNA,DNM1L mRNA,and MFN1 mRNA of thyroid tissues increased in the group B(all P<0.05),and the relative expression levels of DNM1L mRNA and MFN2 mRNA in the thyroid tissues decreased in the group C(all P<0.05).Conclusions Rats with intraperitoneal injection of PCB118 showed impaired thyroid function,higher degree of thyroid tissue aging and increased mRNA expression levels of senescence genes p16,p21,SASP factors IL-6,TNFα,MMP13,and mitochondria dynamics regulators PINK1,DNM1L,MFN1.PCB118 exposure could accelerate senescence and damage function in thyroid tissues of rats via disturbing mitochondrial dynamics,up-regulating senescence gene expression,and promoting SASP.