自噬在多氯联苯118致大鼠甲状腺组织损伤中的作用机制  

Mechanism of autophagy in PCB118-induced thyroid tissue injury in rats

作　　者：王莉[1] 许文立 朱晓霞 段宇[1] WANG Li;XU Wenli;ZHU Xiaoxia;DUAN Yu(The First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院,南京210029

出　　处：《山东医药》2021年第15期26-31,共6页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81670724)。

摘　　要：目的探讨自噬在慢性低浓度[0~1 000μg/(kg·d)]多氯联苯118(PCB118)导致大鼠甲状腺组织损伤中的作用机制。方法将40只雄性Wistar大鼠随机分为A、B、C、D组,每周腹腔注射0、10、100、1 000μg/(kg·d)PCB118 5次,共13周。PCB118处理结束后,处死大鼠取甲状腺组织,用透射电子显微镜观察大鼠甲状腺组织超微结构。采用蛋白免疫印迹法检测甲状腺组织自噬相关蛋白LC3、P62、BECLIN1及β-Ⅲ型微管蛋白(Tubb3)/死亡相关蛋白激酶2(DAPK2)-通路相关蛋白[Tubb3、DAPK2、肌球蛋白调节轻链(MRLC)、磷酸化MRLC(p-MRLC)、自噬相关基因9A(ATG9A)]。采用实时荧光定量PCR法检测Tubb3/DAPK2-通路相关基因[Tubb3、DAPK2、非肌肉肌球蛋白重链IIA(NMMHC-ⅡA)、ATG9A]。结果与A组比较,B、C、D组甲状腺滤泡细胞损伤、部分空泡化,线粒体密度下降、嵴膜消失,伴内质网扩张,且上述改变在C组最为明显,C、D组出现典型的自噬小体。与A组比较,B组LC3Ⅱ/LC3Ⅰ升高(P均<0.05);C、D组LC3Ⅱ/LC3Ⅰ、BECLIN1蛋白表达升高,P62蛋白表达降低(P<0.01)。与B组比较,C组BECLIN1蛋白表达升高(P均<0.01);D组LC3Ⅱ/LC3Ⅰ、BECLIN1蛋白表达升高,P62蛋白表达降低(P均<0.05)。与C组比较,D组LC3Ⅱ/LC3Ⅰ、BECLIN1蛋白表达升高(P均0.01)。与A组比较,C、D组Tubb3蛋白表达降低(P均<0.01),DAPK2蛋白表达、p-MRLC/MRLC升高(P均<0.05)。与B组比较,C组Tubb3蛋白表达降低,DAPK2蛋白表达升高(P均<0.05);D组Tubb3蛋白表达降低,DAPK2、MRLC、p-MRLC蛋白表达及p-MRLC/MRLC升高(P均<0.05)。与C组比较,D组DAPK2蛋白表达降低,MRLC、p-MRLC蛋白表达及p-MRLC/MRLC升高(P均<0.05)。与A组比较,B组Tubb3 mRNA降低,DAPK2、ATG9A mRNA升高(P均<0.05);C组Tubb3 mRNA降低,DAPK2、NMMHC-ⅡA、ATG9A mRNA升高(P均<0.05);D组Tubb3表达降低,NMMHC-ⅡA、ATG9A mRNA升高(P均<0.05)。与B组比较,C组DAPK2、ATG9A mRNA升高(P均<0.01)。与C组比较,D组DAPK2、ATG9A mRNA降低(P均<0.05)。结论�Objective phenyl 118[PCB118;0-1000 μg/(kg·d)]-induced thyroid tissues injury in rats.Methods randomly divided into four groups,including groups A(control group),B,C,and D. Rats were intraperitoneally injected with corresponding concentrations of PCB118[0,10,100 and 1000 μg/(kg·d)],respectively,five days per week for 13 weeks. The rats were euthanized and the thyroid tissues were taken out after the PCB118 treatment. Transmission electron microscopy(TEM)was used to observe ultrastructure changes of rat thyroids treated by PCB118. Western blotting was used to detect the levels of autophagy-related proteins including LC3,P62 and BECLIN1,and β-Ⅲ-tubulin(Tubb3)/death-associated protein kinase 2(DAPK2)-pathway-related proteins[Tubb3,DAPK2,myosin regulatory light chain(MRLC),p-MRLC,and autophagy-related gene 9 A(ATG9 A)]. The qRT-PCR was used to detect the levels of Tubb3/DAPK2-pathway-related genes(Tubb3,DAPK2,NMMHC-ⅡA and ATG9 A).Results the follicular cells were damaged with some vacuolization,mitochondrial density decreased,mitochondrial decidua disappeared,and endoplasmic reticulum distended in three PCB118-treated groups,obviously in the group C;typical autophagosomes were found in the groups C and D. Compared with the group A,LC3Ⅱ/LC3Ⅰ increased in the group B(P<0. 05);the expression of LC3Ⅱ/LC3Ⅰ and BECLIN1 increased,and P62 decreased in the groups C and D(both P<0. 01). Compared with the group B,BECLIN1 increased in the group C(P<0. 01);LC3Ⅱ/LC3Ⅰ and BECLIN1 increased,and P62 decreased in the group D(all P<0. 05). Compared with the group C,LC3Ⅱ/LC3Ⅰ and BECLIN1 increased in the group D(all P<0. 01). Compared with the group A,the expression of Tubb3 protein decreased,while DAPK2 and p-MRLC/MRLC increased in the groups C and D(all P<0. 05). Compared with the group B,Tubb3 protein decreased and DAPK2 increased in the group C(both P<0. 05);Tubb3 decreased,and DAPK2,MRLC,p-MRLC and pMRLC/MRLC increased in the group D(all P<0. 05). Compared with the group C,DAPK2 protein levels decreased,and

关 键 词：环境内分泌干扰物 多氯联苯118 自噬 甲状腺自噬 Tubb3/DAPK2-通路 

分 类 号：R58[医药卫生—内分泌]