亚砷酸钠通过调控ER-PI3K/AKT信号通路影响甲状腺正常细胞增殖与凋亡

Sodium arsenite influences proliferation and apoptosis in normal thyroid cells via modulation of ER-PI3K/AKT signaling pathway

  • 摘要:
    背景 伴随无机砷毒性作用的深入研究发现,砷暴露可对多个内分泌器官产生影响,从而影响其功能,但砷暴露致甲状腺损伤的机制尚不明确。
    目的 基于雌激素受体(ER)-磷脂酰肌醇 3-激酶(PI3K)/蛋白激酶B(AKT)信号通路探讨亚砷酸钠(NaAsO2)对人甲状腺正常细胞(Nthy-ori3-1)的增殖及凋亡的作用机制。
    方法 体外正常培养 Nthy-ori3-1细胞,将其分为对照组(不含药物的完全培养基0 μmol·L−1)、1 μmol·L−1 NaAsO2 组、2 μmol·L−1 NaAsO2组、4 μmol·L−1 NaAsO2组;1 μmol·L−1雌激素受体抑制剂(ICI182780)分别干预同剂量NaAsO2染毒组,分组为1 μmol·L−1 NaAsO2+ICI182780、2 μmol·L−1 NaAsO2+ICI182780、4 μmol·L−1 NaAsO2+ICI182780;采用细胞活性实验筛选NaAsO2的半数致死量;细胞计数试剂盒8(CCK-8)实验检测每组细胞分别在24、36、48 h的细胞活力;平板克隆实验检测每组细胞的集落形成能力;Hoechst33342染色实验检测每组细胞发生凋亡的情况;免疫印迹法(WB)和实时荧光定量PCR(qRT-PCR)实验分别检测每组细胞内ERα、ERβ、c-MYC、Bax、Bcl-2、PI3K、p-PI3K、AKT、p-AKT蛋白及相关基因的表达水平。
    结果 NaAsO2浓度为4.034 μmol·L−1时,达到细胞半数致死量;24、36、48 h时,CCK-8结果显示与对照组相比,1、2、4 μmol·L−1NaAsO2组抑制Nthy-ori3-1细胞增殖能力(P<0.001);平板克隆实验结果表明,其集落形成能力呈浓度梯度降低(P<0.001);1、2、4 μmol·L−1 NaAsO2组经ICI182780干预后,与Nthy-ori3-1细胞的同剂量染NaAsO2组比较,细胞存活率、集落形成能力被恢复(P<0.001,P<0.01);Hoechst 33342染色结果显示,与对照组相比,1、2、4 μmol·L−1 NaAsO2组中凋亡的Nthy-ori3-1细胞核染色逐渐增强,细胞凋亡加重(P<0.001);与Nthy-ori3-1细胞的同剂量染NaAsO2组比较,经ICI182780干预NaAsO2染毒组的细胞凋亡水平降低(P<0.001);WB结果显示,与对照组相比,1、2、4 μmol·L−1NaAsO2组ERα、ERβ、c-MYC、Bcl-2、p-PI3K、p-AKT蛋白表达呈浓度梯度降低(P<0.001,P<0.01),Bax蛋白表达呈浓度梯度增高(P<0.001),而PI3K、AKT蛋白表达差异无统计学意义(P>0.05);与Nthy-ori3-1细胞的同剂量染NaAsO2组比较,经ICI182780干预NaAsO2染毒组的细胞内ERα、ERβ、c-MYC、Bcl-2、p-PI3K、p-AKT蛋白表达升高(P<0.001,P<0.01),Bax蛋白表达降低(P<0.001),而PI3K、AKT蛋白表达无统计学差异(P>0.05);ERαERβc-MYCBcl-2Bax mRNA的表达结果与WB结果一致。
    结论 NaAsO2作用于Nthy-ori3-1细胞后,将抑制Nthy-ori3-1细胞增殖能力,促进其凋亡发生,并表现出剂量依赖;其机制可能是NaAsO2通过抑制ER-PI3K/AKT信号通路,作用于下游增殖/凋亡相关基因而发生。

     

    Abstract:
    Background Recent advances in understanding the toxic effects of inorganic arsenic have revealed that arsenic exposure impacts multiple endocrine organs, thereby altering their functions. However, the mechanisms underlying arsenic-induced thyroid injury remain unclear.
    Objective To investigate the mechanisms by which sodium arsenite (NaAsO₂) affects the proliferation and apoptosis of normal thyroid cells (Nthy-ori3-1) through the estrogen receptor (ER)-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway.
    Methods Nthy-ori3-1 cells were cultured in vitro and divided into the following groups: a control group (complete medium without drugs, 0 μmol·L−1), and NaAsO₂-treated groups at 1, 2, and 4 μmol·L−1. Additionally, 1 μmol·L−1 of the ER inhibitor ICI182780 was used to intervene in the NaAsO₂ exposure groups, resulting in the following combinations: 1 μmol·L−1 NaAsO₂ + ICI182780, 2 μmol·L−1 NaAsO₂ + ICI182780, and 4 μmol·L−1 NaAsO₂ + ICI182780. The median lethal concentration of NaAsO₂ was determined using cell viability assay. Cell viability was assessed at 24, 36, and 48 h using Cell Counting Kit-8 (CCK-8) assay. Colony formation ability was evaluated via plate cloning assay. Apoptosis was detected using Hoechst 33342 staining. Protein and mRNA expression levels of ERα, ERβ, c-MYC, Bax, Bcl-2, PI3K, p-PI3K, AKT, and p-AKT were measured using Western blot (WB) and real-time quantitative PCR (qRT-PCR), respectively.
    Results The median lethal concentration of NaAsO₂ was determined to be 4.034 μmol·L−1. CCK-8 assay at 24, 36, and 48 h revealed that, compared with the control group, the 1, 2, and 4 μmol·L−1 NaAsO₂ groups significantly inhibited Nthy-ori3-1 cell proliferation (P < 0.001). The plate cloning assays demonstrated a concentration-dependent reduction in colony formation ability (P < 0.001). Following the ICI182780 intervention, the cell viability and colony formation ability in the 1, 2, and 4 μmol·L−1 NaAsO₂ groups were significantly restored compared with the corresponding NaAsO₂-only groups (P < 0.001, P < 0.01). The Hoechst 33342 staining indicated that compared with the control group, the nuclear staining intensity and apoptosis levels in the 1, 2, and 4 μmol·L−1 NaAsO₂ groups increased in a concentration-dependent manner (P < 0.001). However, the ICI182780 intervention reduced the apoptosis levels in the NaAsO₂-treated groups compared with their NaAsO₂-only counterparts (P < 0.001). The WB analysis showed that, compared with the control group, the protein expression of ERα, ERβ, c-MYC, Bcl-2, p-PI3K, and p-AKT in the 1, 2, and 4 μmol·L−1 NaAsO₂ groups decreased manner (P < 0.001, P < 0.01), while the Bax expression increased in a concentration-dependent (P < 0.001); the PI3K and AKT protein levels showed no significant differences (P > 0.05). In the ICI182780-treated NaAsO₂ groups, the ERα, ERβ, c-MYC, Bcl-2, p-PI3K, and p-AKT protein expression increased (P < 0.001, P < 0.01), the Bax expression decreased (P < 0.001), and the PI3K and AKT levels remained unchanged (P > 0.05) compared with the corresponding NaAsO₂-only groups. The mRNA expression patterns of ERα, ERβ, c-MYC, Bcl-2, and Bax were consistent with the WB results.
    Conclusion NaAsO₂ inhibits proliferation and promotes apoptosis in Nthy-ori3-1 cells in a dose-dependent manner. The underlying mechanism likely involves NaAsO₂-mediated suppression of the ER-PI3K/AKT signaling pathway, which subsequently regulates downstream proliferation- and apoptosis-related genes.

     

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