活血消瘿方含药血清调控AMPK/mTOR通路对脂多糖诱导的甲状腺滤泡上皮细胞自噬和凋亡的影响

目的 探讨活血消瘿方含药血清对脂多糖(LPS)诱导的甲状腺滤泡上皮细胞(TFECs)自噬和凋亡的影响以及作用机制。方法 光学显微镜观察分离培养的SD大鼠TFECs细胞的形态;免疫荧光染色鉴定TFECs中特异抗原甲状腺球蛋白(Tg)。取对数生长期的TFECs,分为对照组、LPS组、含药血清组、含药血清+compound C(AMPK抑制剂)组、含药血清+MHY1485(mTOR激活剂)组,MTT法检测各组细胞活力;绿色荧光蛋白(GFP)标记质粒转染检测各组细胞自噬小体变化;流式细胞术检测各组细胞凋亡;western blot检测各组细胞中微管相关蛋白1轻链3(LC3)I、LC3II、Beclin1、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)及AMP活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达。结果 光学显微镜观察显示,TFECs形态为梭形、形态均一,且可成簇生长。免疫荧光结果显示,TFECs细胞质中可见较强的Tg荧光染色。与对照组比较,LPS组TFECs细胞活力、自噬小体数量、LC3II/LC3I、Beclin1、Bcl-2、p-AMPK/AMPK蛋白相对表达量显著降低,炎性因子IL-6 、TNF-α水平、细胞凋亡率、Bax、p-mTOR/mTOR蛋白相对表达量显著升高(P< 0.05);与LPS组比较,含药血清组TFECs细胞活力、自噬小体数量、LC3II/LC3I、Beclin1、Bcl-2、p-AMPK/AMPK蛋白相对表达量显著升高,炎性因子IL-6 、 TNF-α水平、细胞凋亡率、Bax、p-mTOR/mTOR蛋白相对表达量显著降低(P< 0.05);与含药血清组比较,含药血清+compound C组、含药血清+MHY1485组TFECs中相应指标与上述趋势相反。结论 活血消瘿方含药血清对LPS诱导的TFECs自噬的促进作用及细胞凋亡的抑制作用可能与激活AMPK/mTOR通路有关。

Effects of Huoxue Xiaoying Recipe-Containing Serum on LPS-Induced Autophagy and Apoptosis of Thyroid Follicular Epithelial Cells by Regulating AMPK/mTOR Pathway

OBJECTIVE To investigate the effects of Huoxue Xiaoying Recipe-containing serum on autophagy and apoptosis of thyroid follicular epithelial cells(TFECs) induced by lipopolysaccharide(LPS) and the mechanism of action. METHODS The morphology of isolated and cultured SD rat TFECs cells was observed with an optical microscope; immunofluorescence staining was used to identify the specific antigen thyroglobulin(Tg) in TFECs. The TFECs in the logarithmic growth phase were divided into control group, LPS group, medicated serum group, medicated serum+compound C(AMPK inhibitor) group, medicated serum+ MHY1485(mTOR activator) group. MTT method was used to detect cell viability of each group; green fluorescent protein(GFP)-labeled plasmid transfection was used to detect autophagosome changes of cells in each group; flow cytometry was used to detect cell apoptosis in each group; Western blot was used to detect the expression of microtubule-associated protein 1 light chain 3(LC3) I, LC3II, Beclin1, Bcl-2 associated X protein(Bax), B cell lymphoma-2(Bcl-2) and AMP-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) pathway related proteins of cells in each group. RESULTS Optical microscope observation showed that TFECs were fusiform and uniform in shape and could grow in clusters. Immunofluorescence analysis showed strong Tg fluorescence staining in the cytoplasm of TFECs. Compared with the control group, the cell viability, number of autophagosomes, and the relative expression of LC3II/LC3I, Beclin1, Bcl-2, and p-AMPK/AMPK proteins in the LPS group were significantly reduced, while the levels of inflammatory factors IL-6 and TNF-α, the rate of apoptosis, and the relative expression of Bax and p-mTOR/mTOR proteins were significantly increased(P<0.05); compared with the LPS group, the cell viability, number of autophagosomes, and the relative expression of LC3II/LC3I, Beclin1, Bcl-2, and p-AMPK/AMPK proteins in the medicated serum group were significantly increased, while the levels of inflammatory factors IL-6 and TNF-α, the rate of apoptosis, and the relative expression of Bax and p-mTOR/mTOR proteins were significantly reduced(P<0.05); compared with the medicated serum group, the corresponding indexes of TFECs in the medicated serum+compound C group, medicated serum+MHY1485 group showed opposite changing trends. CONCLUSION The effects of Huoxue Xiaoying Recipe-containing serum in promoting LPS-induced autophagy and inhibition of apoptosis of TFECs may be related to the activation of AMPK/mTOR pathway.