玉米赤霉烯酮致PC12细胞自噬流阻滞的相关作用机制

Mechanism of Autophagy Block in PC12 Cells Induced by Zearalenone

旨在研究玉米赤霉烯酮(ZEA)对PC12细胞自噬流阻滞的作用机制,以PC12细胞(大鼠肾上腺嗜铬细胞瘤系)为研究材料,以不同浓度的ZEA处理24 h,CCK-8检测细胞存活率,并筛选ZEA最适浓度,分别设置Con组、15μmol·L^(-1) ZEA组、30μmol·L^(-1) ZEA组,Western blot检测自噬相关蛋白ATG5、LC3Ⅱ、p62、Beclin1,溶酶体相关膜蛋白LAMP1,组织蛋白酶B(CTSB)和组织蛋白酶D(CTSD),细胞质及细胞核内TFEB蛋白以及自噬体溶酶体融合相关蛋白STX17和SNAP29的蛋白表达水平;免疫荧光技术检测LC3荧光聚点,TFEB入核情况;mRFP-GFP-LC3检测自噬流情况;AO染色观察细胞内酸性环境变化情况。结果显示,与对照组相比,ZEA处理组中自噬相关蛋白ATG5、LC3Ⅱ、p62、Beclin1蛋白表达水平均显著上升(P<0.05);免疫荧光检测显示LC3荧光聚点逐渐增加;mRFP-GFP-LC3检测表明自噬流阻滞;ZEA处理组中的细胞质蛋白CTSD及CTSB表达水平均显著升高(P<0.05),LAMP1表达水平在ZEA为30μmol·L^(-1)时显著下降(P<0.05);AO染色表明,ZEA可致PC12细胞酸性环境改变,导致溶酶体损伤;p-ERK及核内TFEB蛋白表达水平显著下降(P<0.05),而细胞质内TFEB蛋白表达水平未见显著性变化;SNAP29、STX17蛋白表达水平均显著下降(P<0.05)。综上表明,ZEA可引起PC12细胞自噬水平升高,自噬体增多,并伴随自噬流阻滞,其作用机制与ZEA致溶酶体损伤,抑制TFEB入核,降低自噬体与溶酶体融合相关蛋白表达有关。

This experiment aims to investigate the mechanism of zearalenone(ZEA)on autophagy block of PC12 cells.Taking PC12 cells(rat renal pheochromocytoma)for research materials,treated with different concentration of ZEA for 24 h,Cell Counting Kit-8(CCK-8)was used to detect the cell survival rate.The optimal concentration of ZEA was screened.PC12 cells were divided into control group,15μmol·L^(-1) ZEA group and 30μmol·L^(-1) ZEA group.The two ZEA groups were treated with 15,30μmol·L^(-1) ZEA for 24 h,respectively,and the control group was administered with the same amount of 1640 medium.Western blot was used to detect the expression levels of autophagy associated proteins Atg5,LC3Ⅱ,p62,Beclin1,lysosomal associated membrane proteins LAMP1,cathepsin B(CTSB)and cathepsin D(CTSD),TFEB proteins in cytoplasm and nucleus,autophagosomal and lysosomal fusion associated proteins STX-17 and SNAP29.The immunofluorescence assay was used to detect LC3 fluorescence dots and TFEB nuclear translocation.The mRFP-GFP-LC3 was used to detect autophagy flow.AO staining was used to observe the changes of intracellular acidic environment.The results showed that compared with the control group,the expression levels of Atg5,LC3Ⅱ,p62 and Beclin1 in all ZEA treatment groups were significantly increased(P<0.05).The immunofluorescence showed that LC3 fluorescence dots increased gradually.The mRFP-GFP-LC3 assay showed autophagy flow was blocked.The expression levels of cytoplasmic protein CTSD and CTSB in ZEA-treated group were significantly increased(P<0.0),while the expression level of LAMP1 decreased significantly when ZEA was 30μmol·L^(-1)(P<0.05).The result of AO staining showed that ZEA could damage the lysosome of PC12 cells.The expression level of p-ERK and TFEB protein in nucleus was significantly decreased(P<0.05).However,there was no significant change in the expression levels of TFEB protein in cytoplasm.The expression level of SNAP29 and STX-17 protein decreased significantly(P<0.05).In conclusion,ZEA can increase the autophagy level and increase the number of autophagosomes in PC12 cells,accompanied by autophagy flow block.The mechanism was related to ZEA lysosomal damage,inhibition of TFEB entry into the nucleus,and reduction of autophagosomal-lysosomal fusion related protein expression.