溶酶体损伤在甲基汞诱导SH-SY5Y细胞衰老中的作用及褪黑素干预研究
Role of lysosomal damage in senescence of SH-SY5Y cells induced by methylmercury and intervention of melatonin
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摘要:
背景 甲基汞（MeHg）是一种神经毒物，褪黑素（MT）对神经系统具有保护作用，但其能否拮抗MeHg诱导的神经细胞损伤及其机制尚不清楚。
目的 采用人神经母细胞瘤细胞（SH-SY5Y细胞）为研究对象，通过建立MeHg诱导SH-SY5Y细胞衰老模型，观察自噬相关蛋白、溶酶体数量与功能的改变以及MT在其中的干预作用和机制。
方法 （1）采用不同剂量MeHg（0、0.125、0.25、0.5、1、2、4 μmol·L−1）处理SH-SY5Y细胞48 h，细胞活性检测试剂盒（CCK-8法）检测细胞活性并计算存活率，并采用β-半乳糖苷酶（SA-β-gal）染色法检测衰老细胞情况。（2）筛选出可明显引起SH-SY5Y细胞衰老的MeHg剂量（即0.5 μmol·L−1），并采用其1/2和1/4的剂量（即0.25和0.125 μmol·L−1）分别作为中、低剂量组进行染毒。（3）MT干预实验分为4组：对照组（0.1% DMSO）、染汞组（0.5 μmol·L−1 MeHg）、单纯MT组（1 mmol·L−1 MT）、MT干预组（1 mmol·L−1 MT+0.5 μmol·L−1 MeHg）。MT干预组为1 mmol·L−1 MT预处理细胞24 h后0.5 μmol·L−1 MeHg染毒48 h。（4）SA-β-gal染色观察细胞衰老；Western blotting检测衰老相关蛋白p16，自噬相关蛋白p62、LC3Ⅱ以及溶酶体相关蛋白LAMP1、LAMP2、TFEB的表达水平；溶酶体红色荧光探针染色检测溶酶体数量；溶酶体绿色荧光探针检测溶酶体pH变化；电镜观察溶酶体形态学变化。
结果 CCK-8结果表明，随着染毒浓度增加细胞存活率降低；与对照组相比，0.5 μmol·L−1 MeHg染毒组SA-β-gal染色蓝染细胞率增多了48%（
P < 0.01），p16和p62及LC3Ⅱ蛋白表达水平明显增高（P < 0.05）；LAMP1、LAMP2蛋白水平与溶酶体红色及绿色荧光探针的荧光强度随MeHg浓度的升高而降低（P < 0.05）；电镜结果表明溶酶体体积增大。与MeHg染毒组相比，1 mmol·L−1 MT + 0.5 μmol·L−1 MeHg处理组p16蛋白表达降低及SA-β-gal染色蓝染细胞率降低了19%（P < 0.05），p62及LC3Ⅱ蛋白表达明显降低，LAMP1、LAMP2蛋白表达与溶酶体红色及绿色荧光探针的荧光强度均有所增加，TFEB入核显著增加，差异具有统计学意义（P < 0.05）。结论 MeHg可能通过减少SH-SY5Y细胞溶酶体数量及损伤溶酶体活性致细胞衰老，MT可改善MeHg所致SH-SY5Y细胞溶酶体异常进而干预细胞衰老。
Abstract:Background Methylmercury (MeHg) is a neurotoxin, and melatonin (MT) has a protective effect on the nervous system, but whether it can antagonize MeHg-induced nerve cell damage and the associated mechanism remain unknown.
Objective Human neuroblastoma cells (SH-SY5Y cells) were used as research objects. A MeHg-induced SH-SY5Y cell senescence model was established to observe autophagy related protein, lysosomal number, and function changes, as well as potential intervention role and associated mechanism of MT.
Methods (1) After SH-SY5Y cells were treated with different doses of MeHg (0, 0.125, 0.25, 0.5, 1, 2, and 4 μmol·L−1) for 48 h, the cell viability was detected using a cell viability detection kit (CCK-8 method) and the viability rate was calculated. Senescent cells were detected by an acidic senescence-associated-β-galactosidase (SA-β-gal) staining. (2) A MeHg dose of 0.5 μmol·L−1 that significantly induced senescence of SH-SY5Y cells was screened, and a half and a quarter of the dose (0.25 and 0.125 μmol·L−1) were used for the middle and low dose groups, respectively. (3) In the MT intervention experiments, SH-SY5Y cells were divided into four groups, including control group (0.1% DMSO), MeHg group (0.5 μmol·L−1 MeHg), MT group (1 mmol·L−1 MT), and MT intervention group (1 mmol·L−1 MT+0.5 μmol·L−1 MeHg). In the MT intervention group, cells were exposed to 0.5 μmol·L−1 MeHg for 48 h after 24 h of 1 mmol·L−1 MT pretreatment. (4) SA-β-gal staining was conducted to observe cell senescence; Western blotting for the expression levels of senescence-associated protein p16, autophagy-associated protein p62, LC3Ⅱ, and lysosomal-associated proteins LAMP1, LAMP2, and TFEB; Lyso-Tracker Red for the quantity of lysosomes; LysoSensor Green DND-189 for lysosomal pH changes; electron microscope for the morphological changes of lysosomes.
Results The results of CCK-8 indicated that the viability rate of cells decreased with the increase of MeHg exposure concentration. Compared with the control group, the SA-β-gal positive cell ratio in the 0.5 μmol·L−1 MeHg group increased by 48% (
P <0.01), p16, p62, as well as LC3Ⅱ protein expressions were significantly increased (P <0.05), LAMP1 and LAMP2 protein levels, as well as the fluorescence intensities of lysosomal red and green fluorescent probes decreased with the increase of MeHg concentration (P <0.05), and the volume of lysosomes increased under the electron microscope. Compared with the MeHg group, the expression of p16 protein was decreased in the 1 mmol·L−1 MT + 0.5 μmol·L−1 MeHg group and the SA-β-gal positive cell ratio was significantly decreased by 19% (P <0.05), the protein levels of p62 and LC3Ⅱ were significantly decreased, the LAMP1 and LAMP2 protein levels and the fluorescence intensities of lysosomal red and green fluorescent probes were increased respectively, the nuclear entry of TFEB was significantly increased, and the differences were statistically significant (P <0.05).Conclusion MeHg may cause cellular senescence by reducing the number of lysosomes and impairing lysosomal activity in SH-SY5Y cells, and MT may ameliorate MeHg-induced lysosomal abnormalities in SH-SY5Y cells, thereby intervening cell senescence.
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