程序性坏死抑制剂Nec-1对铅致BV2细胞损伤的影响

易湘; 杨纯; 彭东杰; 区仕燕; 姜岳明; 李少军

doi:10.13213/j.cnki.jeom.2021.21175

摘要: 背景

程序性坏死与神经退行性疾病的发生和发展关系密切，但铅是否引起神经细胞程序性坏死尚未见报道。

目的

探讨程序性坏死在铅诱导神经小胶质细胞（BV2细胞）损伤中的作用及其抑制剂对铅诱导BV2细胞损伤的影响。

方法

取生长良好的对数生长期BV2细胞，经0、1、5、10、25、50、100、200 μmol·L⁻¹醋酸铅分别处理12、24、36、48 h后，采用噻唑蓝法测定细胞存活率。BV2细胞经0、25、50、100 μmol·L⁻¹醋酸铅处理24 h后，分别采用酶联免疫吸附试验法、蛋白质免疫印迹法和流式细胞术测定细胞内肿瘤坏死因子-α （TNF-α）、受体相互作用蛋白激酶3（RIPK3）、受体相互作用蛋白激酶1（RIPK1）和混合谱系激酶结构域样蛋白（MLKL）的表达情况；并经RIPK1抑制剂Nec-1预处理1 h，检测Nec-1对铅诱导BV2细胞损伤的影响。

结果

BV2细胞的存活率随着铅染毒浓度的增加（r_{12 h}=−0.995，r_{24 h}=−0.984，r_{36 h}=−0.983，r_{48 h}=−0.981，均P<0.01）而下降，在5 μmol·L⁻¹醋酸铅染毒下，细胞存活率也随着染毒时间的延长（r=−0.994，P<0.01）而下降。与对照组比较，同一染毒时间，当铅染毒浓度达到10 μmol·L⁻¹时即可引起BV2细胞存活率下降（P<0.01）。与对照组比较，25、50、100 μmol·L⁻¹铅处理组细胞程序性坏死相关蛋白RIPK1、MLKL蛋白表达升高（P<0.05或0.01），并伴有炎症细胞因子TNF-α含量升高，以100 μmol·L⁻¹醋酸铅组最高（P<0.01）；50、100 μmol·L⁻¹醋酸铅处理组p-RIPK1、p-MLKL及RIPK3表达水平均升高（P<0.01）。此外，与相应染铅组比较，Nec-1预处理使染铅BV2细胞活性升高，坏死及晚凋率降低（P<0.05或0.01）。

结论

铅可致BV2细胞活性降低，坏死率升高，并伴有RIPK3和RIPK1、MLKL蛋白及其磷酸化的表达上调，而RIPK1抑制剂Nec-1对铅引起的BV2细胞损伤具有一定的干预作用，提示程序性坏死可能在铅神经毒性中发挥着重要作用。

Abstract: Background

Programmed necrosis is closely related to the occurrence and development of neurodegenerative diseases, but whether lead causes programmed cell necrosis has not been reported.

Objective

This experiment is designed to probe into the function of programmed necrosis and the effect of its inhibitor on lead-induced microglia (BV2 cell) injury.

Methods

The BV2 cells at logarithmic growth phase were treated with 0, 1, 5, 10, 25, 50, 100, and 200 μmol·L⁻¹ lead acetate for 12, 24, 36, and 48 h, respectively, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to determine cell viability. After treatment with 0, 25, 50, and 100 μmol·L⁻¹ lead acetate for 24 h, enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to determine the expressions of tumor necrosis factor-α (TNF-α), receptor-interacting protein kinase 3 (RIPK3), receptor-interacting protein kinase 1 (RIPK1), and mixed lineage kinase domain-like protein (MLKL) in the cells, and the effect of RIPK1 inhibitor Nec-1 pretreatment on lead-induced BV2 cell injury .

Results

The BV2 cell viability decreased with higher lead concentration (r_{12 h}=−0.995, r_{24 h}=−0.984, r_{36 h}=−0.983, r_{48 h}=−0.981, all P<0.01) and time extension (only for 5 μmol·L⁻¹ lead acetate, r=−0.994, P<0.01). Compared with the control group, the BV2 cell viability decreased at the same exposure time when the concentration of lead was above 10 μmol·L⁻¹ (P<0.01). Compared with the control group, the expressions of RIPK1 and MLKL were increased in the 25, 50, and 100 μmol·L⁻¹ lead groups (P<0.05 or 0.01), accompanied by an increase in the contents of inflammatory cytokine TNF-α, especially in the 100 μmol·L⁻¹ lead group, the increment was the highest (P<0.01). The expression levels of p-RIPK1 and p-MLKL in BV2 cells were both increased when the concentration of lead acetate was above 50 μmol·L⁻¹ (P<0.01). In addition, pretreatment with Nec-1 increased the cell viability rate and decreased the necrosis and late apoptosis rate of BV2 cells exposed to lead compared with corresponding lead exposure groups (P<0.05).

Conclusions

Lead can reduce BV2 cell viability, increase necrosis rate, and up-regulate the expressions of RIPK1, RIPK3, amd MLKL, and the phosphorylation levels of RIPK1 and MLKL. The RIPK1 inhibitor Nec-1 has an intervention effect on lead-induced damage in BV2 cells, indicating that programmed necrosis may play a role in lead neurotoxicity.

程序性坏死抑制剂Nec-1对铅致BV2细胞损伤的影响

Effect of programmed necrosis inhibitor Nec-1 on lead-induced BV2 cell injury