姜黄醇通过抑制铁死亡途径减轻HT22小鼠海马神经元细胞损伤

Curcumol Alleviate Erastin-Induced Ferroptosis in HT22 Cells

目的 验证姜黄醇可抑制埃拉斯汀(Erastin)诱导的HT22细胞铁死亡并发挥细胞保护作用。方法 先通过Western blot检测观察不同时间梯度(0、4、8、12、24 h)Erastin对HT22细胞铁死亡相关蛋白表达的影响,再将HT22细胞分为正常对照组、DMSO组、Erastin处理组(0.5μmol·L^(-1) Erastin处理24 h)、姜黄醇组(0.5μmol·L^(-1) Erastin处理24 h+50μmol·L^(-1)姜黄醇处理48 h),通过Western blot检测HT22细胞铁死亡相关蛋白表达水平;细胞铁离子试剂盒、细胞亚铁含量试剂盒检测细胞内总铁、亚铁水平;CCK-8试剂盒检测细胞活力、AnnexinV/PI细胞凋亡试剂盒检测细胞凋亡。结果 Erastin刺激上调了HT22细胞xCT(P<0.05)、NCOA4(P<0.001)、ASCL4(P<0.01)水平,降低GPX4(P<0.001)、FPN(P<0.01)、FTH1(P<0.05)水平。DMSO组与正常对照组间铁蛋白相关蛋白水平、铁离子水平、细胞活力、细胞凋亡率无统计学差异。相比Erastin组,姜黄醇处理组神经元细胞活力显著升高(P<0.001)、细胞GPX4酶升高(P<0.01),细胞死亡率、细胞总铁、亚铁含量降低(P<0.001);xCT(P<0.001)、NCOA4(P<0.001)、ASCL4(P<0.001)水平显著下调,FTH1(P<0.01)、GPX4(P<0.001)蛋白水平显著上调。结论 姜黄醇可抑制Erastin诱导的HT22铁死亡并发挥细胞保护作用。

Objective To verify that curcumol can inhibit erastin-induced ferroptosis in HT22 cells and play a role of cell protection. Methods Firstly, the effect of 0.5 μmol·L-1 Erastin at different time gradients (0, 4, 8, 12, 24 h) on theexpression of ferroptosis-related proteins in HT22 cells were abserved by Western blot, Then HT22 cells were dividedinto normal control group, DMSO group, Erastin treatment group (0.5 μmol·L-1 Erastin treatment for 24), and curcumolgroup (0.5 μmol·L-1 Erastin treatment for 24 h +50 μmol·L-1 curcumin treatment for 48 h ). The expression levels offerroptosis-related proteins in HT22 cells were detected by Western blot, cell iron ion kit and cell ferrous content kitwere used to detect the levels of total iron and ferrous iron in cells, cell viability was detected by CCK-8 kit, and cellapoptosis was detected by Annexin V/PI apoptosis kit. Results After Erastin stimulation, the levels of xCT (P<0.05),NCOA4 (P<0.001) and ACSL4 (P<0.01) were up-regulated, while GPX4 (P<0.001), FPN (P<0.01) and FTH1 (P<0.05)were decreased in HT22 cells. The ferritin-related protein levels, iron levels, cell viability and apoptosis rate of DMSOgroup were basically the same as those of normal control group. Compared with Erastin group, the cell viability ofneurons (P<0.001) and the GPX4 enzyme level of the cells (P<0.01) in the curcumol treatment group was significantlyincreased, the apoptosis rate (P<0.001) and the total iron and ferrous iron contents of the cells (P<0.001) were decreasedoppositely. In addition, the expression levels of Xct (P<0.001), NCOA4 (P<0.001), and ACSL4 (P<0.001) weresignificantly down-regulated, the expression levels of FTH1 (P<0.01) and GPX4 (P<0.001) were significantly upregulated(P<0.01). Conclusion Curcumol could protect HT22 cells by inhibiting erastin-induced ferroptosis.