Shi Lei, Jiang Chen-Chen, Lu Jia-Jun, Li Zi-Xu, Li Wang-Jie, Yin Xiu-Yun, Chen Zhuo, Zhao Xin-Ya, Zhang Hui, Hu Hao-Ran, Zhou Lu-Tan, Han Jun
Third-Grade Pharmacology Laboratory, National Administration of Traditional Chinese Medicine Wuhu 241002, China School of Pharmacy, Drug Research and Development Center, Wannan Medical College Wuhu 241002, China.
Anhui Provincial Engineering Laboratory for Screening and Re-evaluation of Active Compounds of Herbal Medicines in Southern Anhui Wuhu 241002, China Anhui Provincial Engineering Research Center for Polysaccharide Drugs Wuhu 241002, China.
Zhongguo Zhong Yao Za Zhi. 2023 Jun;48(11):3046-3054. doi: 10.19540/j.cnki.cjcmm.20221201.401.
The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.
本研究旨在从铁死亡角度探讨中药当归挥发油主要活性成分藁本内酯减轻PC12细胞氧糖剥夺/复氧再灌注（OGD/R）损伤的机制。体外诱导OGD/R，在再灌注期间加入藁本内酯12 h后，采用细胞计数试剂盒-8（CCK-8）法检测细胞活力。用2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）染色检测细胞内活性氧（ROS）水平。采用蛋白质免疫印迹法检测铁死亡相关蛋白谷胱甘肽过氧化物酶4（GPX4）、转铁蛋白受体1（TFR1）和溶质载体家族7成员11（SLC7A11）以及铁自噬相关蛋白核受体辅激活因子4（NCOA4）、铁蛋白重链1（FTH1）和微管相关蛋白1轻链3（LC3）的表达。通过免疫荧光染色分析LC3蛋白的荧光强度。采用化学发光免疫分析法检测谷胱甘肽（GSH）、丙二醛（MDA）和铁的含量。通过过表达NCOA4基因观察藁本内酯对铁死亡的影响。结果显示，与OGD/R组相比，藁本内酯提高了OGD/R损伤的PC12细胞活力，抑制了ROS释放，降低了铁、MDA含量以及TFR1、NCOA4和LC3的表达，提高了GSH含量以及GPX4、SLC7A11和FTH1的表达。在铁自噬中过表达关键蛋白NCOA4后，藁本内酯对铁死亡的抑制作用部分被逆转，表明藁本内酯可能通过阻断铁自噬进而抑制铁死亡来减轻PC12细胞的OGD/R损伤。藁本内酯减轻PC12细胞OGD/R损伤的机制是抑制了铁自噬相关的铁死亡。
