肝豆灵片通过调控PKCβⅡ/ACSL4/ALOX5信号通路改善肝豆状核变性铁死亡的机制

doi:10.3976/j.issn.1002-4026.20230170

摘要/Abstract

摘要：

基于PKCβⅡ/ACSL4/ALOX5信号通路,探讨肝豆灵片对 TX 小鼠肝豆状核变性铁死亡的影响以及对 CuCl₂诱导的HT22细胞铁死亡的作用机制。将 TX 小鼠分为对照组、模型组、肝豆灵片组、Fer-1组、谷胱甘肽组,HT22细胞分为对照组、模型组、肝豆灵片组、Fer-1组、肝豆灵片+Fer-1 组,采用 HE染色检测各组小鼠海马组织病理学改变,蛋白质免疫印迹检测各组小鼠海马组织与HT22细胞中PKCβⅡ、ACSL4、ALOX5蛋白的表达,以及HT22细胞中SLC7A11、GPX4蛋白的表达,微量法检测各组TX小鼠海马组织中Fe²⁺的浓度,微板法检测各组HT22细胞中SOD(超氧化物歧化酶)、MDA(丙二醛)、GSH-Px 水平,实时荧光定量聚合链式反应检测各组HT22细胞中PKCβⅡ、ACSL4、ALOX5 mRNA 的表达。结果表明:与对照组相比,模型组海马组织出现明显损伤,PKCβⅡ、ACSL4、ALOX5蛋白表达均明显增高,SLC7A11、GPX4蛋白表达明显下降,Fe²⁺的浓度明显升高(P<0.05);与模型组相比,肝豆灵片组、Fer-1组和谷胱甘肽组海马组织的病理损伤均得到改善,且肝豆灵片组效果明显;肝豆灵片组和Fer-1组能抑制HT22细胞铁死亡,PKCβⅡ、ACSL4、ALOX5蛋白与 mRNA 表达较模型组均明显减少,MDA的浓度明显降低(P<0.05),SOD活力及GSH-Px浓度明显增加(P<0.05)。肝豆灵片能减轻 TX 小鼠海马组织铁死亡,抑制CuCl₂诱导的HT22细胞铁死亡,其机制可能与下调PKCβⅡ/ACSL4/ALOX5信号通路,减轻细胞内脂质过氧化有关。

关键词: 肝豆灵片, 肝豆状核变性, PKCβⅡ/ACSL4/ALOX5信号通路, 铁死亡, 机制

Abstract:

This study investigates the effects of Gandouling (GDL) tablets on ferroptosis in hepatolenticular degeneration in TX mice and their mechanism of action on the ferroptosis of HT22 cells induced by CuCl₂, based on the PKCβII/ACSL4/ ALOX5 signaling pathway. TX mice were divided into five groups: control, model, GDL tablet, Fer-1, and Glutathione. HT22 cells were also divided into five groups: control, model, GDL tablet, Fer-1, and GDL tablet + Fer-1. Hematoxylin and eosin staining was used to detect the pathological changes in the hippocampus tissues of the mice. Western blotting was used to detect the expression of PKCβⅡ, ACSL4, and ALOX5 in the hippocampus tissues and HT22 cells of the mice, as well as the expression of SLC7A11 and GPX4 in HT22 cells. The content of Fe²⁺ in the hippocampus tissues of the mice was detected via microassay. The levels of SOD, MDA, and GSH-Px in HT22 cells were detected by microplate assay. Finally, the expression of PKCβⅡ, ACSL4, and ALOX5 mRNA in HT22 cells was detected by quantitative real-time polymerase chain reaction. Compared with the control group, the hippocampus tissues of mice in the model group showed clear damage; the protein expression of PKCβⅡ, ACSL4, and ALOX5 showed a clear increase; the protein expression of SLC7A11 and GPX4 decreased significantly; and Fe²⁺content increased significantly (P<0.05). Compared with the model group, the pathological damage to hippocampus tissues showed improvements in the GDL tablet, Fer-1, and Glutathione group with the effects being noticeable in the GDL tablet group. It was possible to inhibit ferroptosis of HT22 cells in the GDL tablet and Fer-1 group and significantly lower their expression of PKCβⅡ, ACSL4, and ALOX5 protein and mRNA in comparison to the model group(P<0.05). The MDA contentalso decreased significantly (P<0.05) while the SOD activity and the GSH-Px content increased significantly(P<0.05). Thus, GDL tablets can inhibit ferroptosis in hippocampus tissues of TX mice andinhibit ferroptosis induced by CuCl₂ in HT22 cells. Moreover, the ferroptosis mechanism may be related to the down-regulation of the PKCβⅡ, ACSL4, and ALOX5 signaling pathway and the attenuation of intracellular lipid peroxidation.

Key words: Gandouling tablets, hepatolenticular degeneration, PKCβⅡ/ACSL4/ALOX5 signaling pathway, ferroptosis, mechanism of action

中图分类号:

R285

吴博进, 董婷, 闻雨雅, 田丽伟, 赵晨玲. 肝豆灵片通过调控PKCβⅡ/ACSL4/ALOX5信号通路改善肝豆状核变性铁死亡的机制[J]. 山东科学, 2024, 37(4): 34-44.

WU Bojin, DONG Ting, WEN Yuya, TIAN Liwei, ZHAO Chenling. The mechanism by which Gandouling tablets improve ferroptosis in hepatolenticular degeneration through PKCβⅡ/ACSL4/ALOX5 signaling pathway regulation[J]. Shandong Science, 2024, 37(4): 34-44.

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基因名称	引物序列	产物长度/bp
PKCβⅡ	上游5'-ATCGCTGCTGTATGGACTTATTCAC-3'	130
	下游5'-AACACTTGATGGTCTTGGTCTTCTG-3'
ACSL4	上游5'-CCTGAGGGGCTTGAAATTCAC-3'	89
	下游5'-GTTGGTCTACTTGGAGGAACG-3'
ALOX5	上游5'-ACGCATCTGGTGTCTGAGGTG-3'	318
	下游5'-GAGCAGTCCATCATCACGATAGAAG-3'
GPX4	上游5'TGTGCATCCCGCGATGATT-3'	97
	下游5'CCCTGTACTTATCCAGGCAGA-3'
SLC7A11	上游5'GCATTCCCAGGGGCTAACAT-3'	282
	下游5'AATTTCTCCCATGCGGGTGT-3'
GADPH	上游5'GTGTTCCTACCCCCAATGTG-3'	215
	下游5'GTCATTGAGAGCAATGCCAG-3'