| 沈耘,吴佳艺,韩艳艳,等.右美托咪定预处理通过调控 p62/Keap1/Nrf2途径减轻脂多糖诱导的大鼠肝脏氧化应激、炎症和铁死亡[J].安徽医药,2025,29(3):472-481. |
| 右美托咪定预处理通过调控 p62/Keap1/Nrf2途径减轻脂多糖诱导的大鼠肝脏氧化应激、炎症和铁死亡 |
| Dexmedetomidine pretreatment alleviates lipopolysaccharide (LPS)-induced hepatic oxidative stress, inflammation and ferroptosis in rats via the p62/Keap1/Nrf2 signaling pathway |
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| DOI：10.3969/j.issn.1009-6469.2025.03.010 |
| 中文关键词: 肝损伤 右美托咪定 氧化应激 炎症 铁死亡 p62蛋白 /Kelch样 ECH2相关蛋白 1/核因子 E2相关因子 2通路 |
| 英文关键词: Liver injury Dexmedetomidine Oxidative stress Inflammation Ferroptosis p62/Keap1/Nrf2 signaling pathway |
| 基金项目:上海市卫生系统优秀青年医学人才培养项目( PWRq2022-27) |
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| 中文摘要: |
| 目的研究右美托咪定( Dex)对脂多糖( LPS)诱导的肝脏氧化应激、炎症及铁死亡的影响及其可能的机制。方法 2022年 8月至 2023年 6月，腹腔注射 LPS制备大鼠肝损伤模型。 50只雄性 Wistar大鼠按随机数字表法分为五组:对照组、 Dex组、 LPS组、 LPS+Dex组和 LPS+Dex+p62活化抑制剂( XRK3F2)组，每组各 10只。 Kaplan.Meier法进行生存分析；酶联免疫吸附测定( ELISA)大鼠血清中天冬氨酸转氨酶( AST)、丙氨酸转氨酶( ALT)、肿瘤坏死因子 α(TNF-α)、白细胞介素( IL)-1β和 IL-6含量；苏木素 -伊红( HE)染色观察大鼠肝组织的形态学变化；流式细胞术检测肝脏组织中活性氧簇含量；丙二醛、谷胱甘肽和总谷胱甘肽( T-GSH)含量，超氧化物歧化酶( SOD)、过氧化氢酶( CAT)和谷胱甘肽过氧化物酶( GSH-Px)活性及谷胱甘肽 /氧化型谷胱甘肽( GSH/GSSG)比值采用试剂盒进行检测；实时荧光定量 PCR(RT-qPCR)检测炎症因子、铁死亡及信号通路相关分子 mRNA的表达；大鼠肝组织中 Fe2+含量用组织铁测定试剂盒检测；透射电镜观察大鼠肝组织细胞中线粒体形态；蛋白质印迹法检测铁死亡及信号通路相关分子蛋白的表达。结果与对照组相比， LPS组大鼠生存率和体质量明显降低，肝组织湿 /干比及 AST和 ALT酶活力显著增加，肝组织形态发生损坏、炎症和坏死细胞增加；相较于 LPS组， LPS+Dex组大鼠生存率和体质量明显增加，肝水肿、肝功能受损及肝组织病理学损伤明显减轻。与对照组相比， LPS可导致大鼠肝组织中活性氧簇和丙二醛含量增加， SOD、CAT和 GSH-Px酶活性降低，谷胱甘肽和 T-GSH含量和 GSH/GSSG比值减少； Dex预处理则减弱了 LPS诱导的氧化应激参数的改变。与对照组相比， LPS组大鼠血清中 TNF-α、IL-1β和 IL-6含量和肝组织中 TNF-α、IL-1β和 IL-6 mRNA表达升高； Dex则降低了 LPS诱导的炎症因子的表达和释放。与对照组相比， LPS组大鼠肝组织细胞线粒体皱缩、嵴减少、膜密度增加， Fe2+含量增加，谷胱甘肽过氧化物酶 4(GPX4)、溶质载体家族 7成员 11(SLC7A11)和铁蛋白重链(l FTH1)的 mRNA和蛋白表达明显降低，乙酰辅酶 A合成酶长链家族成员 4(ACSL4)和转铁蛋白受体 1(TfR1)的 mRNA和蛋白表达水平显著升高； Dex预处理显著改善线粒体受损并抑制铁死亡。与对照组相比， LPS组大鼠肝组织中 p62表达( 0.44±0.02比 1.00±0.05)减少， Kelch样 ECH2相关蛋白 1(Keap1)(2.98±0.01比 1.00±0.04)和核因子 E2相关因子 2(Nrf2)(2.50±0.06比 1.00±0.03)表达升高； Dex预处理则增加了 p62和核内 Nrf2的表达，减少了 Keap1和胞质内 Nrf2表达。在用 Dex预处理的基础上使用 p62抑制剂( XRK3F2)后，与 LPS+Dex组相比， Dex的抗氧化应激、抗炎症反应及抗铁死亡作用均明显降低。结论 Dex对 LPS诱导的大鼠肝损伤的保护作用与其抗氧化应激、抗炎症反应及抗铁死亡效应有关，其作用机制可能是通过调控 p62/Keap1/Nrf2途径来实现的。 |
| 英文摘要: |
| Objective To explore the effects of dexmedetomidine (Dex) on lipopolysaccharide (LPS)-induced liver oxidative stress, in-flammation, and ferroptosis and its underlying mechanisms.Methods The study was conducted from August 2022 to June 2023, andthe rat model of liver injury was established by intraperitoneal injection of LPS. A total of 50 male Wistar rats were divided into 5groups: control group, Dex group, LPS group, LPS+Dex group, and LPS+Dex+p62 activation inhibitor (XRK3F2) group, each group con-sisting of 10 rats. Kaplan.Meier was performed to analyze survival rate. Enzyme-linked immunosorbent assays (ELISAs) were conduct-ed to measure aspartate transaminase (AST), alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) content in sera of the rats. Hematoxylin-eosin (HE) staining was used to observe the morphological changes ofliver tissue. Reactive oxygen species (ROS) production in the liver was detected by flow cytometry. Malondialdehyde, glutathione and total glutathione (T-GSH) content, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities, as wellas glutathione/reduced glutathione (GSH/GSSG) ratio were measured by commercial kits. The mRNA expressions of inflammatory medi-ators, ferroptosis-related molecules, and signaling pathway-associated molecules were assessed by RT-qPCR analyses. Fe2+ content in the liver was measured by tissue iron assay kit. Transmission electron microscopy was used to observe the mitochondrial morphology.Western blot was conducted to detect the protein levels of ferroptosis-and signal pathway-related molecular proteins in the liver tissues. Results Compared with control group, LPS significantly decreased survival rate and body weight but increased the liver wet/dry ratioand activities of AST and ALT. LPS also led to severe liver damage as shown by increased inflammatory and necrotic cells. Comparedwith LPS group, Dex preconditioning raised survival rate and body weight, alleviated liver edema, liver function impairment and liverhistopathological injury. Compared with control group, LPS resulted in oxidative stress as evidenced by increase in ROS and malondial-dehyde production, decrease in SOD, CAT and GSH-Px activities, glutathione and T-GSH contents and GSH/GSSG ratio. Comparedwith LPS group, Dex pretreatment attenuated the effects of LPS on the alterations of oxidative stress parameters. Compared with controlgroup, LPS increased blood release and mRNA expression in the liver of TNF-α, IL-1β and IL-6, which were counteracted by Dex pre-treatment. In addition, aberrant mitochondria, including reduction even disappearance of mitochondria cristae and rupture of mitochon-drial outer membrane were found in LPS-treated rats. LPS also increased Fe2+ content and the mRNA and protein levels of acyl-CoA synthetase long chain family member 4 (ACSL4) and transferrin receptor 1 (TfR1), but downregulated mRNA and protein expressions ofglutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and ferritin heavy chain (FTH1) in the liver; however,Dex preconditioning significantly improved mitochondrial damage and inhibited ferroptosis. Mechanically, LPS downregulated p62(0.44±0.02 vs. 1.00±0.05) expression but upregulated levels of Kelch-like ECH2 associated protein 1 (Keap1) (2.98±0.01 vs. 1.00± 0.04)] and nuclear factor E2 related factor 2 (Nrf2) (2.50±0.06 vs. 1.00±0.03); nevertheless, Dex led to increase in p62 expression andnucleus levels of Nrf2 and decrease in the levels of Keap1 and Nrf2 in cytosol. Addition of p62 inhibitor (XRK3F2) significantly attenu-ated the anti-oxidative, anti-inflammatory and anti-ferroptosis effects of Dex on LPS-induced liver injury.Conclusion The protection of Dex preconditioning on LPS-induced rat liver damage may be related to its inhibitory effects on oxidative stress, inflammation andferroptosis, involving in the activation of p62/Keap1/Nrf2 signaling pathway. |
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