目的 探讨亚精胺对急性肺损伤（acute lung injury,ALI）小鼠肺组织细胞外基质（extracellular matrix,ECM）重塑的影响及调控机制。方法 构建ALI小鼠模型;采用Masson染色观察肺组织胶原沉积情况;采用Real time-PCR和Western blot分别检测肺组织和巨噬细胞中核因子红系2相关因子（Nuclear factor erythroid-2 related factor 2,Nrf2）、Kelch 样ECH 相关蛋白1（Kelch-like ECH associated protein 1,Keap1）、血红素加氧酶-1（Heme oxygenase-1,HO-1）、磷酸肌醇3-激酶（phosphoinositide 3-kinase,PI3K）、磷酸化的PI3K（phosphorylated PI3K,p-PI3K）、有丝分裂原激活的蛋白激酶激酶1（mitogen-activated protein extracellular signal-regulated kinase kinase 1,MEK1）、磷酸化细胞外信号调节激酶1/2（Phosphorylated Extracellular Signal-Regulated Kinase 1/2,p-ERK1/2）、基质金属蛋白酶-2（matrix metalloproteinase-2,MMP2）、MMP9、转化生长因子-β（transforming growth factor β,TGF-β）、基质金属蛋白酶抑制剂-1（Tissue Inhibitor of Metalloproteinases-1,TIMP-1）、Smad2/3、I型胶原蛋白（collagenI） mRNA和蛋白的表达。结果 （1）Masson染色结果显示与亚精胺+ALI组相比,采用Nrf2激动剂预处理后的ALI组小鼠肺组织的胶原沉积明显减少。PCR结果显示亚精胺可降低ALI肺组织中Mmp2、Mmp9、Tgf-β mRNA的表达,升高Timp-1mRNA的表达;使用Nrf2抑制剂预处理后,ALI小鼠肺组织Mmp2、Mmp9、Tgf-β mRNA的表达增加,而TIMP-1mRNA的表达降低;而使用Nrf2激动剂,可降低肺组织Mmp9 mRNA的表达,增加Timp-1mRNA的表达。（2）亚精胺可增加ALI时肺组织PI3K和MEK蛋白;亚精胺可增加脂多糖（lipopolysaccharide,LPS）诱导的巨噬细胞PI3K、p-PI3K、MEK1、P-ERK1/2的蛋白表达。与亚精胺+LPS组相比较,采用PI3K抑制剂后,亚精胺预处理LPS诱导的巨噬细胞中Nrf2表达降低,Keap1的表达升高,HO-1的表达降低;而使用MAPK抑制剂,LPS诱导的巨噬细胞中Nrf2、HO-1的表达上调;Keap1的表达降低。结论 亚精胺通过PI3K、MAPK等信号通路激活Nrf2-Keap1功能轴,抑制巨噬细胞激活,使细胞外基质生成减少,最终可抑制ALI小鼠肺组织病理性ECM重塑。
Abstract
Objective To investigate the effect and regulatory mechanism of spermidine on extracellular matrix (ECM) remodeling in the lung tissue of acute lung injury (ALI) mice. Method An ALI mouse model was constructed. Masson staining was used to observe collagen deposition in lung tissue. Real-time PCR and Western blot were employed to detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2), Keap1, HO-1, PI3K, p-PI3K, MEK1, p-ERK1/2, MMP2, MMP9, TGF-β, TIMP-1, Smad2/3, and collagen I mRNA and protein in lung tissue and macrophages, respectively. Results (1) Masson staining results showed that compared with the spermidine+ALI group, the collagen deposition in the lung tissue of ALI mice pretreated with an Nrf2 agonist was significantly reduced. PCR results demonstrated that spermidine could reduce the expression of Mmp2, Mmp9, and Tgf-β mRNA in ALI lung tissue while increasing Timp-1 mRNA expression. After pretreatment with Nrf2 inhibitors, the expression of Mmp2, Mmp9, and Tgf-β mRNA in the lung tissue of ALI mice increased, whereas Timp-1 mRNA expression decreased. The use of Nrf2 agonists reduced Mmp9 mRNA expression in lung tissue and increased TIMP-1 mRNA expression. (2) Spermidine was found to increase PI3K and MEK protein levels in lung tissue during ALI. In LPS-induced macrophages, spermidine increased the protein expression of PI3K, p-PI3K, MEK1, and p-ERK1/2. Compared with the spermidine+LPS group, the use of PI3K inhibitors resulted in decreased Nrf2 expression, increased Keap1 expression, and decreased HO-1 expression in LPS-induced macrophages pretreated with spermidine. Furthermore, the use of MAPK inhibitors attenuated the LPS-induced upregulation of Nrf2 and HO-1 expression in macrophages, accompanied by decreased Keap1 expression. Conclusion Spermidine, through PI3K and MAPK signaling pathways, activates the Nrf2-Keap1 functional axis, thereby inhibiting macrophage activation, leading to decreased matrix production, ultimately inhibiting pathological ECM remodeling in the lung tissue of ALI mice.
关键词
急性肺损伤 /
细胞外基质 /
核因子-红细胞相关因子2 /
亚精胺
Key words
acute lung injury /
extracellular matrix /
nuclear factor-erythroid 2 related factor 2 /
spermidine
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参考文献
[1] MEYER NJ, GATTINONI L, CALFEE CS.Acute respiratory distress syndrome[J]. Lancet, 2021, 398(10300): 622-37.
[2] BOS LDJ, WARE LB.Acute respiratory distress syndrome: causes, pathophysiology, and phenotypes[J]. Lancet, 2022, 400(10358): 1145-1156.
[3] RUMENDE CM, SUSANTO EC, SITORUS TP.The Management of Pulmonary Fibrosis in COVID-19[J]. Acta Med Indones, 2021, 53(2): 233-241.
[4] LIU X, ZHANG JQ, XIE W.The role of ferroptosis in acute lung injury[J]. Mol Cell Biochem, 2022, 477(5): 1453-1461.
[5] KOPACZ A, KLOSKA D, FORMAN HJ, et al.Beyond repression of Nrf2: an update on Keap1[J]. Free Radic Biol Med, 2020, 157: 63-74.
[6] SUZUKI T, TAKAHASHI J, YAMAMOTO M.Molecular basis of the KEAP1-Nrf2 signaling pathway[J]. Mol Cells, 2023, 46(3): 133-141.
[7] AUDI SH, JACOBS ER, TAHERI P, et al.Assessment of protection offered by the Nrf2 pathway against hyperoxia-induced acute lung injury in Nrf2 knockout rats[J]. Shock, 2022, 57(2): 274-280.
[8] WANG Y, MA J, JIANG Y.Transcription factor Nrf2 as a potential therapeutic target for COVID-19[J]. Cell Stress Chaperones, 2023, 28(1): 11-20.
[9] RODRIGUES D, MACHADO M R, ALVES J V, et al.Cytokine storm in individuals with severe COVID-19 decreases endothelial cell antioxidant defense via downregulation of the Nrf2 transcriptional factor[J]. Am J Physiol Heart Circ Physiol, 2023, 325(2): H252-H263.
[10] DE ALMEIDA LGN, THODE H, ESLAMBOLCHI Y, et al.Matrix Metalloproteinases: from molecular mechanisms to physiology, pathophysiology, and pharmacology[J]. Pharmacol Rev, 2022, 74(3): 712-768.
[11] LIANG W, YAMAHARA K, HERNANDO-ERHARD C, et al.A reciprocal regulation of spermidine and autophagy in podocytes maintains the filtration barrier[J]. Kidney Int, 2020, 98(6): 1434-1448.
[12] SHI B, WANG W, YE M, et al.Spermidine suppresses the activation of hepatic stellate cells to cure liver fibrosis through autophagy activator MAP1S[J]. Liver Int, 2023, 43(6): 1307-1319.
[13] 粟青. 亚精胺对脂多糖诱导的小鼠急性肺损伤的影响[D]. 湖南师范大学, 2020.
[14] 孙国瑛. ALI时肺内TREMs家族表达谱的变化、TREM-2的保护作用及血管活性肠肽的调控[D]. 中南大学, 2012.
[15] ORONSKY B, LARSON C, HAMMOND TC, et al.A Review of Persistent Post-COVID Syndrome (PPCS)[J]. Clin Rev Allergy Immunol, 2023, 64(1): 66-74.
[16] SHA JF, XIE QM, CHEN N, et al.TLR2-hif1alpha-mediated glycolysis contributes to pyroptosis and oxidative stress in allergic airway inflammation[J]. Free Radic Biol Med, 2023, 200: 102-116.
[17] HONG H, LOU S, ZHENG F, et al.Hydnocarpin D attenuates lipopolysaccharide-induced acute lung injury via MAPK/NF-kappaB and Keap1/Nrf2/HO-1 pathway[J]. Phytomedicine, 2022, 101: 154143.
[18] WU YX, ZHANG YR, JIANG FJ, et al.4-OI ameliorates bleomycin-induced pulmonary fibrosis by activating Nrf2 and suppressing macrophage-mediated epithelial-mesenchymal transition[J]. Inflamm Res, 2023, 72(6): 1133-1145.
[19] ALMUNTASHIRI S, ALHUMAID A, ZHU Y, et al.TIMP-1 and its potential diagnostic and prognostic value in pulmonary diseases[J]. Chin Med J Pulm Crit Care Med, 2023, 1(2): 67-76.
[20] SUN K, LI X, SCHERER P E. Extracellular Matrix (ECM) and Fibrosis in Adipose Tissue: Overview and Perspectives[M]. Comprehensive Physiology, 2023: 4387-4407.
[21] GAO Y, DAI W, OUYANG Z, et al.Dendrimer-mediated intracellular delivery of fibronectin guides macrophage polarization to alleviate acute lung injury[J]. Biomacromolecules, 2023, 24(2): 886-895.
基金
湖南省卫生健康委科研项目“亚精胺对LPS诱导的急性肺损伤小鼠肺内Keap1/Nrf2/ARE功能轴的影响”（D202301019729）; 湖南省自然科学基金面上项目“TREM-2通过PI3K/mTORC1/HIF-1ɑ信号通路下调肺内巨噬细胞糖代谢在急性肺损伤中的作用及机制研究”（2025JJ50623）; 2025年湖南师范大学创新创业训练计划项目（2025285）
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