文献类型：期刊文献

中文题名：党参经PI3K/Akt干预溃疡性结肠炎黏膜细胞铁死亡-线粒体动力学失衡的机制研究

英文题名：Intervention of Codonopsis Radix on ferroptosis and mitochondrial dynamics imbalance in mucosal cells of ulcerative colitis via PI3K/Akt

作者：李芳[1];陈正君[1];葛俊李[1];王春霞[1];梁建庆[2];冯翠娟[3];杨扶德[1]

第一作者：李芳

机构：[1]甘肃中医药大学药学院,甘肃兰州730000;[2]甘肃中医药大学基础医学院,甘肃兰州730000;[3]甘肃卫生职业学院中医药学院,甘肃兰州730300

第一机构：甘肃中医药大学药学院（西北中藏药协同创新中心办公室）

年份：2023

卷号：54

期号：12

起止页码：3865

中文期刊名：中草药

外文期刊名：Chinese Traditional and Herbal Drugs

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD2023_2024】；

基金：国家重点研发计划(2018YFC1706305);甘肃省自然科学基金项目(22JR5RA590);甘肃省教育科技创新项目(2022A-064,2023A-297)。

语种：中文

中文关键词：党参;溃疡性结肠炎;PI3K/Akt;氧化应激;线粒体动力学失衡;铁死亡;党参炔苷;苍术内酯Ⅲ

外文关键词：Codonopsis Radix;ulcerative colitis;PI3K/Akt;oxidative stress;mitochondrial dynamics imbalance;ferroptosis;lobetyolin;atractylenolideⅢ

摘要：目的探究党参经磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)调控Kelch样环氧氯丙烷相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)/核因子E相关因子2(nuclear factor erythroid-2related factor 2,Nrf2)/谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、线粒体动力相关蛋白1(dynamin-related protein1,DRP1)信号通路抑制氧化应激干预溃疡性结肠炎(ulcerative colitis,UC)肠黏膜细胞铁死亡-线粒体动力学失衡的分子机制。方法采用网络药理学京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析预测党参干预UC的关键信号通路。SD大鼠采用2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzene sulphonic acid,TNBS)-乙醇复合方法制备UC模型,随机分为对照组、模型组、柳氮磺胺吡啶(0.3 g/kg)组、铁抑制剂Ferrostatin-1(0.8 mg/kg)组和党参高、中、低剂量(18、9、4.5 g/kg)组,每组12只;给予药物连续干预7 d,收集各组大鼠血清、结肠组织。采用苏木素-伊红(HE)染色进行组织病理观察及结肠黏膜组织学损伤评分(tissue damage index,TDI);ELISA法检测血清中Fe^(2+)、白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、IL-8、GPX4、C-反应蛋白(C-reactive protein,CRP)、D-乳酸含量;生化法检测血清中超氧化物歧化酶(superoxide dismutase,SOD)活力及丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutathione,GSH)含量,检测结肠组织三磷酸腺苷(adenosine triphosphate,ATP)含量及髓过氧化物酶(myeloperoxidase,MPO)、Ca^(2+),Mg^(2+)-ATP酶、Na^(+),K^(+)-ATP酶、ATP酶活力。转录测序分析对照组、模型组、党参高剂量组结肠组织差异表达基因,并进行KEGG通路富集分析获得党参干预UC差异表达信号通路。Western blotting检测结肠组织p-PI3K、PI3K、p-Akt、Akt、Keap1、Nrf2、GPX4、重组人铁蛋白重链(recombinant Human Ferritin heavy chain,FTH1)、DRP1和线粒体转运蛋白(mitochondrial Rho-GTP,MIRO)蛋白表达;qRT-PCR检测结肠组织PI3K、Akt、Keap1、Nrf2、GPX4、FTH1 m RNA表达。结果病理观察及评分结果显示党参有效改善了结肠组织的炎症水肿状态;网络药理学预测结果表明党参干预UC关键靶点显著富集于PI3K/Akt信号通路。ELISA实验结果表明党参可有效降低UC模型大鼠血清中IL-1β、IL-6、IL-8、TNF-α、D-乳酸、CRP水平(P<0.05、0.01、0.001),抑制炎症;降低血清Fe^(2+)、MDA含量及MPO活力(P<0.001),升高血清GPX4、GSH水平及SOD活力(P<0.001),抑制氧化应激;升高结肠组织ATP含量及ATP酶、Ca^(2+),Mg^(2+)-ATP酶、Na^(+),K^(+)-ATP酶活力(P<0.05、0.01、0.001),提升能量代谢。转录组学KEGG通路分析提示对照组vs模型组差异基因及党参高剂量组vs模型组差异基因均显著富集于PI3K/Akt等信号通路。Western blotting实验结果确证党参可有效下调结肠组织p-PI3K/PI3K、p-Akt/Akt、Keap1、MIRO和DRP1蛋白表达(P<0.05、0.01、0.001),上调Nrf2、FTH1和GPX4蛋白表达(P<0.05、0.01、0.001),发挥抗氧化应激、抑制铁死亡、调节线粒体动力学作用。qRT-PCR实验结果亦表明党参可有效下调结肠组织PI3K、Akt、Keap1基因表达(P<0.05、0.001),上调Nrf2、FTH1、GPX4基因表达(P<0.05、0.01、0.001)。结论党参是干预UC的有效药物,其机制可能与经PI3K/Akt干预Keap1/Nrf2/GPX4、DRP1信号通路抑制肠黏膜细胞线粒体动力学失衡-铁死亡氧化应激损伤有关。
Objective To explore the mechanism of Dangshen(Codonopsis Radix)on intervening intestinal mucosal cells ferroptosis-mitochondrial dynamics imbalance in ulcerative colitis(UC)via Kelch like ECH-associated protein 1(Keap1)/nuclear factor E-related factor 2(Nrf2)/glutathione peroxidase 4(GPX4)and mitochondrial related protein 1(DRP1)signaling pathway regulated by phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt).Methods Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis in network pharmacology was used to predict the key signaling pathways of Codonopsis Radix intervention in UC.SD rats were used to prepare UC models by 2,4,6-trinitrobenzene sulfonic acid(TNBS)-ethanol composite method,rats were randomly divided into control group,model group,sulfasalazine tables(0.3 g/kg)group,iron inhibitor Ferrostatin-1(0.8 mg/kg)group,and Codonopsis Radix high-,medium-,and low-dose(18,9,4.5 g/kg)groups,with 12 rats in each group;Drugs were given for 7 d,serum and colon tissue from each group of rats were collected.Histopathological observation and histological damage index(TDI)of colon mucosa were performed by HE staining;ELISA was used to detect Fe^(2+),interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),IL-8,GPX4,C-reactive protein(CRP),D-lactate contents in serum;Superoxide dismutase(SOD)activity and contents of malondialdehyde(MDA),glutathione(GSH)in serum were detected,adenosine triphosphate(ATP)content and activities of myeloperoxidase(MPO),Ca^(2+),Mg^(2+)-ATPase,Na^(+),K^(+)-ATPase and ATPase in colon tissue were detected by biochemical method.Differentially expressed genes in colon tissues of control group,model group,and Codonopsis Radix high-dose group were analyzed by transcription sequencing,and KEGG pathway enrichment analysis were performed to obtain the differentially expressed signal pathway of Codonopsis Radix intervention in UC.Western blotting was used to detect the expressions of p-PI3K,PI3K,p-Akt,Akt,Keap1,Nrf2,GPX4,recombinant human ferritin heavy chain(FTH1),DRP1 and mitochondrial Rho GTP(MIRO)proteins in colon tissue;qRT-PCR was used to detect the mRNA expressions of PI3K,Akt,Keap1,Nrf2,GPX4 and FTH1 in colon tissue.Results The pathological observation and scoring results showed that Codonopsis Radix effectively improved the inflammatory and edema state of colon tissue;The network pharmacology prediction results indicated that the key targets of Codonopsis Radix intervention in UC were significantly enriched in PI3K/Akt signaling pathway.The results of ELISA experiments indicated that Codonopsis Radix effectively reduced IL-1β,IL-6,IL-8,TNF-α,D-lactate and CRP levels in serum of UC model rats to inhibit inflammation(P<0.05,0.01,0.001),reduced Fe^(2+),MDA contents and MPO activity in serum(P<0.001),increased GPX4,GSH levels and SOD activity in serum(P<0.001),and inhibited oxidative stress;Codonopsis Radix elevated the ATP content and activities of ATPase,Ca^(2+),Mg^(2+)-ATPase,Na^(+),K^(+)-ATPase in colon tissue(P<0.05,0.01,0.001),and enhanced energy metabolism.The analysis of KEGG pathway in transcriptome suggested that the differential genes in control group vs model group and Codonopsis Radix high-dose group vs model group were significantly enriched in PI3K/Akt and other signal pathways.The results of Western blotting experiments confirmed that Codonopsis Radix effectively downregulated the expressions of p-PI3K/PI3K,p-Akt/Akt,Keap1,MIRO and DRP1 proteins in colon tissue(P<0.05,0.01,0.001),and upregulated the expressions of Nrf2,FTH1 and GPX4 proteins(P<0.05,0.01,0.001),exerting antioxidant stress,inhibiting iron death,and regulating mitochondrial dynamics.qRT-PCR experiment results also showed that Codonopsis Radix effectively downregulated the expressions of PI3K,Akt and Keap1 genes in colon tissue(P<0.05,0.001),and upregulated the expressions of Nrf2,FTH1 and GPX4 genes(P<0.05,0.01,0.001).Conclusion Codonopsis Radix is an effective drug for intervention in UC,and its mechanism may be related to PI3K/Akt intervention in Keap1/Nrf2/GPX4,DRP1 signaling pathways to inhibit mitochondrial dynamic imbalance-ferroptosis oxidative stress injury in intestinal mucosal cells.