Sijunzi Decoction Reverses Metabolic Adaptation and Induces Ferroptosis in Cisplatin-Resistant Non–small Cell Lung Cancer: An Integrative Metabolomics–Pharmacology Analysis

Abstract

Objective

To investigate the mechanistic role of Sijunzi decoction (SJZD) in overcoming chemoresistance through the suppression of adaptive metabolic responses in non–small cell lung cancer (NSCLC).

Methods

Chemical profiling of SJZD-derived components in systemic circulation was conducted using liquid chromatography–tandem mass spectrometry (LC‒MS/MS) in Sprague–Dawley rats. Multiomics integration and network pharmacology were employed to identify convergent targets shared by the bioactive constituents of SJZD and genes associated with cisplatin resistance. In vitro functional assessments using cisplatin-resistant human lung adenocarcinoma (A549/DDP) cells included the following: quantification of cell viability via Cell Counting Kit-8 (CCK-8) assays; evaluation of mitochondrial bioenergetics through targeted metabolomic profiling; and ultrastructural characterization of ferroptotic morphology via transmission electron microscopy (TEM). Cellular redox homeostasis was dynamically monitored using fluorescent probes, including a DCFH-DA probe for reactive oxygen species (ROS) and a C11-BODIPY^581/591 probe for lipid peroxidation. siRNA-mediated gene silencing and immunohistochemical analysis were performed to elucidate the functional hierarchy of the p62/Keap1/nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant axis. Complementary in vivo validation was performed using BALB/c nude mice bearing A549/DDP xenografts, with longitudinal monitoring of tumor progression under SJZD treatment regimens.

Results

Untargeted metabolomics of SJZD-medicated serum revealed 392 differentially abundant metabolites, with pathway enrichment revealing significant dysregulation of glutamine metabolism. Structural validation confirmed 55 bioactive components of SJZD in serum, including glycyrrhizin, ginsenoside Ro, liquiritigenin, and atractylenolide I. Integration of these components with disease targets yielded 355 overlapping genes associated with both SJZD activity and cisplatin-resistant NSCLC, with significant enrichment in oxidative stress response pathways. Experimental assays confirmed that SJZD induced ferroptosis in cisplatin-resistant A549/DDP cells, as evidenced by disrupted iron homeostasis, lipid peroxidation, and characteristic mitochondrial damage. These effects and subsequent cell death were specifically abrogated by the ferroptosis inhibitor ferrostatin-1 (Fer-1) but not by apoptosis inhibition, confirming that ferroptosis is the primary mechanism of cell death. Mechanistically, the inhibition of p62/Keap1/Nrf2 signaling was involved in the modulation of SJZD-induced ferroptosis both in vitro and in vivo.

Conclusions

SJZD counteracts metabolic adaptation through ferroptosis mediated by the inhibition of p62/Keap1/Nrf2 in cisplatin-resistant NSCLC.

Keywords

Cisplatin resistance / Non-small cell lung cancer / Sijunzi decoction / Metabolic reprogramming / Adaptive response / Ferroptosis

References

[1] Sung H, Ferlay J, Siegel RL, et al.. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin., 2021, 71(3): 209-249

[2] Olaizola I, Odriozola-Gimeno M, Olaizola P, et al.. New platinum derivatives selectively cause double-strand DNA breaks and death in naïve and cisplatin-resistant cholangiocarcinomas. J Hepatol., 2025, 83(5): 1077-1091

[3] Wangpaichitr M, Theodoropoulos G, Nguyen DJM, et al.. Cisplatin resistance and redox metabolic vulnerability: A second alteration. Int J Mol Sci., 2021, 22(14): 7379

[4] Bock FJ, Tait SWG. Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell Biol., 2020, 21(2): 85-100

[5] Pavlova NN, Zhu J, Thompson CB. The hallmarks of cancer metabolism: Still emerging. Cell Metab., 2022, 34(3): 355-377

[6] Pan M, Reid MA, Lowman XH, et al.. Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation. Nat Cell Biol., 2016, 18(10): 1090-1101

[7] McGuirk S, Audet-Delage Y, St-Pierre J. Metabolic fitness and plasticity in cancer progression. Trends Cancer., 2020, 6(1): 49-61

[8] Obrist F, Michels J, Durand S, et al.. Metabolic vulnerability of cisplatin-resistant cancers. EMBO J., 2018, 37(14): Article ID: e98597

[9] Kim J, DeBerardinis RJ. Mechanisms and implications of metabolic heterogeneity in cancer. Cell Metab., 2019, 30(3): 434-446

[10] Liu WJ, Pan PY, Sun Y, et al.. Deferoxamine counteracts cisplatin resistance in A549 lung adenocarcinoma cells by increasing vulnerability to glutamine deprivation-induced cell death. Front Oncol., 2022, 11 Article ID: 794735

[11] Wang H, Fleishman JS, Cheng S, et al.. Epigenetic modification of ferroptosis by non-coding RNAs in cancer drug resistance. Mol Cancer., 2024, 23(1): 177

[12] Wang Y, Hu J, Wu S, et al.. Targeting epigenetic and posttranslational modifications regulating ferroptosis for the treatment of diseases. Signal Transduct Target Ther., 2023, 8(1): 449

[13] Wang Y, Hu J, Fleishman JS, et al.. Inducing ferroptosis by traditional medicines: a novel approach to reverse chemoresistance in lung cancer. Front Pharmacol., 2024, 15: 1290183

[14] Dixon SJ, Lemberg KM, Lamprecht MR, et al.. Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell., 2012, 149(5): 1060-1072

[15] Jiang X, Stockwell BR, Conrad M. Ferroptosis: Mechanisms, biology and role in disease. Nat Rev Mol Cell Biol., 2021, 22(4): 266-282

[16] Mao C, Liu X, Zhang Y, et al.. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer. Nature., 2021, 593(7860): 586-590

[17] Li X, Zhao L, Wang J, et al.. The Mechanism of Sijunzi Decoction Suppresses Gastric Cancer Metastasis via the m6A Methyltransferase METTL14 Based on Untargeted Metabolomics Studies and Network Pharmacology Analysis. Drug Des Devel Ther., 2025, 19: 2369-2392

[18] Shang L, Wang Y, Li J, et al.. Mechanism of Sijunzi Decoction in the treatment of colorectal cancer based on network pharmacology and experimental validation. J Ethnopharmacol., 2023, 302(Pt A): Article ID: 115876

[19] Wang X, Pan S, Chen L, et al.. Sijunzi decoction enhances sensitivity of colon cancer cells to NK cell destruction by modulating P53 expression. J Ethnopharmacol., 2024, 329 Article ID: 118115

[20] Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm., 2016, 7(2): 27-31

[21] Chinese Pharmacopoeia CommissionPharmacopoeia of the People’s Republic of China [S], 20202020BeijingChina Medical Science Press

[22] Sun Y, Liu WJ, Zhao QY, et al.. Down-Regulating the Expression of miRNA-21 Inhibits the Glucose Metabolism of A549/DDP Cells and Promotes Cell Death Through the PI3K/AKT/mTOR/HIF-1α Pathway. Front Oncol., 2021, 11 Article ID: 653596

[23] Shang RX, Xu WD, Xu XQ, et al.. Analysis of the major metabolites of Sijunzi decoction in rat serum based on HPLC-MS. J Jiangsu Univ (Medicine Edition), 2018, 28(6): 529-533

[24] Wang ZH, Li TF, Li RS, et al.. Sijunzi Tang improves gefitinib resistance by regulating glutamine metabolism. Biomed Pharmacother., 2023, 167 Article ID: 115438

[25] Eniafe J, Jiang S. The functional roles of TCA cycle metabolites in cancer. Oncogene., 2021, 40(19): 3351-3363

[26] Tong WH, Rouault TA. Functions of mitochondrial ISCU and cytosolic ISCU in mammalian iron-sulfur cluster biogenesis and iron homeostasis. Cell Metab., 2006, 3(3): 199-210

[27] Jelic MD, Mandic AD, Maricic SM, et al.. Oxidative stress and its role in cancer. J Cancer Res Ther., 2021, 17(1): 22-28

[28] Sánchez-García FJ, Pérez-Hernández CA, Rodríguez-Murillo M, et al.. The Role of Tricarboxylic Acid Cycle Metabolites in Viral Infections. Front Cell Infect Microbiol., 2021, 11 Article ID: 725043

[29] Mainali R, Zabalawi M, Long D, et al.. Dichloroacetate reverses sepsis-induced hepatic metabolic dysfunction. Elife., 2021, 10 Article ID: e64611

[30] Du J, Zhou Y, Li Y, et al.. Identification of Frataxin as a regulator of ferroptosis. Redox Biol., 2020, 32 Article ID: 101483

[31] Gorrini C, Harris IS, Mak TW. Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discovery., 2013, 12(12): 931-947

[32] Best SA, De Souza DP, Kersbergen A, et al.. Synergy between the KEAP1/NRF2 and PI3K pathways drives non-small-cell lung cancer with an altered immune microenvironment. Cell Metab., 2018, 27(4): 935-943.e934

[33] Zhao Y, Lu J, Mao A, et al.. Autophagy inhibition plays a protective role in ferroptosis induced by alcohol via the p62–Keap1–Nrf2 pathway. J Agric Food Chem., 2021, 69(33): 9671-9683

[34] Singh A, Misra V, Thimmulappa RK, et al.. Dysfunctional KEAP1–NRF2 interaction in non-small-cell lung cancer. PLoS Med., 2006, 3(10): Article ID: e420

[35] Fan Z, Wirth AK, Chen D, et al.. Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis. Oncogenesis., 2017, 6(8): Article ID: e371

[36] Best SA, Ding S, Kersbergen A, et al.. Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma. Nat Commun., 2019, 10(1): 4190

[37] Romero R, Sayin VI, Davidson SM, et al.. Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis. Nat Med., 2017, 23(11): 1362-1368

[38] Mitsuishi Y, Taguchi K, Kawatani Y, et al.. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell., 2012, 22(1): 66-79

[39] Wang J, Lu Q, Cai J, et al.. Nestin regulates cellular redox homeostasis in lung cancer through the Keap1–Nrf2 feedback loop. Nat Commun., 2019, 10(1): 5043

[40] Tirumalai R, Rajesh Kumar T, Mai KH, et al.. Acrolein causes transcriptional induction of phase II genes by activation of Nrf2 in human lung type II epithelial (A549) cells. Toxicol Lett., 2002, 132(1): 27-36

[41] Wang ZH. Study on the mechanism of Sijunzi Tang improving gefitinib resistance by regulating glutamine metabolism [Master’s thesis]. Peking Union Medical College; 2022.

[42] Li C, Niu M, Wang R, et al.. The modulatory properties of Si Jun Zi Tang enhancing anticancer of gefitinib by an integrating approach. Biomed Pharmacother., 2019, 111: 1132-1140

[43] Zhao W, Liu Z, Zhang Z, et al.. Si Jun Zi decoction inhibits the growth of lung cancer by reducing the expression of PD-L1 through TLR4/MyD88/NF-κB pathway. J Ethnopharmacol., 2024, 318(Pt A): Article ID: 116948

[44] Zhan L, Zhang H, Zhang Q, et al.. Regulatory role of KEAP1 and NRF2 in PPARγ expression and chemoresistance in human non-small-cell lung carcinoma cells. Free Radical Biol Med., 2012, 53(4): 758-768

Funding

National Natural Science Foundation of China(82174254)

RIGHTS & PERMISSIONS

The Author(s)