Andrographolide blocked the progression of endometriosis by promoting ferroptosis via inhibiting anabolism of serine

Ferroptosis is a potential target for the treatment of endometriosis (EMs). The role of andrographolide (AP) in the ferroptosis has gradually attracted attention, but its mechanism of action in endometriosis has not been clarified. Here we investigated the inhibitory effect of AP on endometriosis and its mechanism. Our results showed that AP treatment inhibited progression of ectopic endometrial 12Z cells. We also demonstrated that AP treatment induced ferroptosis and apoptosis in 12Z cells. Combined metabolomics and transcriptomic analysis indicated that AP might exert its effect by down-regulating PSAT1 and PHGDH to inhibit serine synthesis. Knockdown of PSAT1 expression elevated ferroptosis level and blocked progression of 12Z cells; however, the ferroptosis inhibitor Ferrostatin-1 (Fer-1), phosphoserine, or serine mitigated these phenotypes. We also observed that AP inhibited the EMs progression and either PSAT1 or PHGDH level in mice model. Finally, AP exhibited inhibitory effects on the viability of ectopic endometrial stromal cells (EESCs) and the expression of PSAT1, as well as induced ferroptosis in EESCs. Overall, AP is a new potential drug for the treatment of endometriosis. Similar content being viewed by others Data availability Upon reasonable request, the corresponding author will provide access to any data used or analyzed during this study. Abbreviations - EMs : - Endometriosis - AP : - Andrographolide - Acsl4 : - Acyl-CoA synthetase long-chain family member 4 - GPX4 : - Glutathione peroxidase 4 - C-Caspase 3 : - Cleaved-caspase 3 - C-PARP : - Cleaved-PARP - CCK-8 : - Cell counting kit—8 - Fer-1 : - Ferrostatin-1 - MDA : - Malondialdehyde - ROS : - Reactive oxygen species - LDH : - Lactate dehydrogenase - LPO : - Lipid peroxidation - SiRNA : - Small interfering RNA - SiNC : - Small interfering RNA of negative control - SiPAST1 : - Small interfering RNA of PAST1 - 8-OHdG : - 8-Hydroxydeoxyguanosine - Ser : - Serine - P-Ser : - O-Phospho-l-serine - E2 : - 17β-Estradiol

Allaire C, Bedaiwy MA, Yong PJ (2023) Diagnosis and management of endometriosis. Can Med Assoc J 195(10):E363–E371. https://doi.org/10.1503/cmaj.220637 Chen WF, Tsai SC, Zhang YH, Chang HM, Wu WJ, Su JH, Wu BN, Chen CY, Lin MY, Chen HL, Lee CH (2024) Rhopaloic acid A triggers mitochondria damage-induced apoptosis in oral cancer by JNK/BNIP3/Nix-mediated mitophagy. Phytomedicine 132:155855. https://doi.org/10.1016/j.phymed.2024.155855 Dixon SJ, Olzmann JA (2024) The cell biology of ferroptosis. Nat Rev Mol Cell Biol 25(6):424–442. https://doi.org/10.1038/s41580-024-00703-5 Dong X, Xu L, Wang S, Jiao X, Yan S, Huang Y, Yuan M, Wang G (2023) Endometrial stromal cell autophagy-dependent ferroptosis caused by iron overload in ovarian endometriosis is inhibited by the ATF4-xCT pathway [Journal Article]. Mol Hum Reprod 30(1):10.1093/molehr/gaad046 Du C, Liu C, Yu K, Zhang S, Fu Z, Chen X, Liao W, Chen J, Zhang Y, Wang X, Chen M, Chen F, Shen M, Wang C, Chen S, Wang S, Wang J (2024) Mitochondrial serine catabolism safeguards maintenance of the hematopoietic stem cell pool in homeostasis and injury. Cell Stem Cell 31(10):1484–1500. https://doi.org/10.1016/j.stem.2024.07.009 Jiang X, Stockwell BR, Conrad M (2021) Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22(4):266–282. https://doi.org/10.1038/s41580-020-00324-8 Jiang X, Peng Q, Peng M, Oyang L, Wang H, Liu Q, Xu X, Wu N, Tan S, Yang W, Han Y, Lin J, Xia L, Tang Y, Luo X, Dai J, Zhou Y, Liao Q (2024) Cellular metabolism: a key player in cancer ferroptosis. Cancer Commun 44(2):185–204. https://doi.org/10.1002/cac2.12519 Jiaqi L, Siqing H, Qin W, di Z, Bei Z, Jialin Y (2023) Andrographolide promoted ferroptosis to repress the development of non-small cell lung cancer through activation of the mitochondrial dysfunction [Journal Article]. Phytomedicine, 109, 154601. https://doi.org/10.1016/j.phymed.2022.154601 Koppula P, Zhuang L, Gan B (2021) Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein & Cell 12(8):599–620. https://doi.org/10.1007/s13238-020-00789-5 Kuang M, Cai L, Zhao J, Huang L, Ji Y, Lv B, Kuang W (2023) Identification of potential ferroptosis hub genes in acute-on-chronic liver failure based on bioinformatics analysis and experimental verification. BMC Med Genomics 16(1):52. https://doi.org/10.1186/s12920-023-01480-4 Li Y, Zeng X, Lu D, Yin M, Shan M, Gao Y (2021) Erastin induces ferroptosis via ferroportin-mediated iron accumulation in endometriosis [Journal Article]. Hum Reprod 36(4):951–964. https://doi.org/10.1093/humrep/deaa363 Li W, Fu H, Fang L, Chai H, Ding B, Qian S (2023) Andrographolide induced ferroptosis in multiple myeloma cells by regulating the P38/Nrf2/HO-1 pathway [Journal Article; Research Support, Non-U.S. Gov’t]. Arch Biochem Biophys 742:109622. https://doi.org/10.1016/j.abb.2023.109622 Li R, Wei R, Liu C, Zhang K, He S, Liu Z, Huang J, Tang Y, An Q, Lin L, Gan L, Zhao L, Zou X, Wang F, Ping Y, Ma Q (2024) Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke [Journal Article]. Acta Pharm Sin B 14(9):3983–4000. https://doi.org/10.1016/j.apsb.2024.05.007 Lu J, Ma Y, Wu J, Huang H, Wang X, Chen Z, Chen J, He H, Huang C (2019) A review for the neuroprotective effects of andrographolide in the central nervous system. Biomed Pharmacother 117:109078. https://doi.org/10.1016/j.biopha.2019.109078 Ma R, Shimura T, Yin C, Okugawa Y, Kitajima T, Koike Y, Okita Y, Ohi M, Uchida K, Goel A, Yao L, Zhang X, Toiyama Y (2021) Antitumor effects of Andrographis via ferroptosis-associated genes in gastric cancer [Journal Article]. Oncol Lett 22(1):523. https://doi.org/10.3892/ol.2021.12784 Meresman GF, Gotte M, Laschke MW (2021) Plants as source of new therapies for endometriosis: a review of preclinical and clinical studies [Journal Article; Research Support, Non-U.S. Gov’t; Review]. Hum Reprod Update 27(2):367–392. https://doi.org/10.1093/humupd/dmaa039 Moazen S, Arjmand M (2024) The role of local angiotensin II/angiotensin type 1 receptor in endometriosis: a potential target for new treatment approaches. Curr Mol Pharmacol 17:e1012166503. https://doi.org/10.2174/0118761429315431240712100124 Ng SW, Norwitz SG, Taylor HS, Norwitz ER (2020) Endometriosis: the role of iron overload and ferroptosis [Journal Article; Review]. Reprod Sci 27(7):1383–1390. https://doi.org/10.1007/s43032-020-00164-z Othman NS, Mohd AD (2022) Andrographolide induces G2/M cell cycle arrest and apoptosis in human glioblastoma DBTRG-05MG cell line via ERK1/2 /c-Myc/p53 signaling pathway [Journal Article]. Molecules 27(19):10.3390/molecules27196686 Samartzis EP, Fink D, Stucki M, Imesch P (2019) Doxycycline reduces MMP-2 activity and inhibits invasion of 12Z epithelial endometriotic cells as well as MMP-2 and -9 activity in primary endometriotic stromal cells in vitro. Reprod Biol Endocrinol 17(1):38. https://doi.org/10.1186/s12958-019-0481-z Saunders P, Horne AW (2021) Endometriosis: etiology, pathobiology, and therapeutic prospects. Cell 184(11):2807–2824. https://doi.org/10.1016/j.cell.2021.04.041 Shen L, Zhang J, Zheng Z, Yang F, Liu S, Wu Y, Chen Y, Xu T, Mao S, Yan Y, Li W, Zhang W, Yao X (2022) PHGDH inhibits ferroptosis and promotes malignant progression by upregulating SLC7A11 in bladder cancer. Int J Biol Sci 18(14):5459–5474. https://doi.org/10.7150/ijbs.74546 Stockwell BR (2022) Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. Cell 185(14):2401–2421. https://doi.org/10.1016/j.cell.2022.06.003 Wang L, Liu Y, Du T, Yang H, Lei L, Guo M, Ding HF, Zhang J, Wang H, Chen X, Yan C (2020) ATF3 promotes erastin-induced ferroptosis by suppressing system Xc() [Journal Article; Research Support, N.I.H., Extramural]. Cell Death Differ 27(2):662–675. https://doi.org/10.1038/s41418-019-0380-z Wang S, Li X, Li J, Wang A, Li F, Hu H, Long T, Pei X, Li H, Zhong F, Zhu F (2024) Inhibition of cisplatin-induced Acsl4-mediated ferroptosis alleviated ovarian injury. Chem Biol Interact 387:110825. https://doi.org/10.1016/j.cbi.2023.110825 Wen RJ, Dong X, Zhuang HW, Pang FX, Ding SC, Li N, Mai YX, Zhou ST, Wang JY, Zhang JF (2023) Baicalin induces ferroptosis in osteosarcomas through a novel Nrf2/xCT/GPX4 regulatory axis. Phytomedicine 116:154881. https://doi.org/10.1016/j.phymed.2023.154881 Wu Q, Liang Z, Jiang J, Feng X, Liu J, Zhang Z, Wang H, Wang N, Gou Y, Li Z, Cao Y (2023) Macrophages originated IL-33/ST2 inhibits ferroptosis in endometriosis via the ATF3/SLC7A11 axis. Cell Death Dis 14(10):668. https://doi.org/10.1038/s41419-023-06182-4 Wu YQ, Zhang CS, Xiong J, Cai DQ, Wang CZ, Wang Y, Liu YH, Wang Y, Li Y, Wu J, Wu J, Lan B, Wang X, Chen S, Cao X, Wei X, Hu HH, Guo H, Yu Y, Ghafoor A, Xie C, Wu Y, Xu Z, Zhang C, Zhu M, Huang X, Sun X, Lin SY, Piao HL, Zhou J, Lin SC (2023) Low glucose metabolite 3-phosphoglycerate switches PHGDH from serine synthesis to p53 activation to control cell fate [Journal article; research support, non-U.S. gov’t]. Cell Res 33(11):835–850. https://doi.org/10.1038/s41422-023-00874-4 Yamamoto T, Nakayama J, Urabe F, Ito K, Nishida-Aoki N, Kitagawa M, Yokoi A, Kuroda M, Hattori Y, Yamamoto Y, Ochiya T (2024) Aberrant regulation of serine metabolism drives extracellular vesicle release and cancer progression [Journal article; research support, non-U.S. gov’t]. Cell Rep 43(8):114517. https://doi.org/10.1016/j.celrep.2024.114517 Yang S, Evens AM, Prachand S, Singh AT, Bhalla S, David K, Gordon LI (2010) Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone andrographolide, the active component of Andrographis paniculata [Journal article; research support, N.I.H., extramural]. Clin Cancer Res 16(19):4755–4768. https://doi.org/10.1158/1078-0432.CCR-10-0883 Zhang C, Lu Z, Lyu C, Zhang S, Wang D (2023) Andrographolide inhibits static mechanical pressure-induced intervertebral disc degeneration via the MAPK/Nrf2/HO-1 pathway [Journal article]. Drug Des Devel Ther 17:535–550. https://doi.org/10.2147/DDDT.S392535 Zhang C, Yu JJ, Yang C, Yuan ZL, Zeng H, Wang JJ, Shang S, Lv XX, Liu XT, Liu J, Xue Q, Cui B, Tan FW, Hua F (2023) Wild-type IDH1 maintains NSCLC stemness and chemoresistance through activation of the serine biosynthetic pathway [Journal article]. Sci Transl Med 15(726):eade4113. https://doi.org/10.1126/scitranslmed.ade4113 Zheng Y, Liu X, Guo SW (2012) Therapeutic potential of andrographolide for treating endometriosis [Journal article; research support, non-U.S. gov’t]. Hum Reprod 27(5):1300–1313. https://doi.org/10.1093/humrep/des063 Zhou C, Feng M, Chen Y, Lv S, Zhang Y, Chen J, Zhang R, Huang X (2023) Unraveling immunotherapeutic targets for endometriosis: a transcriptomic and single-cell analysis [Journal article; research support, non-U.S. gov’t]. Front Immunol 14:1288263. https://doi.org/10.3389/fimmu.2023.1288263 Zhu G, Xie Y, Bian Z, Ma J, Zhen N, Chen T, Zhu J, Mao S, Tang X, Liu L, Gu S, Ding M, Pan Q (2024) N6-methyladenosine modification of LATS2 promotes hepatoblastoma progression by inhibiting ferroptosis through the YAP1/ATF4/PSAT1 axis [Journal article]. Int J Biol Sci 20(11):4146–4161. https://doi.org/10.7150/ijbs.92413 Zou W, Wang X, Xia X, Zhang T, Nie M, Xiong J, Fang X (2024) Resveratrol protected against the development of endometriosis by promoting ferroptosis through miR-21-3p/p53/SLC7A11 signaling pathway [Journal Article]. Biochem Biophys Res Commun 692:149338. https://doi.org/10.1016/j.bbrc.2023.149338 Funding This work is financially supported by the National Natural Science Foundation of China (32200703), Key Research Program of Anhui Science and Technology Innovation Platform (202305a12020016), University Natural Foundation of Anhui Educational Committee (2022AH010072) and Key Discipline Construction Fund of the Leading Hospital Project at the First Affiliated Hospital of Anhui Medical University (060201001). Author information Authors and Affiliations Contributions Wei Ye: Investigation; Data curation; methodology. Nie Zhang: Investigation; formal analysis; methodology. Jiaoyu Li: investigation; writing - original draft. Fei Wang: Data curation; Xiaoying Liu: Investigation. Ruixing Zhang: Investigation. Hongxu Chen: Investigation. Linghui Cheng, Conceptualization; methodology, resource. Zhaolian Wei: Conceptualization; writing - review and editing. Fengyu Zhu: Conceptualization; formal analysis; funding acquisition; writing - review and editing. The authors declare that all data were generated in-house and that no paper mill was used. Corresponding authors Ethics declarations Ethical approval Ethical approval for the mice experiments in this study were granted the Ethical Committee and the Institutional Animal Care and Use Committee of Anhui Medical University (license number LLSC20240162). Competing interest The authors declare no competing interests. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Wei Ye, Nie Zhang, and Jiaoyu Li are co-first authors. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Ye, W., Zhang, N., Li, J. et al. Andrographolide blocked the progression of endometriosis by promoting ferroptosis via inhibiting anabolism of serine. Naunyn-Schmiedeberg's Arch Pharmacol 399, 7935–7952 (2026). https://doi.org/10.1007/s00210-025-04905-2 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s00210-025-04905-2