Rehmanniae Radix Praeparata aqueous extract improves hepatic ischemia/reperfusion injury by restoring intracellular iron homeostasis

Abstract

Hepatic ischemia/reperfusion injury (HIRI) is a common pathophysiological condition occurring during or after liver resection and transplantation, leading to hepatic viability impairment and functional deterioration. Recently, ferroptosis, a newly recognized form of programmed cell death, has been implicated in IRI. Rehmanniae Radix Praeparata (RRP), extensively used in Chinese herbal medicine for its hepatoprotective, anti-inflammatory, and antioxidant properties, presents a potential therapeutic approach. However, the mechanisms by which RRP mitigates HIRI, particularly through the regulation of ferroptosis, remain unclear. In this study, we developed a HIRI mouse model and monocrotaline (MCT)- and erastin-induced in vitro hepatocyte injury models. We conducted whole-genome transcriptome analysis to elucidate the protective effects and mechanisms of RRP on HIRI. The RRP aqueous extract was characterized by the presence of acteoside, rehmannioside D, and 5-hydroxymethylfurfural. Our results demonstrate that the RRP aqueous extract ameliorated oxidative stress, reduced intracellular iron accumulation, and attenuated HIRI-induced liver damage. Additionally, RRP significantly inhibited hepatocyte death by restoring intracellular iron homeostasis both in vivo and in vitro. Mechanistically, the RRP aqueous extract reduced intrahepatocellular iron accumulation by inhibiting ZIP14-mediated iron uptake, promoting hepcidin- and ferroportin-mediated iron efflux, and ameliorating mitochondrial iron aggregation through upregulation of Cisd1 expression. Moreover, siRNA-mediated inhibition of hamp synergistically enhanced the RRP aqueous extract’s inhibitory effect on ferroptosis. In conclusion, our study elucidates the mechanisms by which RRP aqueous extracts alleviate HIRI, highlighting the restoration of iron metabolic balance. These findings position RRP as a promising candidate for clinical intervention in HIRI treatment.

Keywords

Hepatic ischemia/reperfusion injury / Rehmanniae Radix Praeparata / Ferroptosis / Intracellular iron homeostasis

References

[1] Nastos C, Kalimeris K, Papoutsidakis N, et al. Global consequences of liver ischemia/reperfusion injury[J]. Oxid Med Cell Longev, 2014, 2014: 906965.

[2] Chen SY, Zhang HP, Li J, et al. Tripartite motif-containing 27 attenuates liver ischemia/reperfusion injury by suppressing transforming growth factor β-activated kinase 1 ( TAK1) by TAK1 binding protein 2/3 degradation[J]. Hepatology, 2021, 73(2): 738-758.

[3] Cannistra M, Ruggiero M, Zullo A, et al. Hepatic ischemia reperfusion injury: a systematic review of literature and the role of current drugs and biomarkers[J]. Int J Surg, 2016, 33: S57-S70.

[4] Han H, Desert R, Das S, et al. Danger signals in liver injury and restoration of homeostasis[J]. J Hepatol, 2020, 73(4): 933-951.

[5] Liu H, Man K. New insights in mechanisms and therapeutics for short- and long-term impacts of hepatic ischemia reperfusion injury post liver transplantation[J]. Int J Mol Sci, 2021, 22(15): 8210.

[6] Capelletti MM, Manceau H, Puy H, et al. Ferroptosis in liver diseases: an overview[J]. Int J Mol Sci, 2020, 21(14): 4908.

[7] Yan HF, Tuo QZ, Yin QZ, et al. The hepathological role of ferroptosis in ischemia/reperfusion-related injury[J]. Zool Res, 2020, 41(3): 220-230.

[8] Liang DG, Minikes AM, Jiang XJ. Ferroptosis at the intersection of lipid metabolism and cellular signaling[J]. Mol Cell, 2022, 82(12): 2215-2227.

[9] Chen H, Zhao WS, Yan XZ, et al. Overexpression of hepcidin alleviates steatohepatitis and fibrosis in a diet-induced nonalcoholic steatohepatitis[J]. J Clin Translat Hepatol, 2022, 10(4): 577-588.

[10] Xin YJ, Gao H, Wang J, et al. Manganese transporter Slc39a14 deficiency revealed its key role in maintaining manganese homeostasis in mice[J]. Cell Discov, 2017, 18(3): 17025.

[11] Sun X, Ou Z, Xie M, et al. HSPB1 as a novel regulator of ferroptotic cancer cell death[J]. Oncogene, 2015, 34(45): 5617-5625.

[12] Zhang RX, Li MX, Jia ZP. Rehmannia glutinosa: review of botany, chemistry and pharmacology[J]. J Ethnopharmacol, 2008, 117(2): 199-214.

[13] Wu PS, Wu SJ, Tsai YH, et al. Hot water extracted Lycium barbarum and Rehmannia glutinosa inhibit liver inflammation and fibrosis in rats[J]. Am J Chin Med, 2011, 39(6): 1173-1191.

[14] Xu J, Wu J, Zhu LY, et al. Simultaneous determination of iridoid glycosides, phenethylalcohol glycosides and furfural derivatives in Rehmanniae Radix by high performance liquid chromatography coupled with triple-quadrupole mass spectrometry[J]. Food Chem, 2012, 135(4): 2277-2286.

[15] Perry B, Zhang J, Saleh T, et al. Liuwei Dihuang, a traditional Chinese herbal formula, suppresses chronic inflammation and oxidative stress in obese rats[J]. J Integr Med, 2014, 12(5): 447-454.

[16] Charlebois E, Pantopoulos K. Iron overload inhibits BMP/ SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes[J]. PLoS One, 2021, 16(6): e0253475.

[17] Zhou F, Ding M, Gu Y, et al. Aurantio-obtusin attenuates non-alcoholic fatty liver disease through AMPK-mediated autophagy and fatty acid oxidation pathways[J]. Front Pharmacol, 2021, 12: 826628.

[18] Wang K, Zhang Z, Tsai HI, et al. Branched-chain amino acid aminotransferase 2 regulates ferroptotic cell death in cancer cells[J]. Cell Death Differ, 2021, 28(4): 1222-1236.

[19] Liu GZ, Xu XW, Tao SH, et al. HBx facilitates ferroptosis in acute liver failure via EZH2 mediated SLC7A11 suppression[J]. J Biomed Sci, 2021, 28(1): 67.

[20] Guo Y, Yang C, Guo R, et al. CHOP regulates endoplasmic reticulum stress-mediated hepatoxicity induced by monocrotaline[J]. Front Pharmacol, 2021, 12: 685895.

[21] L, X, Ge J, Li Y, et al. Integrative lipidomic and transcriptomic study unravels the therapeutic effects of saikosaponins A and D on non-alcoholic fatty liver disease[J]. Acta Pharm Sin B, 2021, 11(11): 3527-3541.

[22] Li YJ, Liu RP, Ding MN, et al. Tetramethylpyrazine prevents liver fibrotic injury in mice by targeting hepatocyte-derived and mitochondrial DNA-enriched extracellular vesicles[J]. Acta Pharm Sin, 2022, 43(8): 2026-2041.

[23] Wu J, Zhou F, Fan G, et al. Ferulic acid ameliorates acetaminophen-induced acute liver injury by promoting AMPK-mediated protective autophagy[J]. IUBMB Life, 2022, 74(9): 880-895.

[24] Chen X, Kang R, Kroemer G, et al. Broadening horizons: the role of ferroptosis in cancer[J]. Nat Rev Clin Oncol, 2021, 18(5): 280-296.

[25] Zhang Z, Tang J, Song J, et al. Elabela alleviates ferroptosis, myocardial remodeling, fibrosis and heart dysfunction in hypertensive mice by modulating the IL-6/STAT3/GPX4 signaling[J]. Free Rad Biol Med, 2022, 181: 130-142.

[26] Cui Y, Zhang Y, Zhao X, et al. ACSL4 exacerbates ischemic stroke by promoting ferroptosis-induced brain injury and neuroinflammation[J]. Brain Behav Immun, 2021, 93: 312-321.

[27] Wang Y, Zhang M, Bi R, et al. ACSL4 deficiency confers protection against ferroptosis-mediated acute kidney injury[J]. Redox Biol, 2022, 51: 102262.

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

[29] Yamada N, Karasawa T, Wakiya T, et al. Iron overload as a risk factor for hepatic ischemia-reperfusion injury in liver transplantation: potential role of ferroptosis[J]. Am J Transplant, 2020, 20(6): 1606-1618.

[30] Yang L, Wang H, Yang X, et al. Auranofin mitigates systemic iron overload and induces ferroptosis via distinct mechanisms[J]. Signal Transduct Tar, 2020, 5(1): 138.

[31] Cheng K, Huang Y, Wang C. 1,25(OH)₂D₃ inhibited ferroptosis in Zebrafish liver cells (ZFL) by regulating Keap1-Nrf2-GPx4 and NF-κB-hepcidin axis[J]. Int J Mol Sci, 2021, 22(21): 11334.

[32] Yu Y, Jiang L, Wang H, et al. Hepatic transferrin plays a role in systemic iron homeostasis and liver ferroptosis[J]. Blood, 2020, 136(6): 726-739.

[33] Eum HA, Lee WY, Kim SH, et al. Anti-inflammatory activity of CML-1: an herbal formulation[J]. Am J Chin Med, 2005, 33(1): 29-40.

[34] Alipieva K, Korkina L, Orhan IE, et al. Verbascoside--a review of its occurrence, (bio)synthesis and pharmacological significance[J]. Biotechnol Adv, 2014, 32(6): 1065-1076.

[35] Chen Q, Qi X, Zhang W, et al. Catalpol inhibits macrophage polarization and prevents postmenopausal atherosclerosis through regulating estrogen receptor alpha[J]. Front Pharmacol, 2021, 12: 655081.

[36] Tang G, Xu Y, Zhang C, et al. Green tea and epigallocatechin gallate (EGCG) for the management of nonalcoholic fatty liver diseases (NAFLD): insights into the role of oxidative stress and antioxidant mechanism[J]. Antioxidants (Basel, Switzerland), 2021, 10(7): 10071076.

[37] Zwolak I. Epigallocatechin gallate for management of heavy metal-induced oxidative stress: mechanisms of action, efficacy, and concerns[J]. Int J Mol Sci, 2021, 22(8): 22084027.

[38] Conrad M, Proneth B. Broken hearts: iron overload, ferroptosis and cardiomyopathy[J]. Cell Res, 2019, 29(4): 263-264.

[39] Mancias JD, Wang X, Gygi SP, et al. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy[J]. Nature, 2014, 509(7498): 105-109.

Funding

Beijing Nova Program(Z201100006820025)

National Natural Science Foundation of China(82274186)

Fundamental Research Funds for the Central Universities(2023-JYB-JBZD-046)

National High-Level Talents Special Support Program to LI Xiaojiaoyan, the National Key Research and Development Program on Modernization of Traditional Chinese Medicine(2022YFC3502100)

Beijing Municipal Science & Technology Commission(7212174)