Ginsenoside Rk2 alleviates hepatic ischemia/reperfusion injury by enhancing AKT membrane translocation and activation

Abstract

Hepatic ischemia–reperfusion injury (IRI) poses a significant threat to clinical outcomes and graft survival during hemorrhagic shock, hepatic resection, and liver transplantation. Current pharmacological interventions for hepatic IRI are inadequate. In this study, we identified ginsenoside Rk2 (Rk2), a rare dehydroprotopanaxadiol saponin, as a promising agent against hepatic IRI through high-throughput screening. The pharmacological effects and molecular mechanisms of Rk2 on hepatic IRI were further evaluated and elucidated in vitro and in vivo. Rk2 significantly reduced inflammation and apoptosis caused by oxygen-glucose deprivation and reperfusion in hepatocytes and dose dependently protected against hepatic I/R-induced liver injury in mice. Integrated approaches, including network pharmacology, molecular docking, transcriptome analysis, and isothermal titration calorimetry, along with experimental validation, indicated that Rk2 protects against hepatic IRI by targeting and activating the AKT (RAC serine/threonine protein kinase) signaling pathway. Pharmacological inhibition of AKT pathway or knockdown of AKT1 effectively diminished protective effects of Rk2. Rk2 directly binds to AKT1, facilitating its translocation from the cytoplasm to plasma membrane. This process markedly enhanced AKT interaction with PDPK1, promoting the activation of AKT1 and its downstream signaling. Our findings demonstrate that Rk2 protects against hepatic IRI by activating AKT signaling through direct binding to AKT1 and facilitating its membrane translocation.

Keywords

AKT signaling / apoptosis / ginsenoside Rk2 / inflammation / ischemia–reperfusion / membrane translocation

References

[1] Heinrich S, Lang H. [Primary liver tumors. Preoperative conditioning of the liver and perioperative management in extended liver resection]. Chirurg. 2015; 86(2): 125-131.

[2] Zhai Y, Petrowsky H, Hong JC, Busuttil RW, Kupiec-Weglinski JW. Ischaemia–reperfusion injury in liver transplantation—From bench to bedside. Nat Rev Gastroenterol Hepatol. 2013; 10(2): 79-89.

[3] Pretzsch E, Nieß H, Khaled NB, et al. Molecular mechanisms of ischaemia-reperfusion injury and regeneration in the liver-shock and surgery-associated changes. Int J Mol Sci. 2022; 23(21): 12942.

[4] Wu MY, Yiang GT, Liao WT, et al. Current mechanistic concepts in ischemia and reperfusion injury. Cell Physiol Biochem. 2018; 46(4): 1650-1667.

[5] Cannistrà M, Ruggiero M, Zullo A, et al. Hepatic ischemia reperfusion injury: a systematic review of literature and the role of current drugs and biomarkers. Int J Surg. 2016; 33(Suppl 1): S57-S70.

[6] Pac-Soo CK, Mathew H, Ma D. Ischaemic conditioning strategies reduce ischaemia/reperfusion-induced organ injury. Br J Anaesth. 2015; 114(2): 204-216.

[7] Mao XL, Cai Y, Chen YH, et al. Novel targets and therapeutic strategies to protect against hepatic ischemia reperfusion injury. Front Med (Lausanne). 2021; 8: 757336.

[8] Tian C, Wang A, Huang H, Chen Y. Effects of remote ischemic preconditioning in hepatectomy: a systematic review and meta-analysis. BMC Anesthesiol. 2024; 24(1): 118.

[9] Chullo G, Panisello-Rosello A, Marquez N, et al. Focusing on ischemic reperfusion injury in the new era of dynamic machine perfusion in liver transplantation. Int J Mol Sci. 2024; 25(2): 1117.

[10] Liu Q, Li J, Wang J, Li J, Janicki JS, Fan D. Effects and mechanisms of chinese herbal medicine in ameliorating myocardial ischemia-reperfusion injury. Evid Based Complement Alternat Med. 2013; 2013: 925625.

[11] Zhu T, Wang L, Wang LP, Wan Q. Therapeutic targets of neuroprotection and neurorestoration in ischemic stroke: applications for natural compounds from medicinal herbs. Biomed Pharmacother. 2022; 148: 112719.

[12] Dossi CG, Vargas RG, Valenzuela R, Videla LA. Beneficial effects of natural compounds on experimental liver ischemia-reperfusion injury. Food Funct. 2021; 12(9): 3787-3798.

[13] Bian XB, Yu PC, Yang XH, et al. The effect of ginsenosides on liver injury in preclinical studies: a systematic review and meta-analysis. Front Pharmacol. 2023; 14: 1184774.

[14] Seyed Reza T, Andarz F-N, Mahdieh Shariat Z, et al. The hepatoprotective effects of ginsenoside from ginseng: a review of molecular mechanisms and therapeutic potentials. Curr Pharm Biotechnol. 2024; 25: 1-16.

[15] Guo Y, Yang T, Lu J, et al. Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway. Life Sci. 2011; 88(13-14): 598-605.

[16] Zhang Y, Ye QF, Lu L, Xu XL, Ming YZ, Xiao JS. Panax notoginseng saponins preconditioning protects rat liver grafts from ischemia/reperfusion injury via an antiapoptotic pathway. Hepatobiliary Pancreat Dis Int. 2005; 4(2): 207-212.

[17] Lin J, Huang HF, Yang SK, et al. The effect of Ginsenoside Rg1 in hepatic ischemia reperfusion (I/R) injury ameliorates ischemia-reperfusion-induced liver injury by inhibiting apoptosis. Biomed Pharmacother. 2020; 129: 110398.

[18] Fan W, Fan L, Wang Z, et al. Rare ginsenosides: a unique perspective of ginseng research. J Adv Res. 2024; 66: 303-328.

[19] Zou J, Yang R, Feng R, Liu J, Wan JB. Ginsenoside Rk2, a dehydroprotopanaxadiol saponin, alleviates alcoholic liver disease via regulating NLRP3 and NLRP6 inflammasome signaling pathways in mice. J Pharm Anal. 2023; 13(9): 999-1012.

[20] Kim JH, Yi YS, Kim MY, Cho JY. Role of ginsenosides, the main active components of Panax ginseng, in inflammatory responses and diseases. J Ginseng Res. 2017; 41(4): 435-443.

[21] Huang X, Xiao J, Wen M, Liang J. Ginsenoside Rk2 protects against ulcerative colitis via inactivating ERK/MEK pathway by SIRT1. J Environ Pathol Toxicol Oncol. 2022; 41(2): 89-98.

[22] Hu JN, Xu XY, Li W, et al. Ginsenoside Rk1 ameliorates paracetamol-induced hepatotoxicity in mice through inhibition of inflammation, oxidative stress, nitrative stress and apoptosis. J Ginseng Res. 2019; 43(1): 10-19.

[23] Lang Z, Yu S, Hu Y, et al. Ginsenoside Rh2 promotes hepatic stellate cell ferroptosis and inactivation via regulation of IRF1-inhibited SLC7A11. Phytomedicine. 2023; 118: 154950.

[24] Jiménez-Castro MB, ME Cornide-Petronio, Gracia-Sancho J, Peralta C. Inflammasome-mediated inflammation in liver ischemia-reperfusion injury. Cells. 2019; 8(10): 1131.

[25] Covington SM, Bauler LD, Toledo-Pereyra LH. Akt: a therapeutic target in hepatic ischemia-reperfusion injury. J Invest Surg. 2017; 30(1): 47-55.

[26] Harada N, Hatano E, Koizumi N, et al. Akt activation protects rat liver from ischemia/reperfusion injury. J Surg Res. 2004; 121(2): 159-170.

[27] Cho H, Thorvaldsen JL, Chu Q, Feng F, Birnbaum MJ. Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem. 2001; 276(42): 38349-38352.

[28] Manning BD, Toker A. AKT/PKB signaling: navigating the network. Cell. 2017; 169(3): 381-405.

[29] Jo H, Mondal S, Tan D, et al. Small molecule-induced cytosolic activation of protein kinase Akt rescues ischemia-elicited neuronal death. Proc Natl Acad Sci U S A. 2012; 109(26): 10581-10586.

[30] Chen J, Huang Q, Li J, et al. Panax ginseng against myocardial ischemia/reperfusion injury: a review of preclinical evidence and potential mechanisms. J Ethnopharmacol. 2023; 300: 115715.

[31] Wang J, Wang H, Mou X, et al. The advances on the protective effects of ginsenosides on myocardial ischemia and ischemia-reperfusion injury. Mini Rev Med Chem. 2020; 20(16): 1610-1618.

[32] Yang PF, Song XY, Chen NH. [Advances in pharmacological studies of Panax notoginseng saponins on brain ischemia-reperfusion injury]. Yao Xue Xue Bao. 2016; 51(7): 1039-1046.

[33] Zheng M, Xin Y, Li Y, et al. Ginsenosides: a potential neuroprotective agent. Biomed Res Int. 2018; 2018: 8174345.

[34] Li J, Li RJ, Lv GY, Liu HQ. The mechanisms and strategies to protect from hepatic ischemia-reperfusion injury. Eur Rev Med Pharmacol Sci. 2015; 19(11): 2036-2047.

[35] Zhang S, Rao S, Yang M, Ma C, Hong F, Yang S. Role of mitochondrial pathways in cell apoptosis during hepatic ischemia/reperfusion injury. Int J Mol Sci. 2022; 23(4): 2357.

[36] Zhang H, Ni M, Wang H, et al. Gsk3β regulates the resolution of liver ischemia/reperfusion injury via MerTK. JCI Insight. 2023; 8(1): e151819.

[37] Zhang X, Tang N, Hadden TJ, Akt RishiAK. FoxO and regulation of apoptosis. Biochim Biophys Acta. 2011; 1813(11): 1978-1986.

[38] Wang M, Zhang J, Gong N. Role of the PI3K/Akt signaling pathway in liver ischemia reperfusion injury: a narrative review. Ann Palliat Med. 2022; 11(2): 806-817.

[39] Zhou H, Yu Y, Zhang J, Zhang Y, Luan Q, Wang G. Protective effects the Akt activator SC79 in hepatic ischemia-reperfusion injury. Med Sci Monit. 2018; 24: 4346-4354.

[40] Izuishi K, Tsung A, Hossain MA, et al. Ischemic preconditioning of the murine liver protects through the Akt kinase pathway. Hepatology. 2006; 44(3): 573-580.

[41] Balasuriya N, Kunkel MT, Liu X, et al. Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity. J Biol Chem. 2018; 293(27): 10744-10756.

[42] Thapa N, Horn HT, Anderson RA. Phosphoinositide spatially free AKT/PKB activation to all membrane compartments. Adv Biol Regul. 2019; 72: 1-6.

[43] Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005; 307(5712): 1098-1101.

[44] Lučić I, Rathinaswamy MK, Truebestein L, Hamelin DJ, Burke JE, Leonard TA. Conformational sampling of membranes by Akt controls its activation and inactivation. Proc Natl Acad Sci U S A. 2018; 115(17): E3940-E3949.

[45] Truebestein L, Hornegger H, Anrather D, et al. Structure of autoinhibited Akt1 reveals mechanism of PIP(3)-mediated activation. Proc Natl Acad Sci U S A. 2021; 118(33): E2101496118.

[46] Qin L, Fan S, Jia R, Liu Y. Ginsenoside Rg1 protects cardiomyocytes from hypoxia-induced injury through the PI3K/AKT/mTOR pathway. Pharmazie. 2018; 73(6): 349-355.

[47] Qin GW, Lu P, Peng L, Jiang W. Ginsenoside Rb1 inhibits cardiomyocyte autophagy via PI3K/Akt/mTOR signaling pathway and reduces myocardial ischemia/reperfusion injury. Am J Chin Med. 2021; 49(8): 1913-1927.

[48] Chen S, Li X, Wang Y, et al. Ginsenoside Rb1 attenuates intestinal ischemia/reperfusion-induced inflammation and oxidative stress via activation of the PI3K/Akt/Nrf2 signaling pathway. Mol Med Rep. 2019; 19(5): 3633-3641.

[49] Li X, Huang Q, Wang M, et al. Compound K inhibits autophagy-mediated apoptosis through activation of the PI3K-Akt signaling pathway thus protecting against ischemia/reperfusion injury. Cell Physiol Biochem. 2018; 47(6): 2589-2601.

[50] Charni-Natan M, Goldstein I. Protocol for primary mouse hepatocyte isolation. STAR Protoc. 2020; 1(2): 100086.

[51] Kadono K, Kageyama S, Nakamura K, et al. Myeloid Ikaros-SIRT1 signaling axis regulates hepatic inflammation and pyroptosis in ischemia-stressed mouse and human liver. J Hepatol. 2022; 76(4): 896-909.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.