Blueberry derived exosome like nanoparticles alleviate CCl₄ induced acute liver injury by inhibiting ACSL4−ALOX15 mediated ferroptosis

Abstract

Blueberry (Vaccinium spp.), a flowering plant in the family Ericaceae, is rich in bioactive compounds such as polyphenols, anthocyanins, and flavonoids, which exhibit anti-inflammatory, antioxidant, and anticancer properties. Increasing evidence suggests that plant-derived exosome-like nanoparticles (PELNs) possess diverse therapeutic activities and hold promise for improving liver function. Acute liver injury (ALI) is a clinical syndrome for which effective pharmacological interventions remain limited. The present study aimed to investigate whether orally administered blueberry-derived exosome-like nanoparticles (BELNs) can ameliorate ALI and to elucidate the underlying mechanisms. Both in vivo and in vitro experiments demonstrated that BELNs significantly attenuated CCl₄-induced ALI. Specifically, BELNs reduced oxidative stress and inhibited the accumulation of lipid peroxidation products. Mechanistic analyses revealed that BELNs alleviated lipid peroxidation and ferroptosis by suppressing the ACSL4–ALOX15 signaling pathway, specifically reducing its expression by approximately 70% (p <0.001) and thereby protecting hepatocytes from injury. Further investigation identified procyanidin B2 (PCB2) as one of the key active components in BELNs responsible for their antioxidant effects. In summary, this study provides new insights into the therapeutic potential of blueberries and their derived natural products and establishes a mechanistic basis for the clinical translation of PELNs.

References

[1] Mustafa AM, Angeloni S, Abouelenein D, Acquaticci L, Xiao J, et al. 2022. A new HPLC—MS/MS method for the simultaneous determination of 36 polyphenols in blueberry, strawberry and their commercial products and determination of antioxidant activity. Food Chemistry 367: 130743

[2] Duan Y, Tarafdar A, Chaurasia D, Singh A, Bhargava PC, et al. 2022. Blueberry fruit valorization and valuable constituents: a review. International Journal of Food Microbiology 381: 109890

[3] Herrera—Balandrano DD, Chai Z, Hutabarat RP, Beta T, Feng J, et al. 2021. Hypoglycemic and hypolipidemic effects of blueberry anthocyanins by AMPK activation: in vitro and in vivo studies . Redox Biology 46: 102100

[4] Yan Y, Zhang F, Chai Z, Liu M, Battino M, et al. 2019. Mixed fermentation of blueberry pomace with L. rhamnosus GG and L. plantarum—1: enhance the active ingredient, antioxidant activity and health—promoting benefits . Food and Chemical Toxicology 131: 110541

[5] Negi T, Kumar Y, Sirohi R, Singh S, Tarafdar A, et al. 2022. Advances in bioconversion of spent tea leaves to value—added products. Bioresource Technology 346: 126409

[6] Kuntz S, Kunz C, Rudloff S. 2017. Inhibition of pancreatic cancer cell migration by plasma anthocyanins isolated from healthy volunteers receiving an anthocyanin—rich berry juice. European Journal of Nutrition 56: 203-214

[7] Whyte AR, Rahman S, Bell L, Edirisinghe I, Krikorian R, et al. 2021. Improved metabolic function and cognitive performance in middle—aged adults following a single dose of wild blueberry. European Journal of Nutrition 60: 1521-1536

[8] Zhuge Q, Zhang Y, Liu B, Wu M. 2020. Blueberry polyphenols play a preventive effect on alcoholic fatty liver disease C57BL/6 J mice by promoting autophagy to accelerate lipolysis to eliminate excessive TG accumulation in hepatocytes. Annals of Palliative Medicine 9: 1045-1054

[9] Wang Y, Cheng M, Zhang B, Nie F, Jiang H. 2013. Dietary supplementation of blueberry juice enhances hepatic expression of metallothionein and attenuates liver fibrosis in rats. PLoS One 8: e58659

[10] Feng J, Wang C, Liu T, Li J, Wu L, et al. 2019. Procyanidin B2 inhibits the activation of hepatic stellate cells and angiogenesis via the Hedgehog pathway during liver fibrosis. Journal of Cellular and Molecular Medicine 23: 6479-6493

[11] Ju S, Mu J, Dokland T, Zhuang X, Wang Q, et al. 2013. Grape exosome—like nanoparticles induce intestinal stem cells and protect mice from DSS—induced colitis. Molecular Therapy: The Journal of the American Society of Gene Therapy 21: 1345-1357

[12] Wang Q, Ren Y, Mu J, Egilmez NK, Zhuang X, et al. 2015. Grapefruit—derived nanovectors use an activated leukocyte trafficking pathway to deliver therapeutic agents to inflammatory tumor sites. Cancer Research 75: 2520-2529

[13] Zhang M, Viennois E, Prasad M, Zhang Y, Wang L, et al. 2016. Edible ginger—derived nanoparticles: a novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis—associated cancer. Biomaterials 101: 321-340

[14] Takakura H, Nakao T, Narita T, Horinaka M, Nakao—Ise Y, et al. 2022. Citrus limon L.—derived nanovesicles show an inhibitory effect on cell growth in p53—inactivated colorectal cancer cells via the macropinocytosis pathway . Biomedicines 10: 1352

[15] Liu R, Zhang F, He X, Huang K. 2025. Plant derived exosome—like nanoparticles and their therapeutic applications in glucolipid metabolism diseases. Journal of Agricultural and Food Chemistry 73: 6385-6399

[16] Thawley V. 2017. Acute liver injury and failure. Veterinary Clinics of North America: Small Animal Practice 47: 617-630

[17] Ramachandran A, Jaeschke H. 2018. Oxidative stress and acute hepatic injury. Current Opinion in Toxicology 7: 17-21

[18] Maiwall R, Kulkarni AV, Arab JP, Piano S. 2024. Acute liver failure. The Lancet 404: 789-802

[19] Lu K, Shen SY, Luo OY, Lu Y, Shi TS, et al. 2022. Manipulating PP2Ac α—ASK—JNK signaling to favor apoptotic over necroptotic hepatocyte fate reduces the extent of necrosis and fibrosis upon acute liver injury . Cell Death & Disease 13: 985

[20] Lin F, Chen W, Zhou J, Zhu J, Yao Q, et al. 2022. Mesenchymal stem cells protect against ferroptosis via exosome—mediated stabilization of SLC7A11 in acute liver injury. Cell Death & Disease 13: 271

[21] De Robertis M, Sarra A, D'Oria V, Mura F, Bordi F, et al. 2020. Blueberry—derived exosome—like nanoparticles counter the response to TNF— α—induced change on gene expression in EA.hy926 cells . Biomolecules 10: 742

[22] Rezakhani L, Alizadeh M, Sharifi E, Soleimannejad M, Alizadeh A. 2021. Isolation and characterization of crab haemolymph exosomes and its effects on breast cancer cells (4T1). Cell Journal 23: 658-664

[23] Wang F, Li L, Deng J, Ai J, Mo S, et al. 2025. Lipidomic analysis of plant—derived extracellular vesicles for guidance of potential anti—cancer therapy. Bioactive Materials 46: 82-96

[24] Tian Y, Liu X, Chen X, Wang B, Dong M, et al. 2024. Integrated untargeted metabolome, full—length sequencing and transcriptome analyses reveal the mechanism of flavonoid biosynthesis in blueberry (Vaccinium spp.) fruit . International Journal of Molecular Sciences 25: 4137

[25] Wang Y, Zhang Q, Kan M, Chang F, He X, et al. 2024. Multi—omics analysis of Au@Pt nanozyme for the modulation of glucose and lipid metabolism. Journal of Nanobiotechnology 22: 524

[26] Sellers RS, Mortan D, Michael B, Roome N, Johnson JK, et al. 2007. Society of toxicologic pathology position paper: organ weight recommendations for toxicology studies. Toxicologic Pathology 35: 751-755

[27] Yi M, Zhang D, Song B, Zhao B, Niu M, et al. 2022. Increased expression of ECT2 predicts the poor prognosis of breast cancer patients. Experimental Hematology & Oncology 11: 107

[28] Liang FG, Zandkarimi F, Lee J, Axelrod JL, Pekson R, et al. 2024. OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response. Molecular Cell 84: 3098-3114.e6

[29] Liu H, Dong T, Dong C, Yang F, Zhou Q, et al. 2025. Plant—derived exosome—like nanovesicles: a novel therapeutic perspective for skin diseases. Journal of Nanobiotechnology 23: 640

[30] Trajkovic K, Hsu C, Chiantia S, Rajendran L, Wenzel D, et al. 2008. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 319: 1244-1247

[31] Yang M, Guo J, Li J, Wang S, Sun Y, et al. 2025. Platycodon grandiflorum—derived extracellular vesicles suppress triple—negative breast cancer growth by reversing the immunosuppressive tumor microenvironment and modulating the gut microbiota. Journal of Nanobiotechnology 23: 92

[32] Tadokoro T, Ikeda M, Ide T, Deguchi H, Ikeda S, et al. 2020. Mitochondria—dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity. JCI Insight 5: e132747

[33] Lian MQ, Chng WH, Liang J, Yeo HQ, Lee CK, et al. 2022. Plant—derived extracellular vesicles: recent advancements and current challenges on their use for biomedical applications. Journal of Extracellular Vesicles 11: e12283

[34] Yuan S, Wei C, Liu G, Zhang L, Li J, et al. 2022. Sorafenib attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis via HIF—1 α/SLC7A11 pathway . Cell Proliferation 55: e13158

[35] You JY, Kang SJ, Rhee WJ. 2021. Isolation of cabbage exosome—like nanovesicles and investigation of their biological activities in human cells. Bioactive Materials 6: 4321-4332

[36] Munagala R, Aqil F, Jeyabalan J, Gupta RC. 2016. Bovine milk—derived exosomes for drug delivery. Cancer Letters 371: 48-61

[37] Gong L, Zhou H, Zhang Y, Wang C, Fu K, et al. 2023. Preparation of Phillygenin—Hyaluronic acid composite milk—derived exosomes and its anti—hepatic fibrosis effect. Materials Today Bio 23: 100804

[38] Liu B, Nguyen PL, Yu H, Li X, Wang H, et al. 2024. Honey vesicle—like nanoparticles protect aged liver from non—alcoholic steatohepatitis. Acta Pharmaceutica Sinica B 14: 3661-3679

[39] Jiang M, Li F, Liu Y, Gu Z, Zhang L, et al. 2023. Probiotic—derived nanoparticles inhibit ALD through intestinal miR194 suppression and subsequent FXR activation. Hepatology 77: 1164-1180

[40] Chen J, Zhong K, Jing Y, Liu S, Qin S, et al. 2023. Procyanidin B2: a promising multi—functional food—derived pigment for human diseases. Food Chemistry 420: 136101

[41] Yang BY, Zhang XY, Guan SW, Hua ZC. 2015. Protective effect of procyanidin B2 against CCl₄—induced acute liver injury in mice . Molecules 20: 12250-12265

[42] Xing YW, Lei GT, Wu QH, Jiang Y, Huang MX. 2019. Procyanidin B2 protects against diet—induced obesity and non—alcoholic fatty liver disease via the modulation of the gut microbiota in rabbits. World Journal of Gastroenterology 25: 955-966

[43] Su H, Li Y, Hu D, Xie L, Ke H, et al. 2018. Procyanidin B2 ameliorates free fatty acids—induced hepatic steatosis through regulating TFEB—mediated lysosomal pathway and redox state. Free Radical Biology and Medicine 126: 269-286

[44] Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, et al. 2012. Ferroptosis: an iron—dependent form of nonapoptotic cell death. Cell 149: 1060-1072

[45] Cruz—Gregorio A, Aranda—Rivera AK. 2023. Quercetin and ferroptosis. Life 13: 1730

[46] Wang Y, Branicky R, Noë A, Hekimi S. 2018. Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling. The Journal of Cell Biology 217: 1915-1928

[47] Su L, Zhang J, Gomez H, Kellum JA, Peng Z. 2023. Mitochondria ROS and mitophagy in acute kidney injury. Autophagy 19: 401-414

[48] Lu S, Wang XZ, He C, Wang L, Liang SP, et al. 2021. ATF3 contributes to brucine—triggered glioma cell ferroptosis via promotion of hydrogen peroxide and iron. Acta Pharmacologica Sinica 42: 1690-1702

[49] Okazaki S, Umene K, Yamasaki J, Suina K, Otsuki Y, et al. 2019. Glutaminolysis—related genes determine sensitivity to xCT—targeted therapy in head and neck squamous cell carcinoma. Cancer Science 110: 3453-3463

[50] Seiler A, Schneider M, Förster H, Roth S, Wirth EK, et al. 2008. Glutathione peroxidase 4 senses and translates oxidative stress into 12/15—lipoxygenase dependent— and AIF—mediated cell death. Cell Metabolism 8: 237-248

[51] Chen X, Yu C, Kang R, Kroemer G, Tang D. 2021. Cellular degradation systems in ferroptosis. Cell Death & Differentiation 28: 1135-1148

[52] Klaassen CD, Reisman SA. 2010. Nrf2 the rescue: effects of the antioxidative/electrophilic response on the liver. Toxicology and Applied Pharmacology 244: 57-65

[53] Yang WH, Lin CC, Wu J, Chao PY, Chen K, et al. 2021. The hippo pathway effector YAP promotes ferroptosis via the E3 ligase SKP2. Molecular Cancer Research 19: 1005-1014

[54] Doll S, Proneth B, Tyurina YY, Panzilius E, Kobayashi S, et al. 2017. ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nature Chemical Biology 13: 91-98

[55] Lei G, Mao C, Yan Y, Zhuang L, Gan B. 2021. Ferroptosis, radiotherapy, and combination therapeutic strategies. Protein & Cell 12: 836-857

[56] Conrad M, Pratt DA. 2019. The chemical basis of ferroptosis. Nature Chemical Biology 15: 1137-1147

[57] Xue P, Zhang J, Wang Z, Wu L, Tang J, et al. 2025. Arachidonic acid induces ferroptosis in hepatocellular carcinoma via the SIRT5—ACSL4/LPCAT3/ALOX15 axis, leading to lipid peroxidation and mitochondrial dysfunction. Phytomedicine 148: 157450

[58] Zhou X, Xu S, Zhang Z, Tang M, Meng Z, et al. 2024. Gouqi—derived nanovesicles (GqDNVs) inhibited dexamethasone—induced muscle atrophy associating with AMPK/SIRT1/PGC1 α signaling pathway . Journal of Nanobiotechnology 22: 276

[59] Yang J, Yun X, Zheng W, Zhang H, Yan Z, et al. 2025. Nanoscale engineered exosomes for dual delivery of Sirtuin3 and insulin to ignite mitochondrial recovery in myocardial ischemia—reperfusion. Journal of Nanobiotechnology 23: 439

[60] Liu J, Ren Z, Yang L, Zhu L, Li Y, et al. 2022. The NSUN5—FTH1/FTL pathway mediates ferroptosis in bone marrow—derived mesenchymal stem cells. Cell Death Discovery 8: 99

[61] Li D, Lu X, Xu G, Liu S, Gong Z, et al. 2023. Dihydroorotate dehydrogenase regulates ferroptosis in neurons after spinal cord injury via the P53—ALOX15 signaling pathway. CNS Neuroscience & Therapeutics 29: 1923-1939

[62] Ren X, Wen Y, Yuan M, Li C, Zhang J, et al. 2024. Cerebroprotein hydrolysate—I ameliorates cognitive dysfunction in APP/PS1 mice by inhibiting ferroptosis via the p53/SAT1/ALOX15 signalling pathway. European Journal of Pharmacology 979: 176820