Panax notoginseng saponins regulate differential miRNA expression in osteoclast exosomes and inhibit ferroptosis in osteoblasts

doi:10.12307/2025.064

Abstract

Abstract: BACKGROUND: Steroid-induced femoral head necrosis is mostly caused by long-term and extensive use of hormones, but its specific pathogenesis is not yet clear and needs further study.
OBJECTIVE: To screen out the differential miRNAs in osteoclast exosomes after the intervention of Panax notoginseng saponins, and on this basis, to further construct an osteogenic-related ferroptosis regulatory network to explore the potential mechanism and research direction of steroid-induced osteonecrosis of the femoral head.
METHODS: MTT assay was used to detect the toxic effects of different concentrations of dexamethasone and different mass concentrations of Panax notoginseng saponins on Raw264.7 cell line. Tartrate resistant acid phosphatase staining and TUNEL assay were used to detect the effects of Panax notoginseng saponins on osteoclast inhibition and apoptosis. Exosomes were extracted from cultured osteoclasts with Panax notoginseng saponins intervention. Exosomes from different groups were sequenced to identify differentially expressed miRNAs. CytoScape 3.9.1 was used to construct and visualize the regulatory network between differentially expressed miRNAs and mRNAs. Candidate mRNAs were screened by GO analysis and KEGG analysis. Finally, the differential genes related to ferroptosis were screened out, and the regulatory network of ferroptosis-related genes was constructed.
RESULTS AND CONCLUSION: (1) The concentration of dexamethasone (0.1 μmol/L) and Panax notoginseng saponins (1 736.85 μg/mL) suitable for intervention of Raw264.7 cells was determined by MTT assay. (2) Panax notoginseng saponins had an inhibitory effect on osteoclasts and could promote their apoptosis. (3) Totally 20 differentially expressed miRNAs were identified from osteoclast-derived exosome samples, and 11 differentially expressed miRNAs related to osteogenesis were predicted by target mRNAs. The regulatory networks of 4 up-regulated differentially expressed miRNAs corresponding to 155 down-regulated candidate mRNAs and 7 down-regulated differentially expressed miRNAs corresponding to 238 up-regulated candidate mRNAs were constructed. (4) Twenty-four genes related to ferroptosis were screened out from the differential genes. Finally, 12 networks were constructed (miR-98-5p/PTGS2, miR-23b-3p/PTGS2, miR-425-5p/TFRC, miR-133a-3p/TFRC, miR-185-5p/TFRC, miR-23b-3p/NFE2L2, miR-23b-3p/LAMP2, miR-98-5p/LAMP2, miR-182-5p/LAMP2, miR-182-5p/TLR4, miR-23b-3p/ZFP36, and miR-182-5p/ZFP36). These results indicate that Panax notoginseng saponins may regulate osteoblast ferroptosis by regulating the expression of miRNAs derived from osteoclast exosomes, thus providing a new idea for the study of the mechanism of steroid-induced femoral head necrosis.

Key words: steroid-induced osteonecrosis of the femoral head, Panax notoginseng saponins, osteoclast, exosomes, ferroptosis, regulatory network

CLC Number:

Tao Hongcheng, Zeng Ping, Liu Jinfu, Tian Zhao, Ding Qiang, Li Chaohui, Wei Jianjie, Li Hao. Panax notoginseng saponins regulate differential miRNA expression in osteoclast exosomes and inhibit ferroptosis in osteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(19): 4011-4021.

Figures/Tables 7

References

[1] XU H, WANG C, LIU C, et al. Cotransplantation of mesenchymal stem cells and endothelial progenitor cells for treating steroid-induced osteonecrosis of the femoral head. Stem Cells Transl Med. 2021;10(5): 781-796.
[2] HUANG C, WEN Z, NIU J, et al. Steroid-Induced Osteonecrosis of the Femoral Head: Novel Insight Into the Roles of Bone Endothelial Cells in Pathogenesis and Treatment. Front Cell Dev Biol. 2021;9:777697.
[3] CHANG C, GREENSPAN A, GERSHWIN ME. The pathogenesis, diagnosis and clinical manifestations of steroid-induced osteonecrosis. J Autoimmun. 2020;110:102460.
[4] KIM HJ, ZHAO H, KITAURA H, et al. Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest. 2006;116(8):2152-2160.
[5] WANG F, MIN HS, SHAN H, et al. IL-34 Aggravates Steroid-Induced Osteonecrosis of the Femoral Head via Promoting Osteoclast Differentiation. Immune Netw. 2022;22(3):e25.
[6] YUE C, JIN H, ZHANG X, et al. Aucubin prevents steroid-induced osteoblast apoptosis by enhancing autophagy via AMPK activation. J Cell Mol Med. 2021;25(21):10175-10184.
[7] YANG JX, XIE P, LI YS, et al. Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2. Cell Signal. 2020;70:109504.
[8] JIANG X, STOCKWELL BR, CONRAD M. Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol. 2021;22(4):266-282.
[9] 梁学振,骆帝,李嘉程,等.激素性股骨头坏死中的PTGS2和STAT3：潜在铁死亡相关诊断生物标志物[J].中国组织工程研究, 2023,27(36):5898-5904.
[10] 章家皓,刘予豪,周驰,等.氧化应激促进成骨细胞铁死亡介导激素性股骨头坏死的病理过程[J].中国组织工程研究,2024,28(20): 3202-3208.
[11] 国家中医心血管病临床医学研究中心,中国医师协会中西医结合医师分会,中国中西医结合学会活血化瘀专业委员会,等.三七总皂苷制剂临床应用中国专家共识[J].中国中西医结合杂志,2021, 41(10):1157-1167.
[12] 韩杰,陈跃平,莫坚,等.三七总皂苷干预激素性股骨头缺血坏死模型兔的超微结构评价[J].中国组织工程研究,2019,23(7): 1035-1039.
[13] 方姝晨,邹季,史政康,等.三七总皂苷对股骨头坏死大鼠股骨头成骨作用影响的实验研究[J].中国中医骨伤科杂志,2020,28(6): 6-9,15.
[14] CHEN H, BOUTROS PC. VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinformatics. 2011;12:35.
[15] PEGTEL DM, GOULD SJ. Exosomes. Annu Rev Biochem. 2019;88: 487-514.
[16] YI J, ZHU J, WU J, et al. Oncogenic activation of PI3K-AKT-mTOR signaling suppresses ferroptosis via SREBP-mediated lipogenesis. Proc Natl Acad Sci U S A. 2020;117(49):31189-31197.
[17] LI S, LEI Z, YANG X, et al. Propofol Protects Myocardium From Ischemia/Reperfusion Injury by Inhibiting Ferroptosis Through the AKT/p53 Signaling Pathway. Front Pharmacol. 2022;13:841410.
[18] ZHOU N, YUAN X, DU Q, et al. FerrDb V2: update of the manually curated database of ferroptosis regulators and ferroptosis-disease associations. Nucleic Acids Res. 2023;51(D1):D571-D582.
[19] NOONIN C, THONGBOONKERD V. Exosome-inflammasome crosstalk and their roles in inflammatory responses. Theranostics. 2021;11(9): 4436-4451.
[20] IRIE N, TAKADA Y, WATANABE Y, et al. Bidirectional signaling through ephrinA2-EphA2 enhances osteoclastogenesis and suppresses osteoblastogenesis. J Biol Chem. 2009;284(21):14637-14644.
[21] SUN W, ZHAO C, LI Y, et al. Osteoclast-derived microRNA-containing exosomes selectively inhibit osteoblast activity. Cell Discov. 2016;2: 16015.
[22] SUN F, ZHOU JL, LIU ZL, et al. Dexamethasone induces ferroptosis via P53/SLC7A11/GPX4 pathway in glucocorticoid-induced osteonecrosis of the femoral head. Biochem Biophys Res Commun. 2022;602: 149-155.
[23] FANG L, ZHANG G, WU Y, et al. SIRT6 Prevents Glucocorticoid-Induced Osteonecrosis of the Femoral Head in Rats. Oxid Med Cell Longev. 2022;2022:6360133.
[24] ZHANG J, GUO F, ZHOU R, et al. Proteomics and transcriptome reveal the key transcription factors mediating the protection of Panax notoginseng saponins (PNS) against cerebral ischemia/reperfusion injury. Phytomedicine. 2021;92:153613.
[25] HU H, CHEN Y, ZOU Z, et al. Panax Notoginseng Saponins Prevent Bone Loss by Promoting Angiogenesis in an Osteoporotic Mouse Model. Biomed Res Int. 2020;2020:8412468.
[26] FAN JZ, WANG Y, MENG Y, et al. Panax notoginseng saponins mitigate ovariectomy-induced bone loss and inhibit marrow adiposity in rats. Menopause. 2015;22(12):1343-1350.
[27] QIANG H, LIU H, LING M, et al. Early Steroid-Induced Osteonecrosis of Rabbit Femoral Head and Panax notoginseng Saponins: Mechanism and Protective Effects. Evid Based Complement Alternat Med. 2015; 2015:719370.
[28] LI R, RUAN Q, YIN F, et al. MiR-23b-3p promotes postmenopausal osteoporosis by targeting MRC2 and regulating the Wnt/β-catenin signaling pathway. J Pharmacol Sci. 2021;145(1):69-78.
[29] ZHENG F, WANG F, XU Z. MicroRNA-98-5p prevents bone regeneration by targeting high mobility group AT-Hook 2. Exp Ther Med. 2019;18(4): 2660-2666.
[30] ZHENG F, ZHANG F, WANG F. Inhibition of miR-98-5p promotes high glucose-induced suppression of preosteoblast proliferation and differentiation via the activation of the PI3K/AKT/GSK3β signaling pathway by targeting BMP2. Mol Med Rep. 2022;26(3):292.
[31] CHEN G, HUANG G, LIN H, et al. MicroRNA-425-5p modulates osteoporosis by targeting annexin A2. Immun Ageing. 2021;18(1):45.
[32] WANG Y, MA J, QIU W, et al. Guanidinoacetic Acid Regulates Myogenic Differentiation and Muscle Growth Through miR-133a-3p and miR-1a-3p Co-mediated Akt/mTOR/S6K Signaling Pathway. Int J Mol Sci. 2018;19(9):2837.
[33] LV H, SUN Y, ZHANG Y. MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells. Med Sci Monit. 2015; 21:1527-1534.
[34] WANG Q, LI Y, ZHANG Y, et al. LncRNA MEG3 inhibited osteogenic differentiation of bone marrow mesenchymal stem cells from postmenopausal osteoporosis by targeting miR-133a-3p. Biomed Pharmacother. 2017;89:1178-1186.
[35] WU Y, JIANG Y, LIU Q, et al. lncRNA H19 promotes matrix mineralization through up-regulating IGF1 by sponging miR-185-5p in osteoblasts. BMC Mol Cell Biol. 2019;20(1):48.
[36] ZHANG J, XU N, YU C, et al. LncRNA PART1/miR-185-5p/RUNX3 feedback loop modulates osteogenic differentiation of bone marrow mesenchymal stem cells. Autoimmunity. 2021;54(7): 422-429.
[37] PAN BL, TONG ZW, LI SD, et al. Decreased microRNA-182-5p helps alendronate promote osteoblast proliferation and differentiation in osteoporosis via the Rap1/MAPK pathway. Biosci Rep. 2018;38(6): BSR20180696.
[38] HU Z, YIN Y, JIANG J, et al. Exosomal miR-142-3p secreted by hepatitis B virus (HBV)-hepatocellular carcinoma (HCC) cells promotes ferroptosis of M1-type macrophages through SLC3A2 and the mechanism of HCC progression. J Gastrointest Oncol. 2022;13(2):754-767.
[39] YANG WS, SRIRAMARATNAM R, WELSCH ME, et al. Regulation of ferroptotic cancer cell death by GPX4. Cell. 2014;156(1-2):317-331.
[40] YI L, HU Y, WU Z, et al. TFRC upregulation promotes ferroptosis in CVB3 infection via nucleus recruitment of Sp1. Cell Death Dis. 2022; 13(7):592.
[41] GAO T, LIN M, WU Y, et al. Transferrin receptor (TFRC) is essential for meiotic progression during mouse spermatogenesis. Zygote. 2021; 29(2):169-175.
[42] SUN H, QIAN X, YANG W, et al. Novel prognostic signature based on HRAS, MAPK3 and TFRC identified to be associated with ferroptosis and the immune microenvironment in hepatocellular carcinoma. Am J Transl Res. 2022;14(10):6924-6940.
[43] 王宁,康华丽,薛金慧,等.铁死亡相关基因TFRC在胃癌组织中的表达及其与化疗药物敏感性的关系[J].现代肿瘤医学,2023, 31(22):4183-4189.
[44] CHEN X, KANG R, KROEMER G, et al. Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol. 2021;18(5):280-296.
[45] WANG K, LI Z, XUAN Y, et al. Pan-cancer analysis of NFE2L2 mutations identifies a subset of lung cancers with distinct genomic and improved immunotherapy outcomes. Cancer Cell Int. 2023;23(1):229.
[46] AROLT C, DUGAN M, WILD R, et al. KEAP1/NFE2L2 Pathway Signature Outperforms KEAP1/NFE2L2 Mutation Status and Reveals Alternative Pathway-Activating Mutations in NSCLC. J Thorac Oncol. 2023;18(11):1550-1567.
[47] GÓMEZ-GARCÍA EF, CORTÉS-SANABRIA L, CUETO-MANZANO AM, et al. Association of Variants of the NFE2L2 Gene with Metabolic and Kidney Function Parameters in Patients with Diabetes and/or Hypertension. Genet Test Mol Biomarkers. 2022;26(7-8): 382-390.
[48] SUN C, ZHANG N, HU Q, et al. Ferroptosis-Related Prognostic Gene LAMP2 Is a Potential Biomarker Differential Expressed in Castration Resistant Prostate Cancer. Dis Markers. 2023;2023:8295113.
[49] BARNDT RJ, LIU Q, TANG Y, et al. Metabolic Maturation Exaggerates Abnormal Calcium Handling in a Lamp2 Knockout Human Pluripotent Stem Cell-Derived Cardiomyocyte Model of Danon Disease. Biomolecules. 2022;13(1):69.
[50] SHALATA A, BAR-SHAI M, HADID Y, et al. Danon Disease: Entire LAMP2 Gene Deletion with Unusual Clinical Presentation-Case Report and Review of the Literature. Genes (Basel). 2023;14(8): 1539.
[51] LIU SP, LI XM, LIU DM, et al. LAMP2 as a Biomarker Related to Prognosis and Immune Infiltration in Esophageal Cancer and Other Cancers: A Comprehensive Pan-Cancer Analysis. Front Oncol. 2022; 12:884448.
[52] XING R, LIU D, CHENG X, et al. MiR-207 inhibits autophagy and promotes apoptosis of cardiomyocytes by directly targeting LAMP2 in type 2 diabetic cardiomyopathy. Biochem Biophys Res Commun. 2019;520(1):27-34.
[53] FU D, WANG S, LUO Y, et al. Identification of a novel splicing-altering LAMP2 variant in a Chinese family with Danon disease. ESC Heart Fail. 2023;10(4):2479-2486.
[54] FRIEDMANN ANGELI JP, KRYSKO DV, CONRAD M. Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer. 2019;19(7):405-414.
[55] FENG R, XIONG Y, LEI Y, et al. Lysine-specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4/NOX4 pathway in mice. J Cell Mol Med. 2022;26(15):4254-4267.
[56] ZANFI ED, FANTINI S, LOTTI R, et al. Wnt/CTNNB1 Signal Transduction Pathway Inhibits the Expression of ZFP36 in Squamous Cell Carcinoma, by Inducing Transcriptional Repressors SNAI1, SLUG and TWIST. Int J Mol Sci. 2020;21(16):5692.
[57] CHEN W, CHEN M, ZHAO Z, et al. ZFP36 Binds With PRC1 to Inhibit Tumor Growth and Increase 5-Fu Chemosensitivity of Hepatocellular Carcinoma. Front Mol Biosci. 2020;7:126.
[58] LYU F, LI Y, YAN Z, et al. Identification of ISG15 and ZFP36 as novel hypoxia- and immune-related gene signatures contributing to a new perspective for the treatment of prostate cancer by bioinformatics and experimental verification. J Transl Med. 2022; 20(1):202.
[59] ZHANG Z, GUO M, LI Y, et al. RNA-binding protein ZFP36/TTP protects against ferroptosis by regulating autophagy signaling pathway in hepatic stellate cells. Autophagy. 2020;16(8):1482-1505.