Mechanism of black phosphorus regulating oxidative stress-inflammation cascade in retarding intervertebral disc degeneration

doi:10.12307/2024.250

Abstract

Abstract: BACKGROUND: Oxidative stress plays a critical role in intervertebral disc degeneration. As a reducing material with good biocompatibility, black phosphorus quantum dots have the potential to resist oxidative stress and retard intervertebral disc degeneration.
OBJECTIVE: To evaluate the effect of black phosphorus quantum dots on scavenging reactive oxygen species in the microenvironment of an intervertebral disc through in vivo and in vitro experiments, and further explore the role of black phosphorus quantum dots in Nrf2/ARE pathway and intervertebral disc inflammation.
METHODS: Black phosphorus quantum dots were prepared by a liquid exfoliation technique. (1) In vitro experiment: The nucleus pulposus cells of SD rats were isolated and extracted, and the passages 2-4 nucleus pulposus cells were cocultured with different solutions, including F12-DMEM medium (blank group), black phosphorus quantum dot solution, hydrogen peroxide solution, hydrogen peroxide+black phosphorus quantum dot solution, hydrogen peroxide+black phosphorus quantum dot+Nrf2 specific inhibitor ML385 solution. Cell live/dead staining and intracellular reactive oxygen species, mitochondrial membrane potential and western blot assay were performed respectively. (2) In vivo experiment: Thirty SD rats were randomly divided into sham operation, puncture and puncture + black phosphorus groups, with 10 rats in each group. A Co7-10 intervertebral disc degeneration model was established using intervertebral disc puncture in the puncture group and the puncture+black phosphorus group. Black phosphorus quantum dot solution was injected in the intervertebral disc after a puncture in the puncture+black phosphorus group. The intervertebral disc tissue imaging and histological staining were evaluated at 4 and 8 weeks after surgery.
RESULTS AND CONCLUSION: (1) In vitro experiment: Live/dead staining revealed that the black phosphorus quantum dots had good biocompatibility, were non-toxic to cells, and had a protective effect on nucleus pulposus cells under oxidative stress. Intracellular reactive oxygen species and JC-1 fluorescent probes showed that black phosphorus quantum dots could regulate the reduction of mitochondrial membrane potential caused by oxidative stress in nucleus pulposus cells and protected cells from hydrogen peroxidation-induced intracellular oxidative stress. Western blot analysis showed that compared with the blank group, the protein expressions of Nrf2, heme oxygenase 1, quinone oxidoreductase and type II collagen were decreased in the hydrogen peroxide group (P < 0.05), while the protein expressions of tumor necrosis factor α, interleukin 1β, matrix metalloproteinase 13 and p65 were increased (P < 0.05). The addition of black phosphorus quantum dots could reverse the inhibitory effect of hydrogen peroxide on the Nrf2 pathway and reduce the inflammatory response caused by oxidative stress, but NrF2-specific inhibitors could cancel this effect. (2) In vivo experiment: X-ray and MRI demonstrated that at 4 and 8 weeks after surgery, the intervertebral disc height and water content of nucleus pulposus in the puncture group were lower than those in the sham operation group (P < 0.05), and the intervertebral disc height and water content of nucleus pulposus in the puncture+black phosphorus group were higher than those in the puncture group (P < 0.05). Histological staining exhibited that the degree of intervertebral disc degeneration in the puncture+black phosphorus group was less than that in the puncture group, and the expression of heme oxygenase 1 protein was higher than that in the puncture+black phosphorus group. (3) Our results have indicated that black phosphorus quantum dots can exert an antioxidant effect and delay intervertebral disc degeneration by regulating Nrf2/ARE pathway.

Key words: black phosphorus quantum dot, antioxidant stress, Nrf2/ARE pathway, inflammation, intervertebral disc degeneration

CLC Number:

Kou Yu, Gu Yong, Chen Liang. Mechanism of black phosphorus regulating oxidative stress-inflammation cascade in retarding intervertebral disc degeneration[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(15): 2338-2345.

Figures/Tables 8

References

[1] CIEZA A, CAUSEY K, KAMENOV K, et al. Global estimates of the need for rehabilitation based on the Global Burden of Disease study 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2021;396(10267):2006-2017.
[2] ADAMS MA, ROUGHLEY PJ. What is intervertebral disc degeneration, and what causes it? Spine (Phila Pa 1976). 2006;31(18):2151-2161.
[3] 解志锋,刘清,刘冰,等.腰椎间盘疲劳损伤的生物力学特性[J].中国组织工程研究, 2021,25(3):339-343.
[4] LE MAITRE CL, FREEMONT AJ, HOYLAND JA. Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res Ther. 2007;9(3):R45.
[5] DING F, SHAO ZW, XIONG LM. Cell death in intervertebral disc degeneration. Apoptosis. 2013; 18(7):777-785.
[6] DIMOZI A, MAVROGONATOU E, SKLIROU A, et al. Oxidative stress inhibits the proliferation, induces premature senescence and promotes a catabolic phenotype in human nucleus pulposus intervertebral disc cells. Eur Cell Mater. 2015;30:89-102; discussion 103.
[7] SONG D, GE J, WANG Y, et al. Tea Polyphenol Attenuates Oxidative Stress-Induced Degeneration of Intervertebral Discs by Regulating the Keap1/Nrf2/ARE Pathway. Oxid Med Cell Longev. 2021; 2021:6684147.
[8] LU Y, ZHOU L, HE S, et al. Lycopene alleviates disc degeneration under oxidative stress through the Nrf2 signaling pathway. Mol Cell Probes. 2020;51:101559.
[9] VO N, NIEDERNHOFER LJ, NASTO LA, et al. An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints. J Orthop Res. 2013;31(6):831-837.
[10] NAVARRO-YEPES J, BURNS M, ANANDHAN A, et al. Oxidative stress, redox signaling, and autophagy: cell death versus survival. Antioxid Redox Signal. 2014;21(1):66-85.
[11] FENG C, YANG M, LAN M, et al. ROS: Crucial Intermediators in the Pathogenesis of Intervertebral Disc Degeneration. Oxid Med Cell Longev. 2017;2017:5601593.
[12] MA Q. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol. 2013;53:401-426.
[13] ESPINOSA-DIEZ C, MIGUEL V, MENNERICH D, et al. Antioxidant responses and cellular adjustments to oxidative stress. Redox Biol. 2015;6:183-197.
[14] MOTOHASHI H, KATSUOKA F, ENGEL JD, et al. Small Maf proteins serve as transcriptional cofactors for keratinocyte differentiation in the Keap1-Nrf2 regulatory pathway. Proc Natl Acad Sci U S A. 2004;101(17):6379-6384.
[15] ZHANG X, LIANG S, GAO X, et al. Protective Effect of Chitosan Oligosaccharide against Hydrogen Peroxide-Mediated Oxidative Damage and Cell Apoptosis via Activating Nrf2/ARE Signaling Pathway. Neurotox Res. 2021;39(6):1708-1720.
[16] TAO W, SUN W, LIU L, et al. Chitosan Oligosaccharide Attenuates Nonalcoholic Fatty Liver Disease Induced by High Fat Diet through Reducing Lipid Accumulation, Inflammation and Oxidative Stress in C57BL/6 Mice. Mar Drugs. 2019;17(11):645.
[17] SALOMONE F, GODOS J, ZELBER-SAGI S. Natural antioxidants for non-alcoholic fatty liver disease: molecular targets and clinical perspectives. Liver Int. 2016;36(1):5-20.
[18] ZHANG Y, AHMAD KA, KHAN FU, et al. Chitosan oligosaccharides prevent doxorubicin-induced oxidative stress and cardiac apoptosis through activating p38 and JNK MAPK mediated Nrf2/ARE pathway. Chem Biol Interact. 2019;305:54-65.
[19] LI W, KHOR TO, XU C, et al. Activation of Nrf2-antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis. Biochem Pharmacol. 2008;76(11):1485-1489.
[20] MA Q, KINNEER K, YE J, et al. Inhibition of nuclear factor kappaB by phenolic antioxidants: interplay between antioxidant signaling and inflammatory cytokine expression. Mol Pharmacol. 2003;64(2):211-219.
[21] ZHANG GZ, LIU MQ, CHEN HW, et al. NF-κB signalling pathways in nucleus pulposus cell function and intervertebral disc degeneration. Cell Prolif. 2021;54(7):e13057.
[22] FU HD, LI ZB, XIE HH, et al. Different-sized black phosphorus nanosheets with good cytocompatibility and high photothermal performance. Rsc Adv. 2017;7(24):14618-14624.
[23] BRENT JR, GANGULI AK, KUMAR V, et al. On the stability of surfactant-stabilised few-layer black phosphorus in aqueous media. Rsc Adv. 2016;6(90):86955-86958.
[24] HOU J, WANG H, GE Z, et al. Treating Acute Kidney Injury with Antioxidative Black Phosphorus Nanosheets. Nano Lett. 2020;20(2):1447-1454.
[25] CHENG L, CHEN Z, CAI Z, et al. Bioinspired Functional Black Phosphorus Electrospun Fibers Achieving Recruitment and Biomineralization for Staged Bone Regeneration. Small. 2020;16(50):e2005433.
[26] RAUCCI MG, FASOLINO I, CAPORALI M, et al. Exfoliated Black Phosphorus Promotes in Vitro Bone Regeneration and Suppresses Osteosarcoma Progression through Cancer-Related Inflammation Inhibition. ACS Appl Mater Interfaces. 2019;11(9):9333-9342.
[27] WANG Z, LIU Z, SU C, et al. Biodegradable Black Phosphorus-based Nanomaterials in Biomedicine: Theranostic Applications. Curr Med Chem. 2019;26(10):1788-1805.
[28] BIAN J, CAI F, CHEN H, et al. Modulation of Local Overactive Inflammation via Injectable Hydrogel Microspheres. Nano Lett. 2021;21(6): 2690-2698.
[29] XU YC, GU Y, CAI F, et al. Metabolism Balance Regulation via Antagonist-Functionalized Injectable Microsphere for Nucleus Pulposus Regeneration. Adv Funct Mater. 2020;30(52): 14.
[30] 叶旭文,顾勇,陈亮.负载姜黄素可注射微球延缓椎间盘的退变[J].中国组织工程研究, 2023,27(12):1884-1891.
[31] MASUDA K, AOTA Y, MUEHLEMAN C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976). 2005;30(1):5-14.
[32] CHEN H, LIU Z, WEI B, et al. Redox responsive nanoparticle encapsulating black phosphorus quantum dots for cancer theranostics. Bioact Mater. 2021;6(3):655-665.
[33] GÓMEZ-PÉREZ J, BARNA B, TÓTH IY, et al. Quantitative Tracking of the Oxidation of Black Phosphorus in the Few-Layer Regime. ACS Omega. 2018;3(10):12482-12488.
[34] BELLEZZA I, GIAMBANCO I, MINELLI A, et al. Nrf2-Keap1 signaling in oxidative and reductive stress. Biochim Biophys Acta Mol Cell Res. 2018;1865(5):721-733.
[35] HAN B, ZHU K, LI FC, et al. A simple disc degeneration model induced by percutaneous needle puncture in the rat tail. Spine (Phila Pa 1976). 2008;33(18):1925-1934.
[36] HUA W, LI S, LUO R, et al. Icariin protects human nucleus pulposus cells from hydrogen peroxide-induced mitochondria-mediated apoptosis by activating nuclear factor erythroid 2-related factor 2. Biochim Biophys Acta Mol Basis Dis. 2020;1866(1):165575.
[37] Tang Z, Hu B, Zang F, et al. Nrf2 drives oxidative stress-induced autophagy in nucleus pulposus cells via a Keap1/Nrf2/p62 feedback loop to protect intervertebral disc from degeneration. Cell Death Dis. 2019;10(7):510.
[38] HE R, CUI M, LIN H, et al. Melatonin resists oxidative stress-induced apoptosis in nucleus pulposus cells. Life Sci. 2018;199:122-130.
[39] KIM KW, CHUNG HN, HA KY, et al. Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs. Spine J. 2009;9(8):658-666.
[40] FINKEL T. Signal transduction by reactive oxygen species. J Cell Biol. 2011;194(1):7-15.
[41] THURAKKAL S, ZHANG X. Recent Advances in Chemical Functionalization of 2D Black Phosphorous Nanosheets. Adv Sci (Weinh). 2020;7(2):1902359.
[42] WANG H, YANG X, SHAO W, et al. Ultrathin Black Phosphorus Nanosheets for Efficient Singlet Oxygen Generation. J Am Chem Soc. 2015;137(35):11376-11382.
[43] DING F, SHAO ZW, YANG SH, et al. Role of mitochondrial pathway in compression-induced apoptosis of nucleus pulposus cells. Apoptosis. 2012;17(6):579-590.
[44] NASTO LA, ROBINSON AR, NGO K, et al. Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration. J Orthop Res. 2013;31(7):1150-1157.
[45] BUENDIA I, MICHALSKA P, NAVARRO E, et al. Nrf2-ARE pathway: An emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacol Ther. 2016;157: 84-104.
[46] WANG P, ZHANG S, LIU W, et al. Selenium Attenuates TBHP-Induced Apoptosis of Nucleus Pulposus Cells by Suppressing Mitochondrial Fission through Activating Nuclear Factor Erythroid 2-Related Factor 2. Oxid Med Cell Longev. 2022;2022:7531788.
[47] LI Y, CHEN L, GAO Y, et al. Oxidative Stress and Intervertebral Disc Degeneration: Pathophysiology, Signaling Pathway, and Therapy. Oxid Med Cell Longev. 2022;2022:1984742.
[48] FENG C, ZHANG Y, YANG M, et al. Oxygen-Sensing Nox4 Generates Genotoxic ROS to Induce Premature Senescence of Nucleus Pulposus Cells through MAPK and NF-κB Pathways. Oxid Med Cell Longev. 2017;2017:7426458.
[49] 杨林,石军,郭中华,等.基于高分子材料的椎间盘组织工程：研究重点和热点[J].中国组织工程研究,2021,25(16):2589-2596.