Prim-O-glucosylcimifugin ameliorates aging-impaired endogenous tendon regeneration by rejuvenating senescent tendon stem/progenitor cells

Abstract

Adult tendon stem/progenitor cells (TSPCs) are essential for tendon maintenance, regeneration, and repair, yet they become susceptible to senescence with age, impairing the self-healing capacity of tendons. In this study, we employ a recently developed deep-learning-based efficacy prediction system to screen potential stemness-promoting and senescence-inhibiting drugs from natural products using the transcriptional signatures of stemness. The top-ranked candidate, prim-O-glucosylcimifugin (POG), a saposhnikovia root extract, could ameliorate TPSC senescent phenotypes caused by long-term passage and natural aging in rats and humans, as well as restore the self-renewal and proliferative capacities and tenogenic potential of aged TSPCs. In vivo, the systematic administration of POG or the local delivery of POG nanoparticles functionally rescued endogenous tendon regeneration and repair in aged rats to levels similar to those of normal animals. Mechanistically, POG protects TSPCs against functional impairment during both passage-induced and natural aging by simultaneously suppressing nuclear factor-κB and decreasing mTOR signaling with the induction of autophagy. Thus, the strategy of pharmacological intervention with the deep learning-predicted compound POG could rejuvenate aged TSPCs and improve the regenerative capacity of aged tendons.

References

[1] Nourissat G, Berenbaum F, Duprez D. Tendon injury: from biology to tendon repair. Nat. Rev. Rheumatol., 2015, 11: 223-233

[2] LaPrade CM et al. Return-to-play and performance after operative treatment of Achilles tendon rupture in elite male athletes: a scoping review. Br. J. Sports Med., 2022, 56: 515-520

[3] Teunis T, Lubberts B, Reilly BT, Ring D. A systematic review and pooled analysis of the prevalence of rotator cuff disease with increasing age. J. Shoulder Elb. Surg., 2014, 23: 1913-1921

[4] Lui PPY, Wong CM. Biology of tendon stem cells and tendon in aging. Front. Genet., 2019, 10: 1338

[5] Svensson RB, Heinemeier KM, Couppé C, Kjaer M, Magnusson SP. Effect of aging and exercise on the tendon. J. Appl. Physiol. (1985), 2016, 121: 1237-1246

[6] Marqueti RC et al. Effects of aging and resistance training in rat tendon remodeling. FASEB J., 2018, 32: 353-368

[7] Lee CH et al. Harnessing endogenous stem/progenitor cells for tendon regeneration. J. Clin. Investig., 2015, 125: 2690-2701

[8] Zhou Z et al. Tendon-derived stem/progenitor cell aging: defective self-renewal and altered fate. Aging Cell, 2010, 9: 911-915

[9] Birch HL, Peffers MJ, Clegg PD. Influence of ageing on tendon homeostasis. Adv. Exp. Med. Biol., 2016, 920: 247-260

[10] Chakkalakal JV, Jones KM, Basson MA, Brack AS. The aged niche disrupts muscle stem cell quiescence. Nature, 2012, 490: 355-360

[11] Sousa-Victor P et al. Geriatric muscle stem cells switch reversible quiescence into senescence. Nature, 2014, 506: 316-321

[12] Sousa-Victor P, García-Prat L, Serrano AL, Perdiguero E, Muñoz-Cánoves P. Muscle stem cell aging: regulation and rejuvenation. Trends Endocrinol. Metab., 2015, 26: 287-296

[13] Sarkar TJ et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat. Commun., 2020, 11

[14] Ocampo A et al. In vivo amelioration of age-associated hallmarks by partial reprogramming. Cell, 2016, 167: 1719-1733.e1712

[15] Hu YF et al. Biomaterial-induced conversion of quiescent cardiomyocytes into pacemaker cells in rats. Nat. Biomed. Eng., 2022, 6: 421-434

[16] Zhang J, Li B, Wang JH. The role of engineered tendon matrix in the stemness of tendon stem cells in vitro and the promotion of tendon-like tissue formation in vivo. Biomaterials, 2011, 32: 6972-6981

[17] Zhang C et al. Histone deacetylase inhibitor treated cell sheet from mouse tendon stem/progenitor cells promotes tendon repair. Biomaterials, 2018, 172: 66-82

[18] Li Z et al. 3D culture supports long-term expansion of mouse and human nephrogenic progenitors. Cell Stem Cell, 2016, 19: 516-529

[19] Dou X et al. Short-term rapamycin treatment increases ovarian lifespan in young and middle-aged female mice. Aging Cell, 2017, 16: 825-836

[20] Guo J et al. Aging and aging-related diseases: from molecular mechanisms to interventions and treatments. Signal Transduct. Target. Ther., 2022, 7: 391

[21] Howell K et al. Novel model of tendon regeneration reveals distinct cell mechanisms underlying regenerative and fibrotic tendon healing. Sci. Rep., 2017, 7

[22] Wang Y et al. Functional regeneration and repair of tendons using biomimetic scaffolds loaded with recombinant periostin. Nat. Commun., 2021, 12

[23] Zhu J et al. Prediction of drug efficacy from transcriptional profiles with deep learning. Nat. Biotechnol., 2021, 39: 1444-1452

[24] Zhou J et al. Prim-O-glucosylcimifugin attenuates lipopolysaccharideinduced inflammatory response in RAW 264.7 macrophages. Pharmacogn. Mag., 2017, 13: 378-384

[25] Gao W et al. Prim-O-glucosylcimifugin enhances the antitumour effect of PD-1 inhibition by targeting myeloid-derived suppressor cells. J. Immunother. Cancer, 2019, 7: 231

[26] Wang W et al. A genome-wide CRISPR-based screen identifies KAT7 as a driver of cellular senescence. Sci. Transl. Med., 2021, 13: eabd2655

[27] Deng P et al. Loss of KDM4B exacerbates bone-fat imbalance and mesenchymal stromal cell exhaustion in skeletal aging. Cell Stem Cell, 2021, 28: 1057-1073.e1057

[28] Liao N et al. Antioxidants inhibit cell senescence and preserve stemness of adipose tissue-derived stem cells by reducing ROS generation during long-term in vitro expansion. Stem Cell Res. Ther., 2019, 10: 306

[29] Yin Y, Liu K, Li G. Protective effect of Prim-O-Glucosylcimifugin on ulcerative colitis and its mechanism. Front. Pharmacol., 2022, 13: 882924

[30] Kessler M et al. Chronic Chlamydia infection in human organoids increases stemness and promotes age-dependent CpG methylation. Nat. Commun., 2019, 10

[31] Kessler M et al. The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids. Nat. Commun., 2015, 6

[32] Castilho RM, Squarize CH, Chodosh LA, Williams BO, Gutkind JS. mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging. Cell Stem Cell, 2009, 5: 279-289

[33] Chien Y et al. Control of the senescence-associated secretory phenotype by NF-κB promotes senescence and enhances chemosensitivity. Genes Dev., 2011, 25: 2125-2136

[34] Chen Y et al. Targeted pathological collagen delivery of sustained-release rapamycin to prevent heterotopic ossification. Sci. Adv., 2020, 6: eaay9526

[35] Abraham AC et al. Targeting the NF-κB signaling pathway in chronic tendon disease. Sci. Transl. Med., 2019, 11: eaav4319

[36] Wang C et al. Inhibition of IKKβ/NF-κB signaling facilitates tendinopathy healing by rejuvenating inflamm-aging induced tendon-derived stem/progenitor cell senescence. Mol. Ther. Nucleic Acids, 2022, 27: 562-576

[37] Chen S et al. RelA/p65 inhibition prevents tendon adhesion by modulating inflammation, cell proliferation, and apoptosis. Cell Death Dis., 2017, 8

[38] Yu B et al. Wnt4 signaling prevents skeletal aging and inflammation by inhibiting nuclear factor-κB. Nat. Med., 2014, 20: 1009-1017

[39] Li W et al. Subcutaneously engineered autologous extracellular matrix scaffolds with aligned microchannels for enhanced tendon regeneration: aligned microchannel scaffolds for tendon repair. Biomaterials, 2019, 224: 119488

[40] Best KT et al. NF-κB activation persists into the remodeling phase of tendon healing and promotes myofibroblast survival. Sci. Signal, 2020, 13: eabb7209

[41] Chang J et al. NF-κB inhibits osteogenic differentiation of mesenchymal stem cells by promoting β-catenin degradation. Proc. Natl. Acad. Sci. USA, 2013, 110: 9469-9474

[42] Jurk D et al. Chronic inflammation induces telomere dysfunction and accelerates ageing in mice. Nat. Commun., 2014, 2

[43] Jeon OH et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat. Med., 2017, 23: 775-781

[44] Hou Y et al. NAD(+) supplementation reduces neuroinflammation and cell senescence in a transgenic mouse model of Alzheimer’s disease via cGAS-STING. Proc. Natl. Acad. Sci. USA, 2021, 118: e2011226118

[45] Iglesias M et al. Downregulation of mTOR signaling increases stem cell population telomere length during starvation of immortal planarians. Stem Cell Rep., 2019, 13: 405-418

[46] Wang S et al. Transient activation of autophagy via Sox2-mediated suppression of mTOR is an important early step in reprogramming to pluripotency. Cell Stem Cell, 2013, 13: 617-625

[47] García-Prat L et al. Autophagy maintains stemness by preventing senescence. Nature, 2016, 529: 37-42

[48] Leidal AM, Levine B, Debnath J. Autophagy and the cell biology of age-related disease. Nat. Cell Biol., 2018, 20: 1338-1348

[49] Park JT, Lee YS, Cho KA, Park SC. Adjustment of the lysosomal-mitochondrial axis for control of cellular senescence. Ageing Res. Rev., 2018, 47: 176-182

[50] Young AR et al. Autophagy mediates the mitotic senescence transition. Genes Dev., 2009, 23: 798-803

[51] Leeman DS et al. Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging. Science, 2018, 359: 1277-1283

[52] Debacq-Chainiaux F, Erusalimsky JD, Campisi J, Toussaint O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat. Protoc., 2009, 4: 1798-1806

[53] Aman Y et al. Autophagy in healthy aging and disease. Nat. Aging, 2021, 1: 634-650

[54] Carnio S et al. Autophagy impairment in muscle induces neuromuscular junction degeneration and precocious aging. Cell Rep., 2014, 8: 1509-1521

[55] Kohler J et al. Uncovering the cellular and molecular changes in tendon stem/progenitor cells attributed to tendon aging and degeneration. Aging Cell, 2013, 12: 988-999

[56] Wang Z et al. Functional regeneration of tendons using scaffolds with physical anisotropy engineered via microarchitectural manipulation. Sci. Adv., 2018, 4: eaat4537

[57] Howell KL et al. Macrophage depletion impairs neonatal tendon regeneration. FASEB J., 2021, 35: e21618

[58] Walia B, Li TM, Crosio G, Montero AM, Huang AH. Axin2-lineage cells contribute to neonatal tendon regeneration. Connect. Tissue Res., 2022, 63: 530-543

[59] Murrell GA et al. The Achilles functional index. J. Orthop. Res., 1992, 10: 398-404

[60] Yang S et al. Oriented collagen fiber membranes formed through counter-rotating extrusion and their application in tendon regeneration. Biomaterials, 2019, 207: 61-75

[61] Yin Z et al. Single-cell analysis reveals a nestin(+) tendon stem/progenitor cell population with strong tenogenic potentiality. Sci. Adv., 2016, 2: e1600874

[62] Saraswat K, Rizvi SI. Novel strategies for anti-aging drug discovery. Expert Opin. Drug Discov., 2017, 12: 955-966

[63] Conboy IM, Conboy MJ, Rebo J. Systemic problems: a perspective on stem cell aging and rejuvenation. Aging (Albany NY), 2015, 7: 754-765

[64] Wang R, Wang Y, Zhu L, Liu Y, Li W. Epigenetic regulation in mesenchymal stem cell aging and differentiation and osteoporosis. Stem Cells Int., 2020, 2020: 8836258

[65] Xie C et al. Amelioration of Alzheimer’s disease pathology by mitophagy inducers identified via machine learning and a cross-species workflow. Nat. Biomed. Eng., 2022, 6: 76-93

[66] Stokes JM et al. A deep learning approach to antibiotic discovery. Cell, 2020, 180: 688-702.e613

[67] Chou LY, Ho CT, Hung SC. Paracrine senescence of mesenchymal stromalcells involves inflammatory cytokines and the NF-κB pathway. Cells, 2022, 11: 3324

[68] Di Micco R, Krizhanovsky V, Baker D, d’Adda di Fagagna F. Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat. Rev. Mol. Cell Biol., 2021, 22: 75-95

[69] Zhang X et al. Characterization of cellular senescence in aging skeletal muscle. Nat. Aging, 2022, 2: 601-615

[70] Ji ML et al. Sirt6 attenuates chondrocyte senescence and osteoarthritis progression. Nat. Commun., 2022, 13

[71] Liu F, Wu S, Ren H, Gu J. Klotho suppresses RIG-I-mediated senescence-associated inflammation. Nat. Cell Biol., 2011, 13: 254-262

[72] Li Y et al. Melatonin promotes the restoration of bone defects via enhancement of miR-335-5p combined with inhibition of TNFα/NF-κB signaling. FASEB J., 2023, 37: e22711

[73] Wang N et al. TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin. Open Biol., 2016, 6: 150258

[74] An S et al. Inhibition of 3-phosphoinositide-dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts. Proc. Natl. Acad. Sci. USA, 2020, 117: 31535-31546

[75] Graça AL, Gomez-Florit M, Gomes ME, Docheva D. Tendon aging. Subcell Biochem., 2023, 103: 121-147

[76] Muscat S, Nichols AEC, Gira E, Loiselle AE. CCR2 is expressed by tendon resident macrophage and T cells, while CCR2 deficiency impairs tendon healing via blunted involvement of tendon-resident and circulating monocytes/macrophages. FASEB J., 2022, 36: e22607

[77] Xu H et al. Bioactive glass-elicited stem cell-derived extracellular vesicles regulate M2 macrophage polarization and angiogenesis to improve tendon regeneration and functional recovery. Biomaterials, 2023, 294: 121998

[78] Wang Y et al. Aspirin inhibits adipogenesis of tendon stem cells and lipids accumulation in rat injury tendon through regulating PTEN/PI3K/AKT signalling. J. Cell Mol. Med., 2019, 23: 7535-7544

[79] Li Y et al. Comparative pharmacokinetics of prim-O-glucosylcimifugin and cimifugin by liquid chromatography-mass spectrometry after oral administration of Radix Saposhnikoviae extract, cimifugin monomer solution and prim-O-glucosylcimifugin monomer solution to rats. Biomed. Chromatogr., 2012, 26: 1234-1240

[80] Gu Y, Piao X, Zhu D. Simultaneous determination of calycosin, prim-O-glucosylcimifugin, and paeoniflorin in rat plasma by HPLC-MS/MS: application in the pharmacokinetic analysis of HQCF. J. Int. Med. Res., 2020, 48: 300060520972902

Funding

National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)(81871492)