大黄酸对衰老小鼠成肌细胞系C2C12的成肌诱导活性和机制探讨

doi:10.19439/j.sjos.2026.01.004

摘要/Abstract

摘要： 目的体外构建衰老小鼠成肌细胞系C2C12模型,明确大黄酸的抗炎功能,探讨大黄酸对衰老C2C12的成肌诱导活性和相关免疫调节机制。方法采用CCK-8法评估不同浓度过氧化氢（H₂O₂）预处理C2C12细胞不同时间后的细胞增殖率,利用实时荧光定量反转录PCR (RT-qPCR)观察成肌、衰老相关基因变化。检测活性氧（reactive oxygen species,ROS）和衰老β-半乳糖苷酶（senescence-associated β-galactosidase, SA-β-gal）表达情况,利用免疫荧光实验比较形成的肌管直径。采用CCK-8法测定3天内不同浓度大黄酸（0、1、2、4、8、16、32 μmol/L）对小鼠巨噬细胞系RAW264.7增殖的影响,通过RT-qPCR 和免疫荧光染色检测RAW264.7细胞中极化相关因子的表达。利用RT-qPCR检测相关基因表达,通过ROS、SA-β-gal染色,免疫荧光染色等探讨大黄酸诱导下的巨噬细胞来源条件培养基（conditioned medium,CM）对衰老C2C12成肌细胞成肌功能和衰老相关表型的影响。结果与对照组相比,200和400 μmol/L H₂O₂处理C2C12细胞24 h,显著降低成肌细胞的增殖能力但不造成细胞凋亡,增强衰老相关基因、ROS和SA-β-gal的表达,降低成肌相关基因的表达,抑制肌管形成,且400 μmol/L作用效果更显著（P<0.05）。1 μmol/L浓度的大黄酸对RAW264.7细胞无细胞毒性,降低M1相关表型（TNF-α、IL-1β、iNOS、IL-6）表达,促进M2相关表型（IL-1Ra、CD206）表达（P<0.05）。大黄酸诱导下的巨噬细胞CM提高了衰老C2C12细胞中成肌基因的表达和肌管直径,并降低衰老相关基因、ROS和SA-β-gal的表达（P<0.05）。结论 H₂O₂作用于C2C12可以模拟体内衰老肌肉干细胞,而大黄酸可通过调节巨噬细胞M2型极化提高衰老肌肉成肌能力。

关键词: 大黄酸, 老龄化, 骨骼肌再生, 巨噬细胞, M1/M2 极化

Abstract: PURPOSE: To establish an in vitro model of aged murine myogenic cell line C2C12, clarify rhein's anti-inflammatory function, and explore its effects on myogenic induction activity and related immunomodulatory mechanisms in aged C2C12 cells. METHODS: CCK-8 assay was used to assess cell proliferation rates of C2C12 cells pretreated with different concentrations of hydrogen peroxide(H₂O₂) for varying duration. Real-time quantitative reverse transcription PCR (RT-qPCR) was employed to observe myogenic and aged-related gene expression changes. The expression of reactive oxygen species(ROS) and senescence-associated β-galactosidase (SA-β-gal) was measured. Immunofluorescence was used to compare the diameter of the formed myotubes. The effects of different concentrations of rhein(0, 1, 2, 4, 8, 16, 32 μmol/L) on the proliferation of murine macrophage cell line RAW264.7 over 3 days were measured using CCK-8 assay. The expression of polarization-related factors in RAW264.7 cells was detected by RT-qPCR and immunofluorescence. The effects of macrophage-conditioned medium(CM) induced by rhein on myogenic function and age-related phenotypes in aged C2C12 cells were explored through experiments including RT-qPCR to detect relevant gene expression, ROS and SA-β-gal staining, and immunofluorescence staining. RESULTS: Compared with the control group, treatment of C2C12 cells with 200 and 400 μmol/L H₂O₂ for 24 hours significantly reduced the proliferation ability of C2C12 without causing apoptosis, increased the expression of age-related genes, ROS, and SA-β-gal, decreased the expression of myogenic genes, and inhibited myotube formation, with more pronounced effects at 400 μmol/L(P<0.05). Rhein at a concentration of 1 μmol/L was non-cytotoxic to RAW264.7 cells, reduced the expression of M1-related phenotypes (TNF-α, IL-1β, iNOS, IL-6), and promoted the expression of M2-related phenotypes (IL-1Ra, CD206)(P<0.05). Macrophage-conditioned medium (CM) induced by rhein enhanced the expression of myogenic genes in aged C2C12 cells, increased the diameter of myotube, and reduced the expression of age-related genes, ROS, and SA-β-gal(P<0.05). CONCLUSIONS: An in vitro model of aged muscle stem cells can be established using H₂O₂. Rhein can enhance the myogenic capacity of aged muscle, likely through the regulation of macrophage M2 polarization.

Key words: Rhein, Aged, Skeletal muscle regeneration, Macrophages, M1/M2 polarization

中图分类号:

R739.8

泮灿灿, 祁磊, 司家文, 沈国芳. 大黄酸对衰老小鼠成肌细胞系C2C12的成肌诱导活性和机制探讨[J]. 上海口腔医学, 2026, 35(1): 19-28.

Pan Cancan, Qi Lei, Si Jiawen, Shen Guofang. Myogenic induction activity and mechanism of rhein in aged mouse myoblasts C2C12 cell line[J]. Shanghai Journal of Stomatology, 2026, 35(1): 19-28.

参考文献

[1] Yang Y, Zhou M, Zeng X, et al.The burden of oral cancer in China, 1990-2017: an analysis for the Global Burden of Disease, Injuries, and Risk Factors Study 2017[J]. BMC Oral Health, 2021, 21(1): 44.
[2] Bray F, Laversanne M, Sung H, et al.Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2024, 74(3): 229-263.
[3] GBD 2019 Chronic Respiratory Diseases Collaborators. Global burden of chronic respiratory diseases and risk factors, 1990-2019: an update from the Global Burden of Disease Study 2019BD 2019 Chronic Respiratory Diseases Collaborators. Global burden of chronic respiratory diseases and risk factors, 1990-2019: an update from the Global Burden of Disease Study 2019[J]. EClinicalMedicine, 2023, 59: 101936.
[4] Archontaki M, Athanasiou A, Stavrianos SD, et al.Functional results of speech and swallowing after oral microvascular free flap reconstruction[J]. Eur Arch Otorhinolaryngol, 2010, 267(11): 1771-1777.
[5] Yuan S, Larsson SC.Epidemiology of sarcopenia: prevalence, risk factors, and consequences[J]. Metabolism, 2023, 144: 155533.
[6] Liu J, Saul D, Böker KO, et al.Current methods for skeletal muscle tissue repair and regeneration[J]. Biomed Res Int, 2018: 1984879.
[7] Li MT, Willett NJ, Uhrig BA, et al.Functional analysis of limb recovery following autograft treatment of volumetric muscle loss in the quadriceps femoris[J]. J Biomech, 2014, 47(9): 2013-2021.
[8] Eugenis I, Wu D, Rando TA.Cells, scaffolds, and bioactive factors: engineering strategies for improving regeneration following volumetric muscle loss[J]. Biomaterials, 2021, 278: 121173.
[9] Yeh TS, Lei TH, Barnes MJ, et al.Astragalosides supplementation enhances intrinsic muscle repair capacity following eccentric exercise-induced injury[J]. Nutrients, 2022, 14(20): 4339.
[10] Song W, Liu M, Wu J, et al.Preclinical pharmacokinetics of triptolide: a potential antitumor drug[J]. Curr Drug Metab, 2019, 20(2): 147-154.
[11] Pan Y, Song D, Zhou W, et al.Baicalin inhibits C2C12 myoblast apoptosis and prevents against skeletal muscle injury[J]. Mol Med Rep, 2019, 20(1): 709-718.
[12] Huang Z, Fang Q, Ma W, et al.Skeletal muscle atrophy was alleviated by salidroside through suppressing oxidative stress and inflammation during denervation[J]. Front Pharmacol, 2019, 10: 997.
[13] Sheng X, Wang M, Lu M, et al.Rhein ameliorates fatty liver disease through negative energy balance, hepatic lipogenic regulation, and immunomodulation in diet-induced obese mice[J]. Am J Physiol Endocrinol Metab, 2011, 300(5): E886-E893.
[14] Wang J, Zhang J, Guo Z, et al.Targeting HSP70 chaperones by rhein sensitizes liver cancer to artemisinin derivatives[J]. Phytomedicine, 2024, 122: 155156.
[15] Liu X, Wu J, Tian R, et al.Targeting foam cell formation and macrophage polarization in atherosclerosis: the therapeutic potential of rhubarb[J]. Biomed Pharmacother, 2020, 129: 110433.
[16] Deng T, Du J, Yin Y, et al.Rhein for treating diabetes mellitus: a pharmacological and mechanistic overview[J]. Front Pharmacol, 2022, 13: 1106260.
[17] Zhao W, Zhang X, Zhang R, et al.Self-assembled herbal medicine encapsulated by an oxidation-sensitive supramolecular hydrogel for chronic wound treatment[J]. ACS Appl Mater Interfaces, 2020, 12(51): 56898-56907.
[18] Yin C, Han X, Lu Q, et al.Rhein incorporated silk fibroin hydrogels with antibacterial and anti-inflammatory efficacy to promote healing of bacteria-infected burn wounds[J]. Int J Biol Macromol, 2022, 201: 14-19.
[19] Jia Z, Yang C, Jiao J, et al.Rhein and polydimethylsiloxane functionalized carbon/carbon composites as prosthetic implants for bone repair applications[J]. Biomed Mater, 2017, 12(4): 045004.
[20] Zhou F, Li M, Chen M, et al.Redox homeostasis strategy for inflammatory macrophage reprogramming in rheumatoid arthritis based on ceria oxide nanozyme-complexed biopolymeric micelles[J]. ACS Nano, 2023, 17(5): 4358-4372.
[21] Zheng J, Fan R, Wu H, et al.Directed self-assembly of herbal small molecules into sustained release hydrogels for treating neural inflammation[J]. Nat Commun, 2019, 10(1): 1604.
[22] Chazaud B.Inflammation and skeletal muscle regeneration: leave it to the macrophages![J]. Trends Immunol, 2020, 41(6): 481-492.
[23] Tidball JG.Regulation of muscle growth and regeneration by the immune system[J]. Nat Rev Immunol, 2017, 17(3): 165-178.
[24] Zhang J, Muri J, Fitzgerald G, et al. Endothelial lactate controls muscle regeneration from ischemia by inducing M2-like macrophage polarization [J]. Cell Metab, 2020, 31(6): 1136-1153.e7.
[25] Lin B, Lin J, Wang F, et al.Computed tomography-defined sarcopenia as a risk factor for short-term postoperative complications in oral cancer patients with free flap reconstruction: a retrospective population-based cohort study[J]. Head Neck, 2023, 45(10): 2555-2570.
[26] Ye C, Zheng X, Aihemaitijiang S, et al.Sarcopenia and catastrophic health expenditure by socio-economic groups in China: an analysis of household-based panel data[J]. J Cachexia Sarcopenia Muscle, 2022, 13(3): 1938-1947.
[27] Domingues-Faria C, Vasson MP, Goncalves-Mendes N, et al.Skeletal muscle regeneration and impact of aging and nutrition[J]. Ageing Res Rev, 2016, 26: 22-36.
[28] Shaw AC, Goldstein DR, Montgomery RR.Age-dependent dysregulation of innate immunity[J]. Nat Rev Immunol, 2013, 13(12): 875-887.
[29] Tobin SW, Alibhai FJ, Wlodarek L, et al.Delineating the relationship between immune system aging and myogenesis in muscle repair[J]. Aging Cell, 2021, 20(2): e13312.
[30] Sousa NS, Brás MF, Antunes IB, et al.Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration[J]. Nat Aging, 2023, 3(5): 585-599.
[31] Sousa-Victor P, García-Prat L, Muñoz-Cánoves P.Control of satellite cell function in muscle regeneration and its disruption in ageing[J]. Nat Rev Mol Cell Biol, 2022, 23(3): 204-226.
[32] Huang W, Hickson LJ, Eirin A, et al.Cellular senescence: the good, the bad and the unknown[J]. Nat Rev Nephrol, 2022, 18(10): 611-627.
[33] Debacq-Chainiaux F, Erusalimsky JD, Campisi J, et al.Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo[J]. Nat Protoc, 2009, 4(12): 1798-1806.
[34] Boengler K, Kosiol M, Mayr M, et al.Mitochondria and ageing: role in heart, skeletal muscle and adipose tissue[J]. J Cachexia Sarcopenia Muscle, 2017, 8(3): 349-369.
[35] Wu X, Zhu N, He L, et al.5′-cytimidine monophosphate ameliorates H2O2-induced muscular atrophy in C2C12 myotubes by activating IRS-1/Akt/S6K pathway[J]. Antioxidants (Basel), 2024, 13(2): 249.
[36] Jung S, Ahn B.Filbertone reduces senescence in C2C12 myotubes treated with doxorubicin or H₂O₂ through MuRF1 and myogenin[J]. Nutrients, 2024, 16(18): 3177-3177.
[37] Kim A, Park SM, Kim NS, et al.Ginsenoside Rc, an active component of Panax ginseng, alleviates oxidative stress-induced muscle atrophy via improvement of mitochondrial biogenesis[J]. Antioxidants (Basel), 2023, 12(8): 1576.
[38] Pillon NJ, Bilan PJ, Fink LN, et al.Cross-talk between skeletal muscle and immune cells: muscle-derived mediators and metabolic implications[J]. Am J Physiol Endocrinol Metab, 2013, 304(5): E453-E465.
[39] Tidball JG, Flores I, Welc SS, et al.Aging of the immune system and impaired muscle regeneration: a failure of immunomodulation of adult myogenesis[J]. Exp Gerontol, 2021, 145: 111200.
[40] Fountain WA, Naruse M, Claiborne A, et al.Controlling inflammation improves aging skeletal muscle health[J]. Exerc Sport Sci Rev, 2023, 51(2): 51-56.
[41] Franceschi C, Campisi J.Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases[J]. J Gerontol A Biol Sci Med Sci, 2014, 69(Suppl 1): S4-S9.
[42] Bian AL, Hu HY, Rong YD, et al.A study on relationship between elderly sarcopenia and inflammatory factors IL-6 and TNF-α[J]. Eur J Med Res, 2017, 22(1): 25.
[43] Kedlian VR, Wang Y, Liu T, et al.Human skeletal muscle aging atlas[J]. Nat Aging, 2024, 4(5): 727-744.
[44] Wang Y, Wehling-Henricks M, Welc SS, et al.Aging of the immune system causes reductions in muscle stem cell populations, promotes their shift to a fibrogenic phenotype, and modulates sarcopenia[J]. FASEB J, 2019, 33(1): 1415-1427.
[45] Zhang W, Chen H, Zhao J, et al.Body temperature-induced adhesive hyaluronate/gelatin-based hybrid hydrogel dressing for promoting skin regeneration[J]. Int J Biol Macromol, 2023, 253(Pt 3): 126848.
[46] Liang X, Ding L, Ma J, et al.Enhanced mechanical strength and sustained drug release in carrier-free silver-coordinated anthraquinone natural antibacterial anti-inflammatory hydrogel for infectious wound healing[J]. Adv Healthc Mater, 2024, 13(23): e2400841.
[47] Zhang Z, Peng S, Xu T, et al.Retinal microenvironment-protected Rhein-GFFYE nanofibers attenuate retinal ischemia-reperfusion injury via inhibiting oxidative stress and regulating microglial/macrophage M1/M2 polarization[J]. Adv Sci (Weinh), 2023, 10(30): e2302909.
[48] Zhou Y, Gao C, Vong C T, et al.Rhein regulates redox-mediated activation of NLRP3 inflammasomes in intestinal inflammation through macrophage-activated crosstalk[J]. Br J Pharmacol, 2022, 179(9): 1978-1997.
[49] Smerdu V, Karsch-Mizrachi I, Campione M, et al.Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle[J]. Am J Physiol, 1994, 267(6 Pt 1): C1723-1728.
[50] Paulin D, Li Z.Desmin: a major intermediate filament protein essential for the structural integrity and function of muscle[J]. Exp Cell Res, 2004, 301(1): 1-7.
[51] Hasty P, Bradley A, Morris JH, et al.Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene[J]. Nature, 1993, 364(6437): 501-506.
[52] Zammit PS.Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis[J]. Semin Cell Dev Biol, 2017, 72: 19-32.
[53] Clavel S, Coldefy AS, Kurkdjian E, et al.Atrophy-related ubiquitin ligases, atrogin-1 and MuRF1 are up-regulated in aged rat tibialis anterior muscle[J]. Mech Ageing Dev, 2006, 127(10): 794-801.
[54] Bodine SC, Latres E, Baumhueter S, et al.Identification of ubiquitin ligases required for skeletal muscle atrophy[J]. Science, 2001, 294(5547): 1704-1708.
[55] Wang Y, Welc SS, Wehling-Henricks M, et al.Myeloid cell-derived tumor necrosis factor-alpha promotes sarcopenia and regulates muscle cell fusion with aging muscle fibers[J]. Aging Cell, 2018, 17(6): e12828.
[56] Langen RC, Van Der Velden JL, Schols AM, et al. Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization[J]. FASEB J, 2004, 18(2): 227-237.
[57] Parameswaran N, Patial S.Tumor necrosis factor-α signaling in macrophages[J]. Crit Rev Eukaryot Gene Expr, 2010, 20(2): 87-103.
[58] Shepherd VL, Hoidal JR.Clearance of neutrophil-derived myeloperoxidase by the macrophage mannose receptor[J]. Am J Respir Cell Mol Biol, 1990, 2(4): 335-340.
[59] Deng X, He X, Huang G, et al.The mechanism of rhein ameliorating the survival rate of transplanted mesenchymal stem cells by improving myocardial microenvironment in acute myocardial infarction[J]. J Biomater Tissue Eng, 2021, 11(4): 684-689.
[60] López-Otín C, Blasco MA, Partridge L, et al.Hallmarks of aging: an expanding universe[J]. Cell, 2023, 186(2): 243-278.