二甲双胍干预骨关节炎模型小鼠早期骨关节炎软骨及软骨下骨变化
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摘要
背景:有研究表明,二甲双胍具有抗炎的作用,能够有效抑制白细胞介素1β、肿瘤坏死因子α等炎症因子的表达,可通过激活AMPK信号通路影响骨骼的代谢,但其对关节软骨及软骨下骨的作用尚未明确。目的:探讨二甲双胍对小鼠早期骨关节炎关节软骨的损伤及软骨下骨病理改变的影响。方法:SPF级雄性C57小鼠,出生3 d内乳鼠(用于原代软骨细胞培养)均由南方医科大学实验动物中心提供,实验方案经南方医科大学第三附属医院动物实验伦理委员会批准。①造模实验:将8周龄C57雄性小鼠随机分成模型对照组和造模+二甲双胍组,均建立创伤性骨关节炎模型;造模+二甲双胍组小鼠造模后用二甲双胍200 mg/(kg·d)灌胃给药;模型对照组灌胃等量生理盐水。用药后5周小鼠麻醉后取膝关节标本,番红O快速绿染色、OARSI评分评估小鼠关节软骨的损伤情况及软骨下骨硬化程度;免疫组织化学染色检测基质金属蛋白酶13、Ⅱ型胶原、RUNX2、骨钙素蛋白的表达。②细胞实验:取出生3 d内乳鼠原代软骨细胞培养,先用10μg/L白细胞介素1β诱导,再用二甲双胍(5,10 mmol/L)共培养,Western Blot检测原代软骨细胞Ⅱ型胶原、RUNX2、骨钙素蛋白的表达。结果与结论:①番红O快速绿染色及OARSI评分显示:模型对照组的关节软骨损伤、软骨下骨的硬化程度与造模+二甲双胍组相比更加严重;②免疫组化的结果显示:模型对照组基质金属蛋白酶13蛋白表达显著高于造模+二甲双胍组;造模+二甲双胍组Ⅱ型胶原的表达呈上升趋势,成骨蛋白RUNX2、骨钙素表达呈下降趋势(P <0.05);③原代软骨细胞培养结果:单纯白细胞介素1β刺激下,软骨细胞基质金属蛋白酶13及RUNX2的表达明显增加,Ⅱ型胶原的表达则明显下降;加入二甲双胍后,随着其浓度的增加,基质金属蛋白酶13及RUNX2的表达呈浓度依赖性的下降,而Ⅱ型胶原的表达随二甲双胍表达呈浓度依赖性增加;④结果提示,二甲双胍能够缓解早期骨关节炎软骨及软骨基质的降解,同时能够抑制软骨下骨的硬化和重塑,从而发挥缓解早期骨关节炎进展的作用。
BACKGROUND: Metformin has been shown to hold anti-inflammatory effect, inhibit the expression of interleukin1β and tumor necrosis factor α, and affect bone metabolism by activating AMPK signaling pathway. However, the effects of metformin on cartilage and subchondral bone remain obscure. OBJECTIVE: To investigate the effects of metformin on articular cartilage injury and pathological changes of subchondral bone in early osteoarthritis in mice. METHODS: C57 mice, SPF grade and postnatal 3 day neonate mice(used for primary chondrocyte culture) were provided by Laboratory Animal Center of Southern Medical University, and the experimental protocol was approved by the Animal Ethics Committee of the Third Affiliated Hospital of Southern Medical University.(1) Modeling: Eight-week-old C57 male mice were randomly divided into control and experimental groups. The traumatic osteoarthritis model was established in all mice, the experimental group was treated with 200 mg/(kg·d) metformin by intragastric administration, and the control group the same volume of normal saline. The knee joint specimens were taken for histological analysis after 5 weeks of treatment. Safranin O fast green staining and Osteoarthritis Research Society International score were used to evaluate the damage of articular cartilage and the degree of subchondral osteosclerosis. The expression of matrix metalloproteinase 13, collagen type Ⅱ, Runt-related transcription factor 2 and osteocalcin protein was detected by immunohistochemical staining.(2) Cytological experiment: Primary chondrocytes were isolated from neonate mice born within three days, induced by 10 μg/L interleukin 1β, and co-cultured with metformin(5, 10 mmol/L). The expression levels of collagen type Ⅱ, Runt-related transcription factor 2 and osteocalcin protein in chondrocytes were detected by western blot assay. RESULTS AND CONCLUSION:(1) The results of safranin O fast green staining and Osteoarthritis Research Society International score showed that the degree of articular cartilage damage and subchondral osteosclerosis in the control group was more serious than that in the experimental group.(2) The immunohistochemical staining results revealed that the expression level of matrix metalloproteinase 13 in the control group was significantly higher than that in the experimental group. In the experimental group, the level of collagen type Ⅱ was on a rise, and the levels of Runt-related transcription factor 2 and osteocalcin proteins were on a decline(P < 0.05).(3) The expression levels of matrix metalloproteinase 13 and Runt-related transcription factor 2 were significantly increased after interleukin 1β induction, and the expression level of collagen type Ⅱ was significantly decreased. After metformin added, the expression levels of matrix metalloproteinase 13 and Runt-related transcription factor 2 were significantly decreased, and the expression level of collagen type Ⅱ was significantly increased with metformin concentration increasing.(4) In summary, metformin can alleviate the degradation of cartilage and cartilage matrix of early osteoarthritis and inhibit the sclerosis and remodeling of subchondral bone, thereby playing a role in delaying the progress of early osteoarthritis.
BACKGROUND: Metformin has been shown to hold anti-inflammatory effect, inhibit the expression of interleukin1β and tumor necrosis factor α, and affect bone metabolism by activating AMPK signaling pathway. However, the effects of metformin on cartilage and subchondral bone remain obscure. OBJECTIVE: To investigate the effects of metformin on articular cartilage injury and pathological changes of subchondral bone in early osteoarthritis in mice. METHODS: C57 mice, SPF grade and postnatal 3 day neonate mice(used for primary chondrocyte culture) were provided by Laboratory Animal Center of Southern Medical University, and the experimental protocol was approved by the Animal Ethics Committee of the Third Affiliated Hospital of Southern Medical University.(1) Modeling: Eight-week-old C57 male mice were randomly divided into control and experimental groups. The traumatic osteoarthritis model was established in all mice, the experimental group was treated with 200 mg/(kg·d) metformin by intragastric administration, and the control group the same volume of normal saline. The knee joint specimens were taken for histological analysis after 5 weeks of treatment. Safranin O fast green staining and Osteoarthritis Research Society International score were used to evaluate the damage of articular cartilage and the degree of subchondral osteosclerosis. The expression of matrix metalloproteinase 13, collagen type Ⅱ, Runt-related transcription factor 2 and osteocalcin protein was detected by immunohistochemical staining.(2) Cytological experiment: Primary chondrocytes were isolated from neonate mice born within three days, induced by 10 μg/L interleukin 1β, and co-cultured with metformin(5, 10 mmol/L). The expression levels of collagen type Ⅱ, Runt-related transcription factor 2 and osteocalcin protein in chondrocytes were detected by western blot assay. RESULTS AND CONCLUSION:(1) The results of safranin O fast green staining and Osteoarthritis Research Society International score showed that the degree of articular cartilage damage and subchondral osteosclerosis in the control group was more serious than that in the experimental group.(2) The immunohistochemical staining results revealed that the expression level of matrix metalloproteinase 13 in the control group was significantly higher than that in the experimental group. In the experimental group, the level of collagen type Ⅱ was on a rise, and the levels of Runt-related transcription factor 2 and osteocalcin proteins were on a decline(P < 0.05).(3) The expression levels of matrix metalloproteinase 13 and Runt-related transcription factor 2 were significantly increased after interleukin 1β induction, and the expression level of collagen type Ⅱ was significantly decreased. After metformin added, the expression levels of matrix metalloproteinase 13 and Runt-related transcription factor 2 were significantly decreased, and the expression level of collagen type Ⅱ was significantly increased with metformin concentration increasing.(4) In summary, metformin can alleviate the degradation of cartilage and cartilage matrix of early osteoarthritis and inhibit the sclerosis and remodeling of subchondral bone, thereby playing a role in delaying the progress of early osteoarthritis.
引文
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[2]Dieppe PA,Lohmander LS.Pathogenesis and management of pain in osteoarthritis.Lancet.2005; 365(9463):965-973.
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[6]Coxib and traditional NSAID Trialists’(CNT)Collaboration, Bhala N,Emberson J,Merhi A,et al. Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs:meta-analyses of individual participant data from randomised trials.Lancet.2013; 382:769–779.
[7]Pap T, Korb-Pap A. Cartilage damage in osteoarthritis and rheumatoid arthritis—two unequal siblings. Nat Rev Rheumatol. 2015;11(10):606-15.
[8]Wojdasiewicz P, Poniatowski?A, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis,Mediators Inflamm. 2014(2014)561459.
[9]Blasioli DJ, Kaplan DL.The roles of catabolic factors in the development of osteoarthritis, Tissue Eng. Tissue Eng Part B Rev. 2014;20(4):355-363.
[10] Wang M, Shen J, Jin H, et al.Recent progress in understanding molecular mechanisms of cartilage degeneration during osteoarthritis. Ann N Y Acad Sci. 2011;1240:61-69.
[11]范梅琳,刘云峰,章毅,等;二甲双胍对脂多糖诱导的THP-1细胞相关炎症因子及凋亡的影响[J].中华内分泌代谢杂志,2013, 29(9):801-805.
[12] Sag D,Carling D,Stout RD,et al.Adenosine 5'-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype.J Immunol.2008; 181(12):8633-8641.
[13] Glasson SS,Chambers MG,Van Den Berg WB,et al.The OARSI histopathology initiative e recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage 2010;18 Suppl 3 S17-23.
[14] Zhen G, Wen C, Jia X,et al. Inhibition of TGF-beta signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat Med.2013;19:70412.
[15] Poulet B,de Souza R,Kent AV,et al.Intermittent applied mechanical loading induces subchondral bone thickening that may be intensified locally by contiguous articular cartilage lesions. Osteoarthritis Cartilage.2015; 23:9408.
[16] Botter SM, van Osch GJ, Clockaerts S,et al.Osteoarthritis induction leads to early and temporal subchondral plate porosity in the tibial plateau of mice:an in vivo microfocal computed tomography study.Arthritis Rheum.2011; 63:26909.
[17] Chen LY, Wang Y, Terkeltaub R,et al. Activation of AMPK-SIRT3 Signaling is Chondroprotective by Preserving Mitochondrial DNA Integrity and Function.Osteoarthritis Cartilage. 2018 Nov;26(11):1539-1550.
[18] Sebastian Alers, Antje S. L?ffler, Sebastian Wesselborg et al. Role of AMPK-mT OR-Ulk1/2 in the Regulation of Autophagy:Cross Talk, Shortcuts,and Feedbacks. Mol. Cell. Biol. 2012, 32(1):2.
[19] Sahu BD, Kuncha M, Sindhura GJ, et al. Hesperidin attenuates cisplatin-induced acute renal injury by decreasing oxidative stress,inflammation and DNA damage. Phytomedicine. 2013;20(5):453-460.
[20] Liu XX, Yu DD, Chen MJ, et al.Hesperidin ameliorates lipopolysaccharideinduced acute lung injury in mice by inhibiting HMGB1 release. Int Immunopharmacol. 2015;25(2):370-376..
[21] Guilak F.Biomechanical factors in osteoarthritis. Best Pract Res Clin Rheumatol. 2011;25(6):815-823.
[22] Scholtes S, Kr?mer E, Weisser M, et al.Global chondrocyte gene expression after a single anabolic loading period:Time evolution and reinducibility of mechano-responses, J Cell Physiol 233(2018)699-711.
[23] O'Conor CJ, Leddy HA, Benefield HC,et al.TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading, Proc Natl Acad Sci U S A 111(2014)1316-1321.
[24] Praxenthaler H, Kr?mer E, Weisser M, et al.Extracellular matrix content and WNT/β-catenin levels of cartilage determine the chondrocyte response to compressive load. Biochim Biophys Acta Mol Basis Dis. 2018;1864(3):851-859.
[25] Bay-Jensen AC,Hoegh-Madsen S,Dam E,et al.Which elements are involved in reversible and irreversible cartilage degradation in osteoarthritis?Rheumatol Int 2010;30:435-442.
[26] Reimann I,Mankin HJ,Trahan C.Quantitative histologic analyses of articular-cartilage and subchondral bone from osteoarthritic and normal human hips.Acta Orthop Scand.1977; 48:63-73.
[27] Vela-Aneroá, Hermida-Gómez T, Gato-Calvo L, et al.Long-term effects of hydrogen sulfide on the anabolic-catabolic balance of articular cartilage in vitro.Nitric Oxide.2017; 70:42-50.
[28] Bauge C, Girard N, Leclercq S,et al. Regulatory mechanism of transforming growth factor beta receptor type II degradation by interleukin-1 in primary chondrocytes. Biochim Biophys Acta.2012; 1823:983-986.
[29] Song YM, Lee YH, Kim JW,et al.Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway, Autophagy, 2015 January 11:1, 46-59.
[30] Lacourt M,Gao C,Li A,et al.Relationship between cartilage and subchondral bone lesions in repetitive impact trauma-induced equine osteoarthritis.Osteoarthritis Cartilage. 2012; 20:572-583.
[31] Sharma AR, Jagga S, Lee SS, Nam JS.Interplay between cartilage and subchondral bone contributing to pathogenesis of osteoarthritis. Int J Mol Sci.2013;14:19805-19830.
[32] Yuan XL, Meng HY, Wang YC,et al.Bonecartilage interface crosstalk in osteoarthritis:Potential pathways and future therapeutic strategies.Osteoarthritis Cartilage.2014; 22:1077-1089.
[2]Dieppe PA,Lohmander LS.Pathogenesis and management of pain in osteoarthritis.Lancet.2005; 365(9463):965-973.
[3]Hootman JM,Helmick CG.Projections of US prevalence of arthritis and associated activity limitations. Arthritis Rheum.2006;54(1):226-229.
[4]Woolf AD,Pfleger B.Burden of major musculoskeletal conditions. Bull World Health Organ.2003; 81:646–656.
[5]Murray CJ,Vos T,Lozano R,et al.Disability-adjusted life years(DALYs)for 291diseases and injuries in 21 regions,1990–2010:a systematic analysis for the Global Burden of Disease Study 2010.Lancet 2012; 380:2197–223.
[6]Coxib and traditional NSAID Trialists’(CNT)Collaboration, Bhala N,Emberson J,Merhi A,et al. Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs:meta-analyses of individual participant data from randomised trials.Lancet.2013; 382:769–779.
[7]Pap T, Korb-Pap A. Cartilage damage in osteoarthritis and rheumatoid arthritis—two unequal siblings. Nat Rev Rheumatol. 2015;11(10):606-15.
[8]Wojdasiewicz P, Poniatowski?A, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis,Mediators Inflamm. 2014(2014)561459.
[9]Blasioli DJ, Kaplan DL.The roles of catabolic factors in the development of osteoarthritis, Tissue Eng. Tissue Eng Part B Rev. 2014;20(4):355-363.
[10] Wang M, Shen J, Jin H, et al.Recent progress in understanding molecular mechanisms of cartilage degeneration during osteoarthritis. Ann N Y Acad Sci. 2011;1240:61-69.
[11]范梅琳,刘云峰,章毅,等;二甲双胍对脂多糖诱导的THP-1细胞相关炎症因子及凋亡的影响[J].中华内分泌代谢杂志,2013, 29(9):801-805.
[12] Sag D,Carling D,Stout RD,et al.Adenosine 5'-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype.J Immunol.2008; 181(12):8633-8641.
[13] Glasson SS,Chambers MG,Van Den Berg WB,et al.The OARSI histopathology initiative e recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage 2010;18 Suppl 3 S17-23.
[14] Zhen G, Wen C, Jia X,et al. Inhibition of TGF-beta signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat Med.2013;19:70412.
[15] Poulet B,de Souza R,Kent AV,et al.Intermittent applied mechanical loading induces subchondral bone thickening that may be intensified locally by contiguous articular cartilage lesions. Osteoarthritis Cartilage.2015; 23:9408.
[16] Botter SM, van Osch GJ, Clockaerts S,et al.Osteoarthritis induction leads to early and temporal subchondral plate porosity in the tibial plateau of mice:an in vivo microfocal computed tomography study.Arthritis Rheum.2011; 63:26909.
[17] Chen LY, Wang Y, Terkeltaub R,et al. Activation of AMPK-SIRT3 Signaling is Chondroprotective by Preserving Mitochondrial DNA Integrity and Function.Osteoarthritis Cartilage. 2018 Nov;26(11):1539-1550.
[18] Sebastian Alers, Antje S. L?ffler, Sebastian Wesselborg et al. Role of AMPK-mT OR-Ulk1/2 in the Regulation of Autophagy:Cross Talk, Shortcuts,and Feedbacks. Mol. Cell. Biol. 2012, 32(1):2.
[19] Sahu BD, Kuncha M, Sindhura GJ, et al. Hesperidin attenuates cisplatin-induced acute renal injury by decreasing oxidative stress,inflammation and DNA damage. Phytomedicine. 2013;20(5):453-460.
[20] Liu XX, Yu DD, Chen MJ, et al.Hesperidin ameliorates lipopolysaccharideinduced acute lung injury in mice by inhibiting HMGB1 release. Int Immunopharmacol. 2015;25(2):370-376..
[21] Guilak F.Biomechanical factors in osteoarthritis. Best Pract Res Clin Rheumatol. 2011;25(6):815-823.
[22] Scholtes S, Kr?mer E, Weisser M, et al.Global chondrocyte gene expression after a single anabolic loading period:Time evolution and reinducibility of mechano-responses, J Cell Physiol 233(2018)699-711.
[23] O'Conor CJ, Leddy HA, Benefield HC,et al.TRPV4-mediated mechanotransduction regulates the metabolic response of chondrocytes to dynamic loading, Proc Natl Acad Sci U S A 111(2014)1316-1321.
[24] Praxenthaler H, Kr?mer E, Weisser M, et al.Extracellular matrix content and WNT/β-catenin levels of cartilage determine the chondrocyte response to compressive load. Biochim Biophys Acta Mol Basis Dis. 2018;1864(3):851-859.
[25] Bay-Jensen AC,Hoegh-Madsen S,Dam E,et al.Which elements are involved in reversible and irreversible cartilage degradation in osteoarthritis?Rheumatol Int 2010;30:435-442.
[26] Reimann I,Mankin HJ,Trahan C.Quantitative histologic analyses of articular-cartilage and subchondral bone from osteoarthritic and normal human hips.Acta Orthop Scand.1977; 48:63-73.
[27] Vela-Aneroá, Hermida-Gómez T, Gato-Calvo L, et al.Long-term effects of hydrogen sulfide on the anabolic-catabolic balance of articular cartilage in vitro.Nitric Oxide.2017; 70:42-50.
[28] Bauge C, Girard N, Leclercq S,et al. Regulatory mechanism of transforming growth factor beta receptor type II degradation by interleukin-1 in primary chondrocytes. Biochim Biophys Acta.2012; 1823:983-986.
[29] Song YM, Lee YH, Kim JW,et al.Metformin alleviates hepatosteatosis by restoring SIRT1-mediated autophagy induction via an AMP-activated protein kinase-independent pathway, Autophagy, 2015 January 11:1, 46-59.
[30] Lacourt M,Gao C,Li A,et al.Relationship between cartilage and subchondral bone lesions in repetitive impact trauma-induced equine osteoarthritis.Osteoarthritis Cartilage. 2012; 20:572-583.
[31] Sharma AR, Jagga S, Lee SS, Nam JS.Interplay between cartilage and subchondral bone contributing to pathogenesis of osteoarthritis. Int J Mol Sci.2013;14:19805-19830.
[32] Yuan XL, Meng HY, Wang YC,et al.Bonecartilage interface crosstalk in osteoarthritis:Potential pathways and future therapeutic strategies.Osteoarthritis Cartilage.2014; 22:1077-1089.
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