Potential mechanism of advanced glycation end products inducing inflammatory reactions in chondrocytes

doi:10.3969/j.issn.2095-4344.0826

Abstract

Abstract:

BACKGROUND: Preliminary experimental studies have shown that after rabbit chondrocytes are induced with advanced glycation end products (AGEs), the expression levels of tumor necrosis factor-α, matrix metalloproteinase 13 and matrix-degrading enzymes are significantly increased. However, the underling mechanism of AGEs-induced chondrocyte injury remains unclear.
OBJECTIVE: To explore the mechanism of AGEs-induced chondrocyte injury by observing the expression levels of inflammatory factors, matrix-degrading degrading enzymes and nuclear factor (NF) κB.
METHODS: The human chondrocytes were harvested by mechanical separation and improved two-step enzyme digestion method, and then identified by collagen type II immunohistochemical staining and immunocytochemistry. Human chondrocytes were stimulated with AGEs (1, 10, 25, 50 and 100 mg/L) for 24 hours, and those incubated with BSA and normal chondrocytes were as controls. Subsequently, the mRNA and protein expression levels of interleukin-1, matrix metalloproteinase 13 and tumor necrosis factor-α were detected by real-time PCR and western blot assay. The contents of inhibitor protein of NF-kB α (IκBα) in cytoplasm and NF-kB (p65) in nuclei were detected by western blot assay.
RESULTS AND CONCLUSION: Compared with control groups, AGEs, especially at the concentration of 100 mg/L, significantly up-regulated the expression levels of tumor necrosis factor-α, matrix metalloproteinase 13, interleukin-1 α and interleukin-1 β in the chondrocytes in a concentration-dependent manner. Moreover, AGEs significantly decreased the content of IκBα in cytoplasm and significantly increased the content of NF-kB (p65) in nucleus. To conclude, AGEs enhance the expression levels of interleukin-1, matrix metalloproteinase 13 and tumor necrosis factor-α probably via NF-KB signaling pathway.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Chondrocytes, Glycosylation End Products, Advanced, Osteoarthritis, NF-kappa B, Tissue Engineering

CLC Number:

R318

Zhao Jun1, Ma Chi1, Yang Na-na2, Tang Xin1, Liu Tang-hao1, Tan Yang-fan1, Peng Xiu-juan1, Chen Cheng3. Potential mechanism of advanced glycation end products inducing inflammatory reactions in chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(24): 3786-3791.

Figures/Tables 1

2.1 人软骨细胞培养及鉴定结果 Ⅱ型胶原免疫组织化学染色可见获得细胞胞质中含有大量棕黄色沉积物，着色较深，说明Ⅱ型胶原免疫组织化学染色为阳性(图1A)，提示获得的细胞具有人软骨细胞表型。为进一步验证上述结果，采用细胞免疫荧光化学方法进一步对细胞进行鉴定，荧光显微镜下Ⅱ型胶原被染为绿色(图1B)，进一步提示所获得的细胞具有软骨细胞表型。 2.2 晚期糖基化终末产物对人软骨细胞肿瘤坏死因子α、基质金属蛋白酶13、白细胞介素1表达的影响随着晚期糖基化终末产物质量浓度增高，肿瘤坏死因子α、基质金属蛋白酶13、白细胞介素1α、白细胞介素1β mRNA表达水平逐渐增多(图2)。在100 mg/L 晚期糖基化终末产物处理组中，白细胞介素1α mRNA表达水平是正常对照组的4.22倍，软骨细胞白细胞介素1β mRNA表达水平是正常对照组的9.39倍，基质金属蛋白酶13 mRNA表达水平是正常对照组的8.71倍，肿瘤坏死因子α mRNA表达水平是正常对照组的6.13倍，差异有显著性意义(P < 0.05)。运用Western blot检测肿瘤坏死因子α、基质金属蛋白酶13、白细胞介素1α、白细胞介素1β蛋白表达水平，结果发现与对照组比较，晚期糖基化终末产物可诱导人关节软骨细胞肿瘤坏死因子α、基质金属蛋白酶13、白细胞介素1α、白细胞介素1α表达增多(P < 0.05)，且以100 mg/L 晚期糖基化终末产物组作用最明显(图3)。 2.3 晚期糖基化终末产物对人软骨细胞核因子κB活化水平的影响与对照组比较，晚期糖基化终末产物能诱导促进软骨细胞核核因子κB p65水平升高，且细胞核核因子κB p65水平随着晚期糖基化终末产物质量浓度的升高而升高 (P < 0.05)，见图4。然而，晚期糖基化终末产物处理组软骨细胞胞浆中IKBα水平明显低于正常对照组(P < 0.05)，且其含量与晚期糖基化终末产物质量浓度呈负相关(见图5)。"

References

[1] Li MH, Xiao R, Li JB, et al.Regenerative approaches for cartilage repair in the treatment of osteoarthritis. Osteoarthritis Cartilage. 2017;25(10):1577-1587.

[2] Flemming, DJ,Gustas-French CN. Rapidly Progressive Osteoarthritis: a Review of the Clinical and Radiologic Presentation.Curr Rheumatol Rep. 2017;19(7):42.

[3] Chen C, Ma C, Zhang Y,et al.Pioglitazone inhibits advanced glycation end product-induced TNF-alpha and MMP-13 expression via the antagonism of NF-kappaB activation in chondrocytes.Pharmacology.2014;94(5-6):265-272.

[4] Yang Q, Chen C, Wu S, et al.Advanced glycation end products downregulates peroxisome proliferator-activated receptor gamma expression in cultured rabbit chondrocyte through MAPK pathway.Eur J Pharmacol. 2010;649(1-3): 108-114.

[5] Yan JY, Tian FM, Wang WY, et al. Age dependent changes in cartilage matrix, subchondral bone mass, and estradiol levels in blood serum, in naturally occurring osteoarthritis in Guinea pigs.Int J Mol Sci.2014;15(8):13578-13595.

[6] Musumeci G,Szychlinska MA,Mobasheri A. Age-related degeneration of articular cartilage in the pathogenesis of osteoarthritis: molecular markers of senescent chondrocytes. Histol Histopathol.2015;30(1):1-12.

[7] Li Y,Wei X,Zhou J,et al.The age-related changes in cartilage and osteoarthritis.Biomed Res Int.2013;2013:916530.

[8] Serrao PR,Vasilceac FA,Gramani-Say K,et al.Expression of receptors of advanced glycation end product (RAGE) and types I, III and IV collagen in the vastus lateralis muscle of men in early stages of knee osteoarthritis. Connect Tissue Res.2014;55(5-6):331-338.

[9] Yang Q,Guo S,Wang S,et al.Advanced glycation end products-induced chondrocyte apoptosis through mitochondrial dysfunction in cultured rabbit chondrocyte. Fundam Clin Pharmacol.2015;29(1):54-61.

[10] DeGroot J, Verzijl N, Wenting-van Wijk MJ, et al.Accumulation of advanced glycation end products as a molecular mechanism for aging as a risk factor in osteoarthritis.Arthritis Rheum.2004;50(4):1207-1215.

[11] Leong DJ,Sun HB.Events in articular chondrocytes with aging. Curr Osteoporos Rep.2011;9(4):196-201.

[12] Chen YJ,Sheu ML,Tsai KS,et al.Advanced glycation end products induce peroxisome proliferator-activated receptor gamma down-regulation-related inflammatory signals in human chondrocytes via Toll-like receptor-4 and receptor for advanced glycation end products.PLoS One.2013;8(6):e66611.

[13] 向登,蒋涛,贺军,等.塞来昔布对不同程度骨关节炎患者TNF-α、IL-1β及PGE-2的影响[J].中国骨与关节损伤杂志,2016,31(05): 496-498.

[14] 陈铖,马翅,张莹,等.AGEs对兔软骨细胞TNF-α和MMP-13表达的影响及其机制研究[J].湖南师范大学学报:医学版,2013,(04): 23-28

[15] Chen JR,Takahashi M,Suzuki M,et al.Comparison of the concentrations of pentosidine in the synovial fluid, serum and urine of patients with rheumatoid arthritis and osteoarthritis. Rheumatology (Oxford).1999;38(12):1275-1278.

[16] Aldini G,Vistoli G, Stefek M, et al. Molecular strategies to prevent, inhibit, and degrade advanced glycoxidation and advanced lipoxidation end products. Free Radic Res.2013;47 Suppl 1:93-137.

[17] Nowotny K,Jung T,Hohn A, et al. Advanced Glycation End Products and Oxidative Stress in Type 2 Diabetes Mellitus. Biomolecules.2015;5(1):194-222.

[18] Lee JJ,Wang PW,Yang IH,et al.Amyloid-beta mediates the receptor of advanced glycation end product-induced pro-inflammatory response via toll-like receptor 4 signaling pathway in retinal ganglion cell line RGC-5.Int J Biochem Cell Biol.2015;64:1-10.

[19] Badger AM, Griswold DE, Kapadia R, et al. Disease- modifying activity of SB 242235, a selective inhibitor of p38 mitogen-activated protein kinase, in rat adjuvant-induced arthritis. Arthritis Rheum. 2000; 43(1):175-183.

[20] Mavunkel BJ, Chakravarty S, Perumattam JJ, et al. Indole-based heterocyclic inhibitors of p38alpha MAP kinase: designing a conformationally restricted analogue. Bioorg Med Chem Lett. 2003;13(18):3087-309.