Synergistic promotion of inflammatory chondrocyte reverse differentiation by the combination of curcumin and magnesium sulfate

doi:10.3969/j.issn.2095-4344.1594

Abstract

Abstract:

BACKGROUND: Osteoarthritis is a common cause of pain and disability in the elderly. The underlying cause is the combination of inappropriate mechanical stress, inflammatory mediators and biochemical factors. Curcumin has been shown to have anti-oxidant, anti-inflammatory, anti-bacterial, anti-tumor, neuroprotective, and cardioprotective effects, radiation protection and therapeutic effects on osteoarthritis. Similarly, magnesium sulfate can relieve joint pain and inhibit joint destruction.
OBJECTIVE: To investigate the mechanism by which the combined use of curcumin and magnesium sulfate exerts synergistic effect to promote inflammatory chondrocyte reverse differentiation.
METHODS: Primary cultured inflammatory chondrocytes were subjected to monolayer culture in vivo. Cell proliferation assay (MTS) was used to detect the proliferation of inflammatory chondrocytes under in vitro monolayer culture conditions. The experimental cells were divided into four groups and underwent 3D induced reverse differentiation culture for 18 days: single culture of chondrocytes (chondrocyte group), inflammatory chondrocyte cultured with curcumin (curcumin group), inflammatory chondrocytes cultured with magnesium sulfate (magnesium sulfate group), and inflammatory chondrocytes cultured with curcumin and magnesium sulfate (combination group).
RESULTS AND CONCLUSION: MTS proliferation experiments showed that inflammatory chondrocytes at passage 3 had a higher rate of early proliferation and a lower degree of differentiation. Quantitative PCR results showed that the mRNA levels of type II collagen, proteoglycan and SOX9 were significantly higher in the combination group than in the curcumin group or magnesium sulfate group (P < 0.01). The size of the gross specimens and the positive area of chondrocyte reverse differentiation for alcian blue staining and safranin O staining in the combination group were significantly larger than those in the other three groups (P < 0.01). TUNEL staining results indicated that the positive area of apoptosis-specific staining in the combination group and magnesium sulfate group was significantly smaller than that in the other two groups (P < 0.01). Therefore, the combined use of curcumin and magnesium sulfate has the synergistic effect to promote the reverse differentiation of inflammatory chondrocytes.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Curcumin, Magnesium, Chondrocytes, Tissue Engineering

CLC Number:

R453
R364

Wang Yang, Song Zhuoyue, Li Guangheng. Synergistic promotion of inflammatory chondrocyte reverse differentiation by the combination of curcumin and magnesium sulfate[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(9): 1410-1415.

Figures/Tables 2

2.1 第2，3代原代细胞培养及其细胞增殖情况取患者炎性软骨行原代培养发现，呈现为短梭形多分枝的第3代炎性软骨细胞较第2代炎性软骨细胞多。通过MTS细胞增殖实验发现，从第4天开始第2代炎性软骨细胞的增殖速率开始明显大于第3代炎性软骨细胞(P < 0.05)，见图1，说明细胞早期增殖速率较高，分化程度较低。 2.2 软骨分化能力蛋白水平和基因水平差异免疫荧光显示在联合治疗组、姜黄素组以及MgSO4组Ⅱ型胶原免疫荧光染色均好于炎性软骨细胞组。实验通过定量分析发现Ⅱ型胶原蛋白在联合治疗组、姜黄素组以及MgSO4组的免疫荧光表达量均高于炎性软骨细胞组(P < 0.01)，而联合治疗组要高于姜黄素组和MgSO4组(P < 0.05)，见图2。通过定量PCR对联合治疗组及姜黄素组和MgSO4组进行成软骨标志物Ⅱ型胶原、SOX9及蛋白聚糖在mRNA水平检测，见图3，各组基因mRNA表达差异有显著性意义(P < 0.01)，而联合治疗组要高于姜黄素和MgSO4组(P < 0.05)。另外MgSO4组在基因SOX9的表达上好于姜黄素组(P < 0.01)。这表明联合治疗组相应的软骨逆分化能力强，可诱导更多的炎性软骨细胞逆分化为正常的软骨细胞。并且MgSO4较姜黄素调节SOX9能力更强。 2.3 3D培养细胞团块的软骨逆分化 3D培养至18 d的4组细胞团块大体观，可以看出18 d时4组细胞团块大小依次是：联合治疗组>MgSO4组>姜黄素组>炎性软骨细胞组(P < 0.05)。4组细胞团块石蜡切片后进行阿尔新蓝染色和番红O染色经定量分析发现染色发现区域面积大小：联合治疗组>MgSO4组>姜黄素组>炎性软骨细胞组(P < 0.01)，见图4。结果显示，在更接近人体的体外3D培养下，两者协同作用促进软骨细胞逆分化。 2.4 3D培养细胞团的凋亡切片后行TUNEL细胞凋亡检测，可以看出镜下凋亡的面积大小依次是炎性软骨细胞组>姜黄素组>MgSO4组>联合治疗组(P < 0.01)，见图5。"

References

[1] Johnson VL, Hunter DJ. The epidemiology of osteoarthritis.Best Pract Res Clin Rheumatol. 2014;28(1):5-15.

[2] Samuels J, Krasnokutsky S, Abramson SB. Osteoarthritis:a tale of three tissues. Bull NYU Hosp Jt Dis. 2008;66(3):244-250.

[3] Takayama K, Ishida K, Matsushita T, et al. SIRT1 regulation of apoptosis of human chondrocytes. Arthritis Rheum. 2009;60(9):2731-2740.

[4] 李云泽,赵序利.骨性关节炎发病机制研究进展[J].中国疼痛医学杂志,2016, 22(10):728-733.

[5] 王亚非,丁建中.骨性关节炎治疗的研究进展[J].北京中医药,2010, 29(11):886-888.

[6] Kalpravidh RW, Siritanaratkul N, Insain P, et al. Improvement in oxidative stress and antioxidant parameters in beta-thalassemia/Hb E patients treated with curcuminoids. Clin Biochem. 2010;43(4-5):424-429.

[7] Khan MA, El-Khatib R, Rainsford KD, et al. Synthesis and anti-inflammatory properties of some aromatic and heterocyclic aromatic curcuminoids. Bioorg Chem. 2012;40(1):30-38.

[8] Ye MX, Zhao YL, Li Y, et al. Curcumin reverses cis-platin resistance and promotes human lung adenocarcinoma A549/DDP cell apoptosis through HIF-1α and caspase-3 mechanisms. Phytomedicine. 2012; 19(8-9):779-87.

[9] Campos CA, Gianino JB, Bailey BJ, et al. Design, synthesis, and evaluation of curcumin-derived arylheptanoids for glioblastoma and neuroblastoma cytotoxicity. Bioorg Med Chem Lett. 2013;23(24): 6874-6878.

[10] Hong D, Zeng X, Xu W, et al. Altered profiles of gene expression in curcumin-treated rats with experimentally induced myocardial infarction. Pharmacol Res. 2010;61(2):142-148.

[11] Phillips J, Moore-Medlin T, Sonavane K, et al. Curcumin inhibits UV radiation-induced skin cancer in SKH-1 mice. Otolaryngol Head Neck Surg. 2013;148(5):797-803.

[12] 孙宗建,何琨,张东,等.姜黄素预先给药对兔呼吸机相关性肺损伤时Nrf2蛋白表达的影响[J].中华麻醉学杂志,2014,34(2):237-240.

[13] 王健,王祥,马捷,岳冰,等.姜黄素对白细胞介素1β诱导兔骨关节炎标志物的作用[J].中华关节外科杂志(电子版),2016,10(3):312-318.

[14] Park S, Lee LR, Seo JH, et al. Curcumin and tetrahydrocurcumin both prevent osteoarthritis symptoms and decrease the expressions of pro-inflammatory cytokines in estrogen-deficient rats. Genes Nutr. 2016; 11:2.

[15] Mathy-Hartert M, Jacquemond-Collet I, Priem F, et al. Curcumin inhibits pro-inflammatory mediators and metalloproteinase-3 production by chondrocytes. Inflamm Res. 2009;58(12):899-908.

[16] Zhang Z, Leong DJ, Xu L, et al. Curcumin slows osteoarthritis progression and relieves osteoarthritis-associated pain symptoms in a post-traumatic osteoarthritis mouse model. Arthritis Res Ther. 2016; 18(1):128.

[17] Delhumeau A, Granry JC, Monrigal JP, et al. Indications for the use of magnesium in anesthesia and intensive care. Ann Fr Anesth Reanim. 1995;14(5):406-416.

[18] Lee CH, Wen ZH, Chang YC, et al. Intra-articular magnesium sulfate (MgSO4) reduces experimental osteoarthritis and nociception: association with attenuation of N-methyl-D-aspartate (NMDA) receptor subunit 1 phosphorylation and apoptosis in rat chondrocytes. Osteoarthritis Cartilage. 2009;17(11):1485-1493.

[19] Baker JF, Walsh PM, Byrne DP, et al. In vitro assessment of human chondrocyte viability after treatment with local anaesthetic, magnesium sulphate or normal saline. Knee Surg Sports Traumatol Arthrosc. 2011; 19(6):1043-1046.

[20] Nielsen FH. Magnesium deficiency and increased inflammation: current perspectives. J Inflamm Res. 2018;11:25-34.

[21] Hu T, Xu H, Wang C, et al. Magnesium enhances the chondrogenic differentiation of mesenchymal stem cells by inhibiting activated macrophage-induced inflammation. Sci Rep. 2018;8(1):3406.

[22] Feyerabend F, Witte F, Kammal M, et al. Unphysiologically high magnesium concentrations support chondrocyte proliferation and redifferentiation. Tissue Eng. 2006;12(12):3545-3556.

[23] Charlier E, Relic B, Deroyer C, et al. Insights on molecular mechanisms of chondrocytes death in osteoarthritis. Int J Mol Sci. 2016;17(12):E2146.

[24] Jain SK, Rains J, Croad J, Larson B, Jones K. Curcumin supplementation lowers TNF-alpha, IL-6, IL-8, and MCP-1 secretion in high glucose-treated cultured monocytes and blood levels of TNF-alpha, IL-6, MCP-1, glucose, and glycosylated hemoglobin in diabetic rats. Antioxid Redox Signal. 2009;11(2):241-249.

[25] Wojdasiewicz P, Poniatowski ?A, Szukiewicz D. The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of osteoarthritis. Mediators Inflamm. 2014;2014:561459.

[26] Park JG, Yi YS, Hong YH, et al. Tabetri™ (tabebuia avellanedae ethanol extract) ameliorates osteoarthritis symptoms induced by monoiodoacetate through its anti-inflammatory and chondroprotective activities. Mediators Inflamm. 2017;2017:3619879.

[27] Goldring MB, Otero M. Inflammation in osteoarthritis. Curr Opin Rheumatol. 2011;23(5):471-478.

[28] Hsu YH, Yang YY, Huwang MH, et al. Anti-IL-20 monoclonal antibody inhibited inflammation and protected against cartilage destruction in murine models of osteoarthritis. PLoS One. 2017; 12(4):e0175802.

[29] Berenbaum F. Osteoarthritis year 2010 in review: pharmacological therapies. Osteoarthritis Cartilage. 2011;19(4):361-365.

[30] Hawker GA, Mian S, Bednis K, et al. Osteoarthritis year 2010 in review: non-pharmacologic therapy. Osteoarthritis Cartilage. 2011;19(4):366-374.

[31] Payr S, Tichy B, Atteneder C, et al. Redifferentiation of aged human articular chondrocytes by combining bone morphogenetic protein-2 and melanoma inhibitory activity protein in 3D-culture. PLoS One. 2017; 12(7):e0179729.

[32] Samavedi S, Diaz-Rodriguez P, Erndt-Marino JD, et al. A three-dimensional chondrocyte-macrophage coculture system to probe inflammation in experimental osteoarthritis. Tissue Eng Part A. 2017; 23(3-4):101-114.