Inhibitory effect of ulinastatin on osteoclast activation and the relationship of ulinastatin to matrix metalloproteinase-2 and matrix metalloproteinase-9: potential of preventing prosthetic osteolysis

doi:10.3969/j.issn.2095-4344.2014.35.011

Abstract

Abstract:

BACKGROUND: It is presumed that urinary trypsin inhibitor could have protective effects on local and systemic tissues and could inhibit osteoclast proliferation and activation under long-term chronic inflammation conditions and in ischemic and anoxic environment which was induced by prosthetic wear.
OBJECTIVE: To investigate the inhibitory effect of ulinastatin on receptor activator for nuclear factor-κb ligand-induced differentiation, proliferation and osteoclastogenesis of RAW264.7 cells and its effects on matrix metalloproteinase-2, matrix metalloproteinase-9 expression level and activity.
METHODS: Mouse monocyte/macrophage cell line RAW264.7 was treated with different concentrations of urinary trypsin inhibitor (0, 500, 5 000 U/mL) for 24, 48 and 72 hours. Experiments were divided into four groups: the blank group (RAW264.7 cells), receptor activator for nuclear factor-κb ligand-induced group (0 U/mL ulinastatin), 500 U/mL ulinastatin group and 5 000 U/mL ulinastatin group.
RESULTS AND CONCLUSION: (1) MTT results indicated that there was no significant difference on the proliferation of RAW264.7 cells treated with urinary trypsin inhibitor at 0-5 000 U/mL (P > 0.05) (2) Tartrate-resistant acid phosphatase staining results revealed that compared with receptor activator for nuclear factor-κb ligand-induced group, the number of tartrate-resistant acid phosphatase-positive cells was significantly less in the ulinastatin group (P < 0.05), showing a time-dose dependent manner. (3) Immunohistochemisical results found that compared with receptor activator for nuclear factor-κb ligand-induced group, the percentage of matrix metalloproteinase-9-positive cells was apparently lower in the ulinastatin group. (4) Western blot assay results demonstrated that matrix metalloproteinase-9 expression was low in the RAW264.7 cells alone. At 48 hours after addition of receptor activator for nuclear factor-κb ligand, matrix metalloproteinase-9 protein expression was large. At 72 hours after culture in the 5 000 U/mL ulinastatin group, matrix metalloproteinase-9 protein expression was evidently reduced. (5) Gelatin zymography results showed that compared with the receptor activator for nuclear factor-κb ligand-induced group, matrix metalloproteinase-9 expression was significantly lower in the 5 000 U/mL ulinastatin group (P < 0.05). Results suggested that urinary trypsin inhibitor inhibited receptor activator for nuclear factor-κb ligand-induced osteoclastogenesis and diminished matrix metalloproteinase-9 expression and activity.

中国组织工程研究杂志出版内容重点：人工关节；骨植入物；脊柱；骨折；内固定；数字化骨科；组织工程

全文链接：

Key words: trypsin inhibitors, RANK ligand, osteoclasts, matrix metalloproteinase 2, matrix metalloproteinase 9

CLC Number:

R318

Ru Jiang-ying, Zhao Jian-ning, Guo Ting, Yu Lei, Ding Hao, Jiang Hui. Inhibitory effect of ulinastatin on osteoclast activation and the relationship of ulinastatin to matrix metalloproteinase-2 and matrix metalloproteinase-9: potential of preventing prosthetic osteolysis[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(35): 5633-5639.

Figures/Tables 2

2.1 乌司他丁对RAW264.7细胞增殖活性的影响将不同浓度及不同时间尿胰蛋白酶抑制剂作用下的RAW264.7细胞进行MTT分析，尿胰蛋白酶抑制剂浓度分别为0，500，5 000 U/mL，MTT法检测加入乌司他丁后24，48，72 h的吸光度值(表1)。0-5 000 U//mL的尿胰蛋白酶抑制剂乌司他丁对RAW264.7细胞增殖无明显影响(P > 0.05)。 2.2 尿胰蛋白酶抑制剂抑制RANKL诱导RAW264.7细胞形成破骨细胞采用TRAP染色法光镜下观察RAW264.7细胞增殖、活化为成熟破骨细胞的情况(图1)。光镜下计数空白组未见TRAP阳性细胞；RANKL诱导RAW264.7细胞48 h后，RAW264.7细胞变大，有伪足伸出，TRAP染色阳性细胞呈酒红色，诱导组成熟破骨细胞数量为(195.701± 5.262)/cm2；加入不同浓度尿胰蛋白酶抑制剂(0，500，5 000 U/mL)24，48，72 h后，TRAP阳性细胞逐渐减少，呈时间剂量依赖关系；加入500，5 000 U/mL的尿胰蛋白酶抑制剂共培养72 h后，光镜下TRAP阳件细胞数分别为(78.802±2.793)/cm2和(23.204±1.405)/cm2；500 U/mL乌司他丁组和5 000 U/mL乌司他丁组分别与与RANKL诱导组比较，差异均有显著性意义(P < 0.05)，且500 U/mL乌司他丁组和5 000 U/mL乌司他丁组之间差异也有显著性意义(P < 0.05，见表2，图2。 2.3 尿胰蛋白酶抑制剂抑制基质金属蛋白酶9的表达和活性免疫组织化学结果：光镜下观察基质金属蛋白酶2、基质金属蛋白酶9阳性细胞的胞浆内有不同程度染色的棕黄色或褐色颗粒(图3)。与RANKL诱导组相比，500 U/mL乌司他丁组中基质金属蛋白酶9染色阳性细胞百分率明显降低(10.911±1.598)%(P < 0.05)，而5 000 U/mL乌司他丁组中基质金属蛋白酶9染色阳性细胞百分率则显著降低(5.509±0.784)%(P < 0.01)；但在不同组间基质金属蛋白酶2阳性细胞百分率差异无显著性意义(P > 0.05)，见表3、图4。 Western blot结果：单独RAW264.7细胞仅表达少量基质金属蛋白酶9，加入RANKL 48 h后可检测到大量基质金属蛋白酶9蛋白表达，加入尿胰蛋白酶抑制剂(5 000 U/mL)共培养72 h后，基质金属蛋白酶9蛋白表达显著减少；而基质金属蛋白酶2在不同组的表达无明显变化(图5)。明胶酶谱分析结果显示，与空白对照组基质金属蛋白酶9活性水平(82.786±0.772)比较，RAW264.7细胞经RANKL诱导48 h后基质金属蛋白酶9活性水平(172.146±0.242)显著增高 (P < 0.01)，而500 U/mL乌司他丁组和5 000 U/mL乌司他丁组的基质金属蛋白酶9活性水平分别为(121.585±0.422)和(98.125±0.373)，较RANKL诱导组均显著降低(P < 0.05)；但不同组间基质金属蛋白酶2活性水平差异无显著性意义(P > 0.05)，见表4、图6。"

References

[1] Athanasou NA,Quinn J,Bulstrode CJ.Resorption of bone byinflammatory cells derived from the joint capsules of hip arthroplasties.J Bone Joint Surg Br.1992;74(1):57-62.
[2] Ulrich SD,Seyler TM,Bennett D,et al.Total hip arthroplasties: What are the reasons for revision? Int Orthop.2008;32(5): 597-604.
[3] Gallo J,Slouf M,Goodman SB.The relationship of polyethylene wear to particle size, distribution, and number: A possible factor explaining the risk of osteolysis after hip arthroplasty.J Biomed Mater Res B Appl Biomater.2010;94(1):171-177.
[4] Abdelmoula LC,Ben M'barek R,Ben Hadj Yahia C,et al. Bisphosphonates: indications in bone diseases other than osteoporosis.Tunis Med. 2011;89(6):511-516.
[5] Wilkinson JM,Little DG.Bisphosphonates in orthopedic applications. Bone.2011;49(1):95-102.
[6] Ren W,Markel DC.Emerging ideas: can erythromycin reduce the risk of aseptic loosening?Clin Orthop Relat Res.2011; 469(8):2399-2403.
[7] Ren W,Zhang R,Hawkins M,et al.Efficacy of periprosthetic erythromycin delivery for wear debris-induced inflammation and osteolysis.Inflamm Res.2010;59(12):1091-1097.
[8] 余剑，赵建宁.破骨细胞活化过程中免疫内酯LY267108对核因子κB的抑制[J].中国组织工程研究, 2014,18(15):2309-2313.
[9] Gu Y,Lee HM,Sorsa T,et al.Doxycyline inhibits mononuclear cell-mediated connective tissue breakdown.FEMS Immunol Med Microbiol.2010;58(2):218-225.
[10] Ong SM,Taylor GJ.Doxycycline inhibits bone resorption by human interface membrane cells from aseptically loose hip replacements.J Bone Joint Surg Br.2003;85(3):456-461.
[11] Wu YJ,Ling Q,Zhou XH,et al.Urinary trypsin inhibitor attenuates Heptic ischemia-reperfusion injury by reducing nuclear factor-kappa B activation.Hepatobiliary Pancreat Dis Int.2009;8(1):53-58.
[12] Inoue K,Takano H,Yanagisawa R, et al.Protective effects of urinary trypsin inhibitor on systemic inflammatory response induced by lipopolysaccharide.J Clin Biochem Nutr,2008; 43(3):139-142.
[13] Inoue K, Takano H. Urinary trypsin inhibitor as a therapeutic option for endotoxin related inflammatory disorders. Expert Opin Investig Drugs,2010,19(4):513-520.
[14] 葛叶盈,徐云,成建庆,等.乌司他丁对老年髋关节置换患者术后并发症影响的病例对照研究[J].中国骨伤,2011,24(6):459-462.
[15] Lee JY,Lee JY,Chon JY,et al.The effect of ulinastatin on hemostasis in major orthopedic surgery.Korean J Anesthesiol. 2010;58(1):25-30.
[16] Hua G,Haiping Z,Baorong H,et al.Effect of Ulinastatin on the Expression of iNOS, MMP-2, and MMP-3 in degenerated nucleus pulposus cells of rabbits.Connective Tissue Res. 2013;54(1):29-33.
[17] Jiang Y,Jia T,Gong W,et al.Titanium particle-challenged osteoblasts promote osteoclastogenesis and osteolysis in a murine model of periprosthestic osteolysis.Acta Biomater. 2013;9(7):7564-7572.
[18] Krane SM,Inada M.Matrix metalloproteinases and bone.Bone. 2008; 43(1):7-18.
[19] Chen D,Zhang X,Guo Y,et al.MMP-9 inhibition suppresses wear debris-induced inflammatory osteolysis through downregulation of RANK/RANKL in a murine osteolysis model.Int J Mol Med.2012;30(6):1417-1423.
[20] Gallo J,Goodman SB,Konttinen YT,et al.Particle disease: biologic mechanisms of periprosthetic osteolysis in total hip arthroplasty. Innate Immun.2013;19(2):213-224.
[21] Yamanaka Y,Clohisy JC,Ito H,et al.Blockade of JNK and NFAT pathways attenuates orthopedic particle-stimulated osteoclastogenesis of human osteoclast precursors and murine calvarial osteolysis. J Orthop Res.2013;31(1):67-72.
[22] Okada Y,Naka K,Kawamura K,et al.Localization of matrix metallo-proteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: implications for bone resorption.Lab Invest.1995;72(3):311-322.
[23] Engsig MT,Chen QJ,Vu TH,et al.Matrix metalloproteinase 9 and vescular endothelial growth factor are essential for osteoclast recruitment into developing long bones.J Cell Biol. 2000;151(4):879-889.