IL-6对RAW264.7细胞成熟分化的体外实验研究
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摘要
目的探讨研究白介素-6(Interleukin-6,IL-6)对核因子NF-κB受体活化因子配体(Receptor activator of nuclear kappa B ligand,RANKL)及对破骨前体细胞的成熟分化和溶骨效应。方法破骨前体细胞RAW264.7细胞经50ng/mL RANKL诱导1 d后将其分为:1、空白对照组(RANKL+PBS)2、低浓度IL-6组(RANKL+50ng/mL IL-6)3、中浓度IL-6组(RANKL+100ng/mL IL-6)4、高浓度IL-6组(RANKL+150ng/mL IL-6)。连续培养9 d后,进行HE染色检测成熟破骨细胞生成量;通过抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase, TRAP)染色法观察TRAP阳性多核细胞的情况;运用扫描电镜检测破骨细胞在骨片上的骨吸收陷窝形成情况。结果 HE染色中,成熟破骨细胞生成量中、高浓度IL-6组明显少于低浓度IL-6组(P<0.05),低浓度IL-6组和空白对照组间无明显差别(P>0.05)。②通过TRAP染色后,经染色阳性区域面积与视野面积的百分比计算,中、高浓度IL-6组与明显少于低浓度和空白对照组(P<0.05)。③扫描电镜观察发现骨吸收陷窝面积与视野面积的百分比随着IL-6浓度的增高,相比空白对照组有显著减少,且高浓度IL-6组中陷窝形成最少(P<0.05)。结论 IL-6能直接作用于经RANKL诱导的RAW264.7细胞,能明显抑制破骨细胞激活分化,并降低破骨细胞所致的骨吸收效应。当IL-6浓度超过50ng/mL时,其抑制破骨细胞的骨吸收效应更加明显。
Objective To investigate the effect of interleukin-6(IL-6), a prototypical cytokine featuring pleiotropic and redundant activity, on the osteoclastic differentiation of RANKL-stimulated RAW264.7 macrophages. Methods RAW264.7 cells were pre-treated with 50ng/mL RANKL for 1 day and followed by IL-6 treatment. Then they were incubated and divided into 4 groups: control group(50ng/mL RANKL + PBS), low(50ng/mL RANKL+ 50ng/mL IL-6), medium(50ng/mL RANKL+ 100ng/mL IL-6), and high concentration(50 ng/mL RANKL+ 150ng/mL IL-6) group for 9 consecutive days. These cells were harvested and stained with hematoxylin-eosin(HE) and tartrate-resistant acid phosphatase(TRAP). The resorption pits of bone slices were observed using scanning electron microscope(SEM). Results HE stains showed that there were increased mature osteoclast cells in the control compared to 100 and 150ng/mL of IL-6 treatment, with a difference being statistically significant(P<0.05). However, the difference did not reach statistical significance between the control and 50ng/mL IL-6 treatment(P>0.05). Similarly, compared to the control, TRAP-positive multinucleated osteoclasts(more than 3 nucleus) significantly decreased under the constant stimuli of 100 and 150ng/mL of IL-6 concentration(P<0.05). Under SEM observation, multiple bone resorption formation was clearly visualized in control group. In contrast, few formations of bone resorption and mature osteoclasts were found when the addition of IL-6 concentration was more than 50ng/mL(P<0.05). Conclusion IL-6 at 100 or 150ng/mL concentrations can powerfully suppress the differentiation of mature osteoclasts, thereby resulting in a remarkably reduction of osteolysis.
Objective To investigate the effect of interleukin-6(IL-6), a prototypical cytokine featuring pleiotropic and redundant activity, on the osteoclastic differentiation of RANKL-stimulated RAW264.7 macrophages. Methods RAW264.7 cells were pre-treated with 50ng/mL RANKL for 1 day and followed by IL-6 treatment. Then they were incubated and divided into 4 groups: control group(50ng/mL RANKL + PBS), low(50ng/mL RANKL+ 50ng/mL IL-6), medium(50ng/mL RANKL+ 100ng/mL IL-6), and high concentration(50 ng/mL RANKL+ 150ng/mL IL-6) group for 9 consecutive days. These cells were harvested and stained with hematoxylin-eosin(HE) and tartrate-resistant acid phosphatase(TRAP). The resorption pits of bone slices were observed using scanning electron microscope(SEM). Results HE stains showed that there were increased mature osteoclast cells in the control compared to 100 and 150ng/mL of IL-6 treatment, with a difference being statistically significant(P<0.05). However, the difference did not reach statistical significance between the control and 50ng/mL IL-6 treatment(P>0.05). Similarly, compared to the control, TRAP-positive multinucleated osteoclasts(more than 3 nucleus) significantly decreased under the constant stimuli of 100 and 150ng/mL of IL-6 concentration(P<0.05). Under SEM observation, multiple bone resorption formation was clearly visualized in control group. In contrast, few formations of bone resorption and mature osteoclasts were found when the addition of IL-6 concentration was more than 50ng/mL(P<0.05). Conclusion IL-6 at 100 or 150ng/mL concentrations can powerfully suppress the differentiation of mature osteoclasts, thereby resulting in a remarkably reduction of osteolysis.
引文
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[11] Xu S, Zhang Y, Liu B, et al. Activation of mTORC1 in B lymphocytes promotes osteoclast formation via regulation of beta-catenin and RANKL/OPG[J]. J Bone Miner Res, 2016, 31(7): 1320-1333.
[12] 王信, 罗艳, 廖文波. 骨保护素体外抑制小鼠单核巨噬细胞成熟分化的实验研究[J]. 中国骨质疏松杂志, 2013, 19(03): 247-250.WANG X, LUO Y, LIAO WB. Study on osteoprotegerin in inhibitation of mouse osteoclast precursor cell differentiation[J]. Chin J Osteoporos, 2013, 19(03): 247-250.(in Chinese)
[13] Scheller J, Garbers C, Rose-John S. Interleukin-6: from basic biology to selective blockade of pro-inflammatory activities[J]. Semin Immunol, 2014, 26(1): 2-12.
[14] Wolf J, Rose-John S, Garbers C. Interleukin-6 and its receptors: a highly regulated and dynamic system[J]. Cytokine, 2014, 70(1): 11-20.
[15] Garbers C, Aparicio-Siegmund S, Rose-John S. The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition[J]. Curr Opin Immunol, 2015, 34: 75-82.
[16] Duplomb L, Baud'huin M, Charrier C, et al. Interleukin-6 inhibits receptor activator of nuclear factor kappaB ligand-induced osteoclastogenesis by diverting cells into the macrophage lineage: key role of Serine727 phosphorylation of signal transducer and activator of transcription 3[J]. Endocrinology, 2008, 149(7): 3688-3697.
[17] Wu Q, Zhou X, Huang D, et al. IL-6 enhances osteocyte-mediated osteoclastogenesis by promoting JAK2 and RANKL activity in vitro[J]. Cell Physiol Biochem, 2017, 41(4): 1360-1369.
[18] Yoshitake F, Itoh S, Narita H, et al. Interleukin-6 directly inhibits osteoclast differentiation by suppressing receptor activator of NF-kappaB signaling pathways[J]. J Biol Chem, 2008, 283(17): 11535-11540.
[2] Wang X, Luo Y, Liao WB, et al. Effect of osteoprotegerin in combination with interleukin-6 on inhibition of osteoclast differentiation[J]. Chin J Traumatol, 2013, 16(5): 277-280.
[3] Iqbal J, Yuen T, Kim SM, et al. Opening windows for bone remodeling through a SLIT[J]. J Clin Invest, 2018, 128(4): 1255-1257.
[4] 王信, 汪洋, 朱勇军, 等. 诱导小鼠破骨前体细胞成熟分化的实验条件探讨[J].中国骨质疏松杂志, 2011, 17(03):190-194.Wang X, Wang Y, Zhu YJ, et al. Study on experimental coditions of mouse osteoclast precursor cell differentiation[J]. Chin J Osteoporos, 2011,17(03):190-194.(in Chinese)
[5] Furuya M, Kikuta J. Direct cell-cell contact between mature osteoblasts and osteoclasts dynamically controls their functions in vivo[J]. Nat Commun, 2018, 9(1): 300.
[6] Zhang L, Liu W, Zhao J, et al. Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/beta-catenin pathway[J]. Biochim Biophys Acta, 2016, 1860(10): 2211-2219.
[7] Wang X, Zhu Y, Zheng S, et al. Amiloride inhibits osteoclastogenesis by suppressing nuclear factor-kappaB and mitogen-activated protein kinase activity in receptor activator of nuclear factor-kappaB-induced RAW264.7 cells[J]. Mol Med Rep, 2015, 11(5): 3451-3456.
[8] Zeng XZ, He LG, Wang S, et al. Aconine inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells by suppressing NF-kappaB and NFATc1 activation and DC-STAMP expression[J]. Acta Pharmacol Sin, 2016, 37(2): 255-263.
[9] Manzano-Moreno FJ, Ramos-Torrecillas J, Melguizo-Rodriguez L, et al. Bisphosphonate modulation of the gene expression of different markers involved in osteoblast physiology: possible implications in bisphosphonate-related osteonecrosis of the jaw[J]. Int J Med Sci, 2018, 15(4): 359-367.
[10] Zanatta LC, Boguszewski CL, Borba VZ, et al. Osteocalcin, energy and glucose metabolism[J]. Arq Bras Endocrinol Metabol, 2014, 58(5): 444-451.
[11] Xu S, Zhang Y, Liu B, et al. Activation of mTORC1 in B lymphocytes promotes osteoclast formation via regulation of beta-catenin and RANKL/OPG[J]. J Bone Miner Res, 2016, 31(7): 1320-1333.
[12] 王信, 罗艳, 廖文波. 骨保护素体外抑制小鼠单核巨噬细胞成熟分化的实验研究[J]. 中国骨质疏松杂志, 2013, 19(03): 247-250.WANG X, LUO Y, LIAO WB. Study on osteoprotegerin in inhibitation of mouse osteoclast precursor cell differentiation[J]. Chin J Osteoporos, 2013, 19(03): 247-250.(in Chinese)
[13] Scheller J, Garbers C, Rose-John S. Interleukin-6: from basic biology to selective blockade of pro-inflammatory activities[J]. Semin Immunol, 2014, 26(1): 2-12.
[14] Wolf J, Rose-John S, Garbers C. Interleukin-6 and its receptors: a highly regulated and dynamic system[J]. Cytokine, 2014, 70(1): 11-20.
[15] Garbers C, Aparicio-Siegmund S, Rose-John S. The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition[J]. Curr Opin Immunol, 2015, 34: 75-82.
[16] Duplomb L, Baud'huin M, Charrier C, et al. Interleukin-6 inhibits receptor activator of nuclear factor kappaB ligand-induced osteoclastogenesis by diverting cells into the macrophage lineage: key role of Serine727 phosphorylation of signal transducer and activator of transcription 3[J]. Endocrinology, 2008, 149(7): 3688-3697.
[17] Wu Q, Zhou X, Huang D, et al. IL-6 enhances osteocyte-mediated osteoclastogenesis by promoting JAK2 and RANKL activity in vitro[J]. Cell Physiol Biochem, 2017, 41(4): 1360-1369.
[18] Yoshitake F, Itoh S, Narita H, et al. Interleukin-6 directly inhibits osteoclast differentiation by suppressing receptor activator of NF-kappaB signaling pathways[J]. J Biol Chem, 2008, 283(17): 11535-11540.