Chinese Journal of Tissue Engineering Research ›› 2021, Vol. 25 ›› Issue (2): 178-185.doi: 10.3969/j.issn.2095-4344.2955
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Geng Bin1, 2, Xia Yayi1, 2
Received:
2020-03-10
Revised:
2020-03-14
Accepted:
2020-04-15
Online:
2021-01-18
Published:
2020-11-21
Contact:
Xia Yayi, MD, Professor, Chief physician, Doctoral supervisor, Department of Joint Surgery, Second Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China; Gansu Provincial Key Laboratory of Bone and Joint Disease Research, Lanzhou 730000, Gansu Province, China
About author:
Geng Bin, MD, Master’s supervisor, Department of Joint Surgery, Second Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China; Gansu Provincial Key Laboratory of Bone and Joint Disease Research, Lanzhou 730000, Gansu Province, China
Supported by:
CLC Number:
Geng Bin, Xia Yayi. Involvement of ERK5 signaling pathway in osteoporosis development in mice[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(2): 178-185.
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2.1 实验动物数量分析 实验选用昆明小鼠12只,分为4组,实验过程无脱失,全部进入结果分析。 2.2 小鼠体内和MC3T3-E1细胞内ERK5的活性 在小鼠体内腹腔注射XMD8-92,骨组织内的ERK5磷酸化水平被明显抑制,见图1 A,B。以往的研究证实ERK5能够被表皮生长因子所激活[30],而且以往的研究证实XMD8-92的最佳浓度为5 μmol/L,且不会影响其他MAPK信号通路[14],此次体外实验表明XMD8-92可以明显抑制表皮生长因子诱导的成骨细胞内ERK5激活,见图1 C,D。 XMD8-92作用于MC3T3-E1细胞的免疫荧光染色结果显示XMD8-92可以明显抑制ERK5核移位的亚细胞转位,见图1E。因为磷酸化的ERK5会转移至核内调控下游靶因子表达[14],XMD8-92可以阻断这一过程,进一步证实XMD8-92可以抑制成骨细胞内ERK5的活性。 "
2.3 小鼠的骨量及骨小梁相关参数变化 小鼠被腹腔注射XMD8-92和/或地塞米松。μ-CT分析显示XMD8-92干预后导致小鼠股骨骨小梁的结构变稀疏,见图2。与对照组相比,骨密度定量显示XMD8-92可使股骨松质骨密度减少约11.4% (P < 0.01,见图2B)。骨体积分数、骨小梁数、骨小梁厚度也分别降低了8.0%、10.7%和5%,而骨小梁离散度和骨小梁模式因子分别增加了17.3%和20.3%(见图2 C-G)。这些结果表明,XMD8-92处理后的小鼠骨体积更小,骨小梁数目和厚度更少,且骨小梁间隙更大。 另外,腹腔注射过量的地塞米松 5周后成功建立小鼠骨质疏松症模型,结果提示XMD8-92能够显著加重塞米松诱导的骨质疏松症(见图2A)。XMD8-92+地塞米松诱导的小鼠股骨骨密度显著降低(-19.4%,P < 0.01),骨体积分数降低(-20.2%,P < 0.001),骨小梁数降低(-16.0%, P < 0.001),骨小 梁厚度(-22.6%,P < 0.001),但骨小梁离散度和骨小梁模式因子增加(+20.6 和+22.5,P < 0.001,见图2 C-G)。 同样,苏木精-伊红染色结果也显示XMD8-92处理后的小鼠、地塞米松处理后的小鼠和XMD8-92+地塞米松诱导后的小鼠其股骨均出现不同程度的骨质流失,见图2 H-O。 结果表明,抑制ERK5活性能够降低小鼠的骨量,并加重地塞米松诱导的骨质疏松症。"
2.5 MC3T3-E1细胞中RANKL/OPG的蛋白表达 RANKL和骨保护素在骨重塑过程中起着重要作用,由成骨细胞合成并分泌,其表达量的变化能够反映出成骨细胞或破骨细胞的活性,间接显示骨形成和骨吸收的动态平衡关系,是检测骨质疏松症常用的生物学指标[1-3]。Western blotting结果显示XMD8-92和地塞米松显著提高了MC3T3-E1细胞中RANKL的蛋白水平(P < 0.05和P < 0.001,见图4 A和B),而且XMD8-92能够进一步促进地塞米松诱导的RANKL上调(P < 0.001,见图4 A和B)。相反,XMD8-92和地塞米松明显下调了骨保护素蛋白的表达,且XMD8-92能够进一步促进地塞米松对骨保护素的下调作用 (P < 0.001,见图4 A和B)。结果提示,ERK5活性的抑制能够明显上调RANKL并下调骨保护素的蛋白表达,XMD8-92可以通过调控RANKL/OPG的蛋白表达来调节骨量的变化。"
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