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通讯作者:

费小明,E-mail:feixiaomingujs@aliyun.com

中图分类号:R733.3

文献标识码:A

文章编号:1007-4368(2023)01-001-08

DOI:10.7655/NYDXBNS20230101

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参考文献 9
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目录contents

    摘要

    目的:研究在体外条件下多西环素(doxycycline,DOX)对前成骨细胞株MC3T3-E1成骨分化的影响及可能机制。方法:在成骨诱导剂体外诱导MC3T3-E1细胞株成骨分化条件下,茜素红染色检测成骨细胞分化;实时定量PCR检测DOX对 MC3T3-E1细胞相关成骨基因OCN、Runx2的影响;用DOX、MEK抑制剂(U0126)单独或联合处理MC3T3-E1细胞后,Western blot 检测N-cadherin、p-MEK及p-ERK等蛋白的表达。结果:在MC3T3-E1细胞株体外成骨诱导分化中,加入DOX可以增强茜素红染色阳性率。DOX上调MC3T3-E1细胞相关成骨基因OCN、Runx2的表达。DOX处理MC3T3-E1细胞后,其N-cadherin蛋白表达水平下降(P < 0.05),p-MEK和p-ERK蛋白的表达增加(P < 0.05)。而MEK拮抗剂(U0126)则显著上调N-cadherin蛋白表达水平,同时降低p-MEK、p-ERK水平。用U0126联合DOX处理MC3T3-E1细胞后,DOX对N-cadherin、p-MEK和p-ERK蛋白水平的影响可被U0126拮抗。在MC3T3-E1细胞株体外成骨诱导分化中,同时存在DOX和U0126时,茜素红染色阳性率低于单独DOX组。结论:DOX可以促进MC3T3-E1细胞株的体外成骨分化;而DOX的这一效应可能是通过MEK-ERK信号通路参与完成的。

    Abstract

    Objective:To investigate the effects of doxycycline on in vitro osteogenic differentiation of pro - osteoblasts cell line MC3T3-E1 and the possible mechanisms. Methods:Alizarin red staining was employed to detect the osteogenic differentiation. Real- time PCR was used to detect the effect of DOX on the expression of OCN and Runx2 in MC3T3-E1 cells. MC3T3-E1 cells were treated with either DOX,MEK inhibitor U0126 alone or in combination,and the levels of N-cadherin,p-MEK and p-ERK were detected by Western blot. Results:When DOX was added during osteogenic induction,alizarin red staining positivity of the cultured MC3T3-E1 was significantly enhanced. DOX increased the expression of OCN and Runx2 in MC3T3-E1 cells. Besides,DOX decreased the levels of N - cadherin and increased the level of p -MEK and p - ERK in MC3T3 - E1(P < 0.05). On the contrary,MEK antagonist U0126 significantly increased the expression of N-cadherin protein and decreased the levels of p-MEK and p-ERK(P < 0.05). When MC3T3-E1 cells were treated with DOX in the presence of MEK inhibitor U0126,the changes in N-cadherin,p-MEK and p-ERK were showed to be reversed comparing with DOX alone. When both U0126 and DOX were present during in vitro osteogenic differentiation of MC3T3-E1, alizarin red staining positivity was less observed than that of DOX alone. Conclusion:In this study,DOX is showed to enhance the in vitro osteogenic differentiation of MC3T3-E1,which is probably associated with MEK/ERK signaling pathway.

  • 多发性骨髓瘤(multiple myeloma,MM)是一种浆细胞恶性肿瘤,在其发生和发展过程中,骨髓瘤细胞与骨髓微环境的相互作用起到非常重要的作用[1-2]。成骨细胞作为为骨髓微环境中的重要细胞成分之一,在MM中可出现明显的异常[3]。MM患者成骨细胞的成骨活性下降,而破骨细胞活性亢进是骨髓瘤患者骨骼病变的核心机制[4-6]。此外,如何纠正 MM 患者的成骨细胞功能障碍,也是 MM 领域的研究热点。多西环素(doxycycline,DOX)是一种临床上常用于抗菌治疗的抗生素。本课题组先前发现DOX在体外有抗骨髓瘤细胞增殖的作用[7-8]。鉴于骨髓微环境中的成骨细胞在MM中的重要作用,本研究拟探索DOX在体外对成骨细胞株MC3T3⁃E1 成骨分化的影响及相应机制。

  • 1 材料和方法

  • 1.1 材料

  • 小鼠前成骨细胞系(MC3T3⁃E1细胞,中科院上海细胞库);α⁃MEM 培养基、胎牛血清(FBS)(Gibco 公司,美国);多西环素(Sigma公司,美国);MEK抑制剂U0126(MCE公司,美国);CCK⁃8试剂盒(同仁公司,日本);PCR 引物(上海生工生物公司);逆转录试剂盒、SYBR Green试剂盒(Takara公司,日本); 引物委托上海生工生物技术有限公司设计并合成。引物序列如下:OCN⁃F5′⁃TGACGAGTTGGCT⁃GACCA⁃3′,OCN⁃R 5′⁃AGGGTGCCTGGAGAGGAG⁃ 3′;Runx2⁃F5′⁃TTGACCTTTGTCCCAATGC⁃3′,Runx2⁃R 5′⁃AGGTTGGAGGCACACATAGG⁃3′;β⁃ac⁃tin⁃F5′⁃CCTGGCACCCAGACAAAT⁃3′,β⁃actin⁃R 5′⁃GGGCCGGACTCGTCATAC⁃3′。兔抗人p⁃ERK1/2抗体、兔抗人p⁃MEK抗体、兔抗人GAPDH抗体等(CST 公司,美国);BCA 蛋白浓度测定试剂盒、蛋白上样缓冲液(5×)、预染 Marker(杭州碧云天生物技术有限公司)。

  • 1.2 方法

  • 1.2.1 细胞培养和药物处理

  • MC3T3⁃E1细胞用含有10% FBS的α⁃MEM培养液培养,取生长状态良好的细胞进行实验。用于实验的 DOX 浓度为 5、10、20、40 mg/L,U0126 浓度为 5、10 μmol/L。

  • 1.2.2 CCK⁃8检测细胞增殖

  • 在96孔板中每孔接种50 μL的细胞悬液(1×105 个/mL),将DOX用培养液配制成所需的浓度,实验组孔加入50 μL药物;对照组加入50 μL培养液。在涡旋振荡器上轻轻摇匀,置于37℃培养箱分别培养 1、2、3 d后,取出96孔板,在每孔中加入10 μL CCK⁃8 试剂,避光孵育约1 h,酶标仪测量在450 nm波长处的吸光值并记录。

  • 1.2.3 Western blot检测蛋白的表达

  • 收集细胞,于冰上裂解,提取总蛋白,用BCA法测定蛋白浓度。配制分离胶以及浓缩胶,按每个加样孔约 30 μg 蛋白量上样。电泳后转膜到孔径为 0.45 μm的PVDF膜上,封闭后将膜放入相应的一抗中,在4℃环境中摇床孵育过夜。洗膜、摇床上孵育二抗,孵育结束洗膜、曝光。用Image J软件进行灰度值分析。

  • 1.2.4 茜素红染色检测成骨

  • 当培养的 MC3T3⁃E1 细胞融合度达到 80%时,消化并调整细胞浓度1×104 个/mL接种至12孔板中的无菌爬片上,每孔培养体系为 1 mL。培养过夜后,不同组别更换培养基,每孔加入1 mL相应培养基。设置阴性对照组(含完全培养基)、诱导组(成骨诱导液)、实验组(含DOX的成骨诱导液)。每3 d 更换新鲜的各组培养基。诱导14、21 d时,经4%多聚甲醛固定30 min,茜素红染色检测成骨,日常光线下标本拍照、倒置显微镜下镜检。

  • 1.2.5 实时定量PCR(qPCR)检测基因表达

  • 收集细胞,冰上裂解,用 TRIzol 提取细胞总 RNA,酶标仪上测定浓度。按照试剂盒说明配制逆转录体系,在PCR扩增仪中进行逆转录;转录完后,将合成的 cDNA 放在冰上冷却。按 SYBR Green 试剂盒说明书在冰上配制 20 μL 反应体系,加入 8 连管中,在仪器中反应,最后进行数据处理分析。

  • 1.3 统计学方法

  • 数据处理、统计分析用GraphPad Prism 5.0软件进行操作;用Image J软件计算蛋白及茜素红染色灰度值。实验数据用均数±标准差(x-±s)表示,数据资料符合正态分布(S⁃W检验),用单因素方差分析方法对各实验组组间进行两两比较(Dunnett⁃t检验)。 P <0.05为差异有统计学意义。

  • 2 结果

  • 2.1 不同浓度 DOX 对前成骨细胞 MC3T3⁃E1 细胞增殖的影响

  • 取生长状态良好的 MC3T3⁃E1 细胞,按实验分组处理 1、2、3 d,发现与对照组相比,5~40 mg/L 的 DOX 对 MC3T3⁃E1 细胞增殖没有明显的抑制作用 (图1)。

  • 图1 CCK⁃8法检测DOX对前成骨细胞MC3T3⁃E1增殖的影响

  • Figure1 Effect of DOX on the proliferation of preosteoblastic MC3T3⁃E1 by CCK⁃8 assay

  • 2.2 茜素红染色检测 DOX 对 MC3T3⁃E1 细胞成骨分化的影响

  • 既往Wiśniewska等[9] 发现DOX本身可以直接抑制 ALP 的活性,对 ALP 染色有干扰,我们前期实验结果与其报道一致,故本研究不采用ALP染色作为成骨分化标志。MC3T3′⁃E1 细胞株体外成骨诱导过程中,在第 14、21 天进行茜素红染色,检测成骨分化的效果。在第 14 天时,肉眼及显微镜下观察茜素红染色3组间没有明显区别(图2A、B)。成骨诱导21 d时,诱导组与实验组均可见明显红染。与诱导组比较,加入DOX的实验组染色更强、钙结节更多(P <0.05,图2C~E)。这一结果提示,在体外成骨诱导分化中,DOX可促进MC3T3⁃E1细胞的成骨分化功能。

  • 2.3 qPCR检测DOX对MC3T3⁃E1细胞相关成骨基因的影响

  • MC3T3⁃E1 细胞经成骨诱导 21 d 后,提取总 RNA 后利用 qPCR 检测相关成骨基因 OCN、Runx2 的相对表达量。与阴性对照组对比,诱导组及 10 mg/L DOX实验组,相关成骨基因均上调,说明成骨诱导成功,但诱导组与实验组间比较,无统计学差异(图3),这提示在成骨诱导的21 d,有DOX与无 DOX组的OCN和Runx2的mRNA水平无差异。

  • 2.4 DOX处理MC3T3⁃E1细胞后,N⁃cadherin、p⁃MEK、 p⁃ERK蛋白的表达情况

  • 用5、10 mg/L 的DOX 分别处理MC3T3⁃E1细胞 2 d,Western blot 法检测 N ⁃cadherin 蛋白的表达情况。结果发现,与对照组比较,不同浓度DOX处理 MC3T3⁃E1细胞2 d后,其N⁃cadherin蛋白表达下降,且差异有统计学意义(P <0.05,图4A)。结合DOX 对细胞增殖和蛋白表达的影响,后续的成骨及Western blot 实验 DOX 浓度均选用 10 mg/L。随后 Western blot 法检测 p ⁃MEK、p ⁃ERK 蛋白水平后发现,与对照组比较,10 mg/L DOX处理2 d后p⁃MEK、 p ⁃ERK 蛋白表达上调,且差异具有统计学意义 (P <0.05,图4B、C)。

  • 2.5 MEK抑制剂U0126处理MC3T3⁃E1细胞,检测 N⁃cadherin蛋白及相关通路蛋白的表达情况

  • 为了进一步探究 DOX 是否对 MEK/ ERK 通路产生影响。用浓度为5、10 μmol/L的MEK的抑制剂 U0126处理MC3T3⁃E1细胞2 d后,Western blot检测 N⁃cadherin、p⁃ERK、p⁃MEK表达量。与对照组比较,不同浓度 U0126 处理 2 d 后 N⁃cadherin 蛋白表达上调(P <0.05,图5A);与此同时,p⁃MEK、p⁃ERK蛋白表达下降,但5 μmol/L的U0126对p⁃ERK的抑制作用更明显(P <0.05,图5B、C),故后续实验U0126选用5 μmol/L。

  • 2.6 U0126联合DOX处理MC3T3⁃E1细胞,检测相关目的蛋白的表达情况

  • 用 5 μmol/L U0126 联合 10 mg/L DOX 处理 MC3T3⁃E1细胞2 d,Western blot法检测N⁃cadherin、 p⁃MEK和p⁃ERK蛋白的表达。与DOX单独处理组比较,联合处理组N⁃cadherin水平较DOX单独处理组升高,且联合处理组p⁃MEK及p⁃ERK蛋白表达较单独处理组降低(图6)。

  • 2.7 U0126联合DOX处理MC3T3⁃E1细胞,茜素红检测MC3T3⁃E1细胞成骨分化的变化

  • 随后的实验探索使用U0126抑制MEK/ERK 通路对DOX促进MC3T3⁃E1细胞成骨分化的作用有何影响。实验共分为5组:①阴性对照组,②阳性成骨对照组,③DOX处理组,④U0126处理组,⑤DOX联合U0126处理组。成骨诱导21 d后,阳性成骨组出现较多的红色钙结节,成不规则片状或散在分布; DOX+U0126 组,红染率低于 DOX 处理组,但高于 U0126 处理组(P <0.05,图7)。这一结果提示, MEK/ERK 通路参与 MC3T3⁃E1 细胞成骨分化的调节,而DOX通过MEK/ERK通路来调节MC3T3⁃E1细胞的成骨分化。

  • 图2 MC3T3⁃E1细胞成骨诱导14、21 d后茜素红染色

  • Figure2 Alizarin red staining of MC3T3⁃E1 cells after osteogenic induction for 14,21 days

  • 图3 成骨诱导21 d后qPCR检测相关成骨基因相对表达量

  • Figure3 Detection of osteogenic gene levels via qRCR after 21 days of osteogenic induction

  • 图4 不同浓度的DOX处理MC3T3⁃E1细胞2 d后Western blot检测蛋白的表达情况

  • Figure4 MC3T3⁃E1 cells were treated with different concentrations of DOX for 2 days and the expression of the indicated proteins was detected by Western blot

  • 图5 用U0126处理MC3T3⁃E1细胞后Western blot检测蛋白的表达情况

  • Figure5 MC3T3⁃E1 cells were treated with U0126 for 2 days and the expression of the indicated proteins was detected by Western blot

  • 3 讨论

  • 在MM细胞与骨骼微环境的相互作用过程中,肿瘤细胞可引起微环境中多种细胞和非细胞成分异常,其中成骨细胞成骨功能和分化的受损尤其明显[210]。即使MM患者经有效治疗后,成骨细胞等的异常仍然存在,并且这些异常与MM细胞的耐药和复发密切相关[4611]。因此,成骨细胞的异常,不但与MM骨骼病变相关,也与MM细胞的生存、耐药等有密切关系。正因为如此,MM骨骼病变的机制和干预方法是MM基础和临床研究的重要方向。

  • 图6 Western blot检测U0126联合DOX处理的MC3T3⁃E1细胞中相关目的蛋白表达

  • Figure6 Expression of the indicated proteins was detected by Western blot in MC3T3⁃E1 cells treated with U0126 combi⁃ nation with DOX

  • 图7 茜素红染色评估DOX联合U0126处理对MC3T3⁃E1细胞成骨分化的影响

  • Figure7 The effect of DOX and U0126 on osteogenic differentiation of MC3T3⁃E1 cells by alizarin red stain

  • 本研究结果提示 DOX 促进前成骨细胞株 MC3T3⁃E1的体外成骨分化,并且对其N⁃cadherin的表达也有影响。既往研究报道DOX有一定的抗实体肿瘤、抑制血管生成等作用[12-13]。另外,也有研究报道DOX促进人和大鼠成骨细胞的增生和矿物质沉积,有正性成骨作用[14-15],这与本研究结果类似。除了作用成骨细胞,DOX还可以通过其他机制来影响骨骼生成。例如动物实验发现,DOX可以抑制基质金属蛋白酶,减轻骨关节炎[16]。此外,DOX 可以抑制大鼠的破骨细胞活性,有助于减少破骨细胞溶骨亢进引起骨骼破坏[17]。在乳腺癌骨转移时,DOX 可以抑制转移灶局部破骨细胞活性及数量,改善溶骨性病变[18]。上述结果提示,DOX可以通过多种机制阻止骨骼破坏,是一个潜在的治疗MM骨骼病变的药物。

  • Gomes 等[15] 研究发现,DOX 可以作用于 Wnt 信号通路,进而影响成骨细胞的分化。而本研究发现, DOX 也可通过 MEK/ERK 信号通路影响 MC3T3⁃E1 细胞的成骨分化。多项研究发现,MEK/ERK信号通路参与成骨细胞的成骨分化的调节[19-21],这与本研究观察到DOX对MEK/ERK信号通路的影响及茜素红染色的结果相一致。除了参与成骨分化调节之外,有研究报道 MEK/ERK 信号通路对成骨细胞的黏附、迁移等细胞行为也有调节作用[22]。本研究还发现,DOX 可以下调 MC3T3⁃E1 细胞株 N⁃cadherin 的表达,并且 MEK/ERK 信号通路参与 DOX 对 N⁃cadherin 下调的作用。既往研究报道 N⁃cadherin 可以与Wnt受体结合,竞争性抑制Wnt/β⁃Catenin信号通路的激活,干扰成骨细胞的成骨分化[23]。有意思的是,N ⁃ cadherin 表达变化后,反过来还会对 PI3K/Akt、MEK/ERK和Wnt/β⁃catenin信号通路产生影响[24-25]。所以,上述研究提示DOX处理MC3T3⁃E1 细胞株后,可以通过MEK/ERK通路下调N⁃cadherin 表达,而N⁃cadherin水平变化后,反过来也可能参与对MEK/ERK信号通路的上调作用。

  • 综上所述,本研究以 MC3T3⁃E1 细胞株为对象证实,DOX 可以促进其成骨分化,并且下调其 N⁃cadherin 蛋白水平。另外初步的机制研究发现, MEK/ERK 信号通路在 DOX 对 MC3T3⁃E1 细胞株成骨分化和N⁃cadherin 表达的影响中起到重要作用。这提示DOX不仅可以直接杀伤MM细胞[7-8],还可能同时通过对骨微环境中成骨细胞的调节作用影响 MM的病理生理过程。DOX对骨微环境的影响有待进一步深入研究。

  • 参考文献

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    • [2] BRIGLE K,ROGERS B.Pathobiology and diagnosis of multiple myeloma[J].Semin Oncol Nurs,2017,33(3):225-236

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    • [5] LENTZSCH S,EHRLICH L A,ROODMAN G D.Patho⁃ physiology of multiple myeloma bone disease[J].Hema⁃ tol Oncol Clin North Am,2007,21(6):1035-1049

    • [6] YEN C H,HSU C M,HSIAO S Y,et al.Pathogenic mech⁃ anisms of myeloma bone disease and possible roles for NRF2[J].Int J Mol Sci,2020,21(18):E6723

    • [7] 颜玲玲,费小明,汤郁,等.多西环素对人多发性骨髓瘤细胞增殖的影响[J].江苏大学学报(医学版),2017,27(3):239-242

    • [8] 李海璐,费小明,汤郁,等.多西环素对骨髓瘤细胞株H929内源性凋亡的影响及其作用机制[J].中国实验血液学杂志,2022,30(2):441-448

    • [9] WI Ś NIEWSKA I E,GAWLIK Z.The effect of doxyc⁃ ycline upon alkaline phosphatase activity in rat kidney(quantitative study using the interferometric technique)[J].Folia Histochem Cytochem,1982,20(3-4):157-162

    • [10] ROODMAN G D.Osteoblast function in myeloma[J].Bone,2010,48(1):135-140

    • [11] LANDGREN O,ISKANDER K.Modern multiple myelo⁃ ma therapy:deep,sustained treatment response and good clinical outcomes[J].J Intern Med,2017,281(4):365-382

    • [12] CLEMENS D L,DURYEE M J,SARMIENTO C,et al.Novel antioxidant properties of doxycycline[J].Int J Mol Sci,2018,19(12):E4078

    • [13] MARKOWSKA A,KAYSIEWICZ J,MARKOWSKA J,et al.Doxycycline,salinomycin,monensin and ivermectin re⁃ positioned as cancer drugs[J].Bioorg Med Chem Lett,2019,29(13):1549-1554

    • [14] SOUSA G P,HELENA F M.Effect of therapeutic levels of doxycycline and minocycline in the proliferation and differentiation of human bone marrow osteoblastic cells[J].Arch Oral Biol,2007,52(3):251-259

    • [15] GOMES K D N,ALVES A P N N,DUTRA P G P,et al.Doxycycline induces bone repair and changes in Wnt sig⁃ nalling[J].Int J Oral Sci,2017,9(3):158-166

    • [16] NGANVONGPANIT K,POTHACHAROEN P,SUWAN ⁃ KONG N,et al.The effect of doxycycline on canine hip osteoarthritis:design of a 6⁃months clinical trial[J].J Vet Sci,2009,10(3):239-247

    • [17] DE FIGUEIREDO F A T,SHIMANO R C,ERVOLINO E,et al.Doxycycline reduces osteopenia in female rats [J].Sci Rep,2019,9(1):15316

    • [18] ZEINA S,GURMIT S.Doxycycline and other tetracy⁃ clines in the treatment of bone metastasis[J].Anti Can⁃ cer Drugs,2003,14(10):773-778

    • [19] LI Y S,VAN LEEUWEN J,et al.ERK activation and al⁃ pha v beta 3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts [J].J Cell Biochem,2002,87(1):85-92

    • [20] WANG N,LI Y,LI Z,et al.Sal B targets TAZ to facilitate osteogenesis and reduce adipogenesis through MEK⁃ERK pathway[J].J Cell Mol Med,2019,23(5):3683-3695

    • [21] WANG F S,WANG C J,SHEEN⁃CHEN S M,et al.Super⁃ oxide mediates shock wave induction of ERK ⁃ dependent osteogenic transcription factor(CBFA1)and mesenchy⁃ mal cell differentiation toward osteoprogenitors[J].J Biol Chem,2002,277(13):10931-10937

    • [22] LAI C F,CHAUDHARY L,FAUSTO A,et al.Erk is es⁃ sential for growth,differentiation,integrin expression,and cell function in human osteoblastic cells[J].J Biol Chem,2001,276(17):14443-14450

    • [23] HAŸ E,LAPLANTINE E,GEOFFROY V,et al.N ⁃cad⁃ herin interacts with axin and LRP5 to negatively regulate Wnt/beta⁃catenin signaling,osteoblast function,and bone formation[J].Mol Cell Biol,2009,29(4):953-964

    • [24] XU L,MENG F,NI M,et al.N⁃cadherin regulates osteo⁃ genesis and migration of bone marrow-derived mesenchy⁃ mal stem cells[J].Mol Biol Rep,2013,40(3):2533-2539

    • [25] HAŸ E,NOURAUD A,MARIE P J.N⁃cadherin negative⁃ ly regulates osteoblast proliferation and survival by antag⁃ onizing Wnt,ERK and PI3K/Akt signalling[J].PLoS One,2009,4(12):e8284

  • 参考文献

    • [1] MICHELS T C,PETERSEN K E.Multiple myeloma:diag⁃ nosis and treatment[J].Am Fam Physician,2017,95(6):373-383

    • [2] BRIGLE K,ROGERS B.Pathobiology and diagnosis of multiple myeloma[J].Semin Oncol Nurs,2017,33(3):225-236

    • [3] 翟妮,王昌敏.多发性骨髓瘤骨病发病机制的研究进展[J].新疆医学,2019,49(10):1033-1036

    • [4] EVANGELOS T,IOANNIS N S,MARIA G,et al.Patho⁃ genesis of bone disease in multiple myeloma:from bench to bedside[J].Blood Cancer J,2018,8(1):7

    • [5] LENTZSCH S,EHRLICH L A,ROODMAN G D.Patho⁃ physiology of multiple myeloma bone disease[J].Hema⁃ tol Oncol Clin North Am,2007,21(6):1035-1049

    • [6] YEN C H,HSU C M,HSIAO S Y,et al.Pathogenic mech⁃ anisms of myeloma bone disease and possible roles for NRF2[J].Int J Mol Sci,2020,21(18):E6723

    • [7] 颜玲玲,费小明,汤郁,等.多西环素对人多发性骨髓瘤细胞增殖的影响[J].江苏大学学报(医学版),2017,27(3):239-242

    • [8] 李海璐,费小明,汤郁,等.多西环素对骨髓瘤细胞株H929内源性凋亡的影响及其作用机制[J].中国实验血液学杂志,2022,30(2):441-448

    • [9] WI Ś NIEWSKA I E,GAWLIK Z.The effect of doxyc⁃ ycline upon alkaline phosphatase activity in rat kidney(quantitative study using the interferometric technique)[J].Folia Histochem Cytochem,1982,20(3-4):157-162

    • [10] ROODMAN G D.Osteoblast function in myeloma[J].Bone,2010,48(1):135-140

    • [11] LANDGREN O,ISKANDER K.Modern multiple myelo⁃ ma therapy:deep,sustained treatment response and good clinical outcomes[J].J Intern Med,2017,281(4):365-382

    • [12] CLEMENS D L,DURYEE M J,SARMIENTO C,et al.Novel antioxidant properties of doxycycline[J].Int J Mol Sci,2018,19(12):E4078

    • [13] MARKOWSKA A,KAYSIEWICZ J,MARKOWSKA J,et al.Doxycycline,salinomycin,monensin and ivermectin re⁃ positioned as cancer drugs[J].Bioorg Med Chem Lett,2019,29(13):1549-1554

    • [14] SOUSA G P,HELENA F M.Effect of therapeutic levels of doxycycline and minocycline in the proliferation and differentiation of human bone marrow osteoblastic cells[J].Arch Oral Biol,2007,52(3):251-259

    • [15] GOMES K D N,ALVES A P N N,DUTRA P G P,et al.Doxycycline induces bone repair and changes in Wnt sig⁃ nalling[J].Int J Oral Sci,2017,9(3):158-166

    • [16] NGANVONGPANIT K,POTHACHAROEN P,SUWAN ⁃ KONG N,et al.The effect of doxycycline on canine hip osteoarthritis:design of a 6⁃months clinical trial[J].J Vet Sci,2009,10(3):239-247

    • [17] DE FIGUEIREDO F A T,SHIMANO R C,ERVOLINO E,et al.Doxycycline reduces osteopenia in female rats [J].Sci Rep,2019,9(1):15316

    • [18] ZEINA S,GURMIT S.Doxycycline and other tetracy⁃ clines in the treatment of bone metastasis[J].Anti Can⁃ cer Drugs,2003,14(10):773-778

    • [19] LI Y S,VAN LEEUWEN J,et al.ERK activation and al⁃ pha v beta 3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts [J].J Cell Biochem,2002,87(1):85-92

    • [20] WANG N,LI Y,LI Z,et al.Sal B targets TAZ to facilitate osteogenesis and reduce adipogenesis through MEK⁃ERK pathway[J].J Cell Mol Med,2019,23(5):3683-3695

    • [21] WANG F S,WANG C J,SHEEN⁃CHEN S M,et al.Super⁃ oxide mediates shock wave induction of ERK ⁃ dependent osteogenic transcription factor(CBFA1)and mesenchy⁃ mal cell differentiation toward osteoprogenitors[J].J Biol Chem,2002,277(13):10931-10937

    • [22] LAI C F,CHAUDHARY L,FAUSTO A,et al.Erk is es⁃ sential for growth,differentiation,integrin expression,and cell function in human osteoblastic cells[J].J Biol Chem,2001,276(17):14443-14450

    • [23] HAŸ E,LAPLANTINE E,GEOFFROY V,et al.N ⁃cad⁃ herin interacts with axin and LRP5 to negatively regulate Wnt/beta⁃catenin signaling,osteoblast function,and bone formation[J].Mol Cell Biol,2009,29(4):953-964

    • [24] XU L,MENG F,NI M,et al.N⁃cadherin regulates osteo⁃ genesis and migration of bone marrow-derived mesenchy⁃ mal stem cells[J].Mol Biol Rep,2013,40(3):2533-2539

    • [25] HAŸ E,NOURAUD A,MARIE P J.N⁃cadherin negative⁃ ly regulates osteoblast proliferation and survival by antag⁃ onizing Wnt,ERK and PI3K/Akt signalling[J].PLoS One,2009,4(12):e8284