«上一篇/Previous Article|本期目录/Table of Contents|下一篇/Next Article»

《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

31卷

期数:

2025年03期

页码:

222-230

栏目:

基础医学

出版日期:

2025-05-20

文章信息/Info

Title:

Experimental study on the inhibition of oxidative stress by bone marrow mesenchymal stem cell-derived exosomes for the treatment of osteoporosis

文章编号:

1006-8147(2025)03-0222-09

作者:

周丽芸¹; 王岩²; 董本超²; 杨培川²; 李光²; 马剑雄²; 马信龙²

（1.天津医科大学骨科临床学院，天津300012；2.天津市骨科研究所，天津市生物力学重点实验室，天津大学天津医院，天津300050）

Author(s):

ZHOU Liyun¹; WANG Yan²; DONG Benchao²; YANG Peichuan²; LI Guang²; MA Jianxiong²; MA Xinlong²

（1.Clinical School/College of Orthopedics，Tianjin Medical University，Tianjin 300012，China；2.Orthopedic Institute，Tianjin Key Laboratory of Orthopedic Biomechanics and Medical Engineering，Tianjin University （Tianjin Hospital），Tianjin 300050，China）

关键词:

骨髓间充质干细胞; 外泌体; 衰老; 成骨分化; 氧化应激

Keywords:

bone marrow mesenchymal stem cells; exosomes; aging; osteogenic differentiation; oxidative stress

分类号:

R683

DOI:

10.20135/j.issn.1006-8147.2025.03.0222

文献标志码:

A

摘要:

目的：探究骨髓间充质干细胞（BMSCs）衍生的外泌体（BMSCs-EXO）治疗骨质疏松症的相关机制。方法：分离并鉴定BMSCs，收集BMSCs上清，提取并鉴定BMSCs-EXO；体外实验采用MC3T3-E1细胞诱导为骨质疏松细胞模型，使用外泌体进行治疗；CCK-8实验和β-半乳糖苷酶（SA-β-gal）染色评估细胞增殖与衰老；检测细胞活性氧簇（ROS）、超氧化物歧化酶（SOD）和丙二醛（MDA）；碱性磷酸酶（ALP）染色和茜素红染色（ARS）观察细胞成骨分化和矿化；Western印迹检测细胞氧化应激和成骨分化相关蛋白；将30只雄性C57BL/6J小鼠通过随机数字表法分为3组：对照组（CON组，n=10），骨质疏松组（D-gal组，n=10），外泌体治疗组（EXO组，n=10）；micro-CT分析骨体积分数（BV/TV）、骨小梁厚度（Tb.Th）、骨小梁数量（Tb.N）和骨小梁分离度（Tb.Sp）相关指标；HE染色评估治疗效果。结果：成功提取并鉴定BMSCs-EXO；细胞实验结果显示，与CON组相比，D-gal组SA-β-gal（t=5.506，P<0.001）、ROS（t=15.11，P<0.0001）、MDA水平显著升高（t=3.356，P<0.01），SOD活性显著降低（t=4.071，P<0.05），核因子E2相关因子2（Nrf-2）、血红素氧合酶（HO）-1蛋白表达显著降低（t=4.503、3.783，均P<0.05），ALP和ARS较浅，Runt相关转录因子2（RUNX2）、骨桥蛋白（OPN）蛋白表达水平显著降低（t=3.721、3.376，均P<0.05）；与D-gal组相比，经EXO治疗后，SA-β-gal（t=3.083，P<0.05）、ROS（t=6.254，P<0.001）、MDA（t=2.519，P<0.01）水平显著降低，Nrf-2、HO-1蛋白表达水平显著升高（t=6.997、3.649，均P<0.05），ALP阳性面积和ARS钙结节沉淀面积显著增加（t=7.912、12.76，均P<0.001），RUNX2、OPN蛋白表达水平显著升高（t=5.731、3.539，均P<0.01）；在动物实验中，与D-gal组相比，EXO组BV/TV、Tb.N、Tb.Th显著增加（t=2.815、4.387、5.497，均P<0.05），Tb.Sp显著减少（t=2.862，P<0.05），骨小梁面积显著增加（t=4.209，P<0.01）。结论：BMSCs-EXO通过Nrf-2/HO-1信号通路缓解氧化应激，减轻D-gal对MC3T3-E1细胞成骨分化的抑制作用，抑制骨质疏松。

Abstract:

Objective： To explore the therapeutic mechanisms of bone marrow mesenchymal stem cell-derived exosomes （BMSCs-EX-O） in osteoporosis treatment. Methods：BMSCs were isolated and identified，BMSCs supernatant was collected，and BMSCs-EXO were extracted and identified. In vitro，MC3T3-E1 cells were induced into osteoporosis cell model and treated with exosomes. Cell proliferation and senescence were evaluated by CCK-8 assay and β-galactosidase （SA-β-gal） staining. Reactive oxygen species （ROS），superoxide dismutase （SOD） and malondialdehyde （MDA） were detected. Alkaline phosphatase（ALP） staining and Alizarin Red S（ARS） staining were used to observe osteogenic differentiation and mineralization of the cells. Western blotting was used to detect the proteins related to oxidative stress and osteogenic differentiation. Thirty male C57BL/6J mice were randomly divided into 3 groups：control group （CON，n=10），osteoporosis group （D-gal，n=10），exosome treatment group （EXO，n=10）. Bone volume fraction （BV/TV），trabecular thickness （Tb.Th），trabecular number （Tb.N） and trabecular separation （Tb.Sp） related indexes were analyzed by micro-CT，and HE staining was used to evaluate the therapeutic effect. Results：BMSCs-EXO were successfully extracted and identified. The results of cell experiments showed that compared with the CON group，the levels of SA-β-gal（t=5.506，P<0.001），ROS （t=15.11，P<0.000 1） and MDA （t=3.356，P<0.01）， in the D-gal group were significantly increased and the activity of SOD was significantly decreased （t=4.071，P<0.01）. The protein expressions of nuclear factor E2 related factor 2 （Nrf-2） and heme oxygenase （HO）-1 were significantly decreased （t=4.503， 3.783，both P<0.05），the staining of ALP and ARS was lighter，and the protein expression levels of RUNX2 and OPN were significantly decreased （t=3.721， 3.376，both P<0.05）. Compared with the D-gal group，the levels of SA-β-gal （t=3.083，P<0.05），ROS （t=6.254，P<0.001） and MDA （t=2.519，P<0.01） were significantly decreased after EXO treatment. The protein expression levels of Nrf-2 and HO-1 were significantly increased （t=6.997，3.649，both P<0.05），ALP positive area and ARS calcium nodule precipitation area were significantly increased （t=7.912，12.76，both P<0.001）. The protein expression levels of RUNX2 and OPN were significantly increased （t=5.731，3.539，both P<0.01）. In animal experiments，BV/TV，Tb.N and Tb.Th were significantly increased （t=2.815，4.387，5.497，all P<0.05），Tb.Sp was significantly decreased （t=2.862，P<0.05），and trabecular bone area was significantly increased （t=4.209，P<0.01） in EXO group compared with D-gal group. Conclusion：BMSCs-EXO alleviates oxidative stress through Nrf-2/HO-1 signaling pathway，alleviates the inhibitory effect of D-gal on osteogenic differentiation of MC3T3-E1 cells，and reduces osteoporosis.

参考文献/References:

[1] LIANG B，BURLEY G，LIN S，et al. Osteoporosis pathogenesis and treatment：existing and emerging avenues[J]. Cell Mol Biol Lett，2022，27（1）：72.
[2] F?魻GER-SAMWALD U，KERSCHAN-SCHINDL K，BUTYLINA M M，et al. Age related osteoporosis：targeting cellular senescence[J]. Int J Mol Sci，2022，23（5）：2701.
[3] PATEL D，WAIRKAR S. Bone regeneration in osteoporosis：opportunities and challenges[J]. Drug Deliv Transl Res，2023，13（2）：419-432.
[4] VEIS D J，O′BRIEN C A. Osteoclasts，master sculptors of bone[J]. Annu Rev Pathol，2023，18：257-281.
[5] NAKAMURA M，AOYAMA N，YAMAGUCHI S，et al. Expression of tartrate-resistant acid phosphatase and cathepsin K during osteoclast differentiation in developing mouse mandibles[J]. Biomed Res，2021，42（1）：13-21.
[6] RUSSELL R G. Pharmacological diversity among drugs that inhibit bone resorption[J]. Curr Opin Pharmacol，2015，22：115-130.
[7] WANG J，FANG C L，NOLLER K，et al. Bone-derived PDGF-BB drives brain vascular calcification in male mice[J]. J Clin Invest，2023，133（23）：e168447.
[8] ZHANG W，WANG T，XUE Y，et al. Research progress of extracellular vesicles and exosomes derived from mesenchymal stem cells in the treatment of oxidative stress-related diseases[J]. Front Immunol，2023，14：1238789.
[9] ALEJANDRO P，CONSTANTINESCU F. A review of osteoporosis in the older adult：an update[J]. Rheum Dis Clin North Am，2018，44（3）：437-451.
[10] RU M，WANG W，ZHAI Z，et al. Nicotinamide mononucleotide supplementation protects the intestinal function in aging mice and D-galactose induced senescent cells[J]. Food Funct，2022，13（14）：7507-7519.
[11] LU S，ZHOU J，YANG C，et al. γ-Glutamylcysteine ameliorates D-gal-induced senescence in PC12 cells and mice via activating AMPK and SIRT1[J]. Food Funct，2022，13（14）：7560-7571.
[12] XIA T S，XU S Y，LAI L Y，et al. Bitter acids from humulus lupulus L. alleviate D-galactose induced osteoblastic senescence and bone loss via regulating AKT/mTOR-mediated autophagy[J]. J Food Drug Anal，2024，32（4）：506-519.
[13] QU Z，ZHANG J，YANG H，et al. Protective effect of tetrahydropalmatine against D-galactose induced memory impairment in rat[J]. Physiol Behav，2016，154：114-125.
[14] TABARA Y，IKEZOE T，YAMANAKA M，et al. Advanced glycation end product accumulation is associated with low skeletal muscle mass，weak muscle strength，and reduced bone density：the Nagahama Study[J]. J Gerontol A Biol Sci Med Sci，2019，74（9）：1446-1453.
[15] HU L，WANG B，FAN Y，et al. Exosomal miR-23b from bone marrow mesenchymal stem cells alleviates oxidative stress and pyroptosis after intracerebral hemorrhage[J]. Neural Regen Res，2023，18（3）：560-567.
[16] YANG H，CHEN J. Bone marrow mesenchymal stem cell-derived exosomes carrying long noncoding RNA ZFAS1 alleviate oxidative stress and inflammation in ischemic stroke by inhibiting microRNA-15a-5p[J]. Metab Brain Dis，2022，37（7）：2545-2557.
[17] LI Y，ZHENG J，WANG X，et al. Mesenchymal stem cell-derived exosomes protect trabecular meshwork from oxidative stress[J]. Sci Rep，2021，11（1）：14863.
[18] YANG J，LIU X，FAN H，et al. Extracellular vesicles derived from bone marrow mesenchymal stem cells protect against experimental colitis via attenuating colon inflammation，oxidative stress and apoptosis[J].PLoS One，2015，10（10）：e0140551.
[19] XU G，LU X，LIU S，et al. MSC-Derived exosomes ameliorate intervertebral disc degeneration by regulating the Keap1/Nrf2 axis[J].Stem Cell Rev Rep，2023，19（7）：2465-2480.
[20] LIU X，CHEN Y，ZHANG T. Mechanism study of BMSC-exosomes combined with hyaluronic acid gel in the treatment of posttraumatic osteoarthritis[J]. Heliyon，2024，10（14）：e34192.
[21] GHEZZI P.Environmental risk factors and their footprints in vivo - a proposal for the classification of oxidative stress biomarkers[J].Redox Biol，2020，34：101442.
[22] SHENG Y，ABREU I，CABELLI D，et al. Superoxide dismutases and superoxide reductases[J]. Chem Rev，2014，114（7）：3854-3918.
[23] DE LOS SANTOS-JIMR?魪NEZ J，CAMPOS-SANDOVAL J，M?魣R-QUEZ-TORRES C，et al. Glutaminase isoforms expression switches microRNA levels and oxidative status in glioblastoma cells[J]. J Biomed Sci，2021，28（1）：14.
[24] JIMENEZ R，TORAL M，G?譫MEZ-GUZM?魣N M，et al. The role of Nrf2 signaling in PPARβ/δ-mediated vascular protection against hyperglycemia-induced oxidative stress[J]. Oxid Med Cell Longev，2018，2018：5852706.
[25] CHANDRA A，RAJAWAT J. Skeletal aging and osteoporosis：mechanisms and therapeutics[J]. Int J Mol Sci，2021，22（7）：3553.
[26] HYEON S，LEE H，YANG Y，et al. Nrf2 deficiency induces oxidative stress and promotes RANKL-induced osteoclast differentiation[J]. Free Radic Biol Med，2013，65：789-799.
[27] HANG Y，ZHONG Z，HOU G，et al. Involvement of oxidative stress in age-related bone loss[J]. J Surg Res，2011，169（1）：e37-e42.

相似文献/References:

[1]张丽梅,贾莉莉 综 述,喻文立 审 校.外泌体在病毒性肝炎、肝硬化和肝癌中的作用研究进展[J].天津医科大学学报,2019,25(03):305.
[2]王雅蕾,王靖怡 综述,齐丽莎 审校.微环境在卵巢癌发生发展中的作用[J].天津医科大学学报,2020,26(03):288.
[3]潘大宇,宁广智,冯世庆.雪旺细胞外泌体影响胶质瘢痕形成修复小鼠脊髓损伤的实验研究[J].天津医科大学学报,2020,26(05):397.
　PAN Da-yu,NING Guang-zhi,FENG Shi-qing.Schwann cell-derived exosomes promote functional recovery of spinal cord injury in mice via affecting glial scar formation[J].Journal of Tianjin Medical University,2020,26(03):397.
[4]张杨,游阿彬,齐寒,等.负载化疗药物的外泌体对肝癌的靶向治疗研究[J].天津医科大学学报,2021,27(03):229.
　ZHANG Yang,YOU A-bin,QI Han,et al.Tumor-derived exosomes mediate targeted therapy in hepatocellular carcinoma mice[J].Journal of Tianjin Medical University,2021,27(03):229.
[5]王敏,邢逸,徐海楠,等.M2型巨噬细胞外泌体对皮肤创面愈合影响的探究[J].天津医科大学学报,2022,28(04):343.
　WANG Min,XING Yi,XU Hai-nan,et al.The effect of M2 macrophage-derived exosomes on skin wound healing[J].Journal of Tianjin Medical University,2022,28(03):343.
[6]吴颖杰,耿梦缘,汪晶,等.不同冻干保护剂在外泌体储存中的研究[J].天津医科大学学报,2022,28(04):353.
　WU Ying-jie,GENG Meng-yuan,WANG Jing,et al.Study on the effect of different lyoprotectants on exosomes[J].Journal of Tianjin Medical University,2022,28(03):353.
[7]索睿,郝天旭,茹仙古丽·吾买尔 综述,等.间充质干细胞来源的外泌体治疗哮喘的研究进展[J].天津医科大学学报,2023,29(06):673.
[8]陈傲,邱帅,李悦,等.功能化外泌体对实验性自身免疫性脑脊髓炎小鼠治疗的研究[J].天津医科大学学报,2024,30(03):212.[doi:10.20135/j.issn.1006-8147.2024.03.0212]
　CHEN Ao,QIU Shuai,LI Yue,et al.Study of functionalized exosomes for treating experimental autoimmune encephalomyelitis in mice[J].Journal of Tianjin Medical University,2024,30(03):212.[doi:10.20135/j.issn.1006-8147.2024.03.0212]
[9]潘金元 综述,蒋丽丽 审校.细胞外囊泡参与乳腺癌转移的研究进展[J].天津医科大学学报,2024,30(03):273.[doi:10.20135/j.issn.1006-8147.2024.03.0270]
[10]吴慧娴,练学淦.低氧预处理骨髓间充质干细胞对小鼠脑缺血再灌注模型中IL-12的影响[J].天津医科大学学报,2024,30(06):522.[doi:10.20135/j.issn.1006-8147.2024.06.0522]
　WU Huixian,LIAN Xuegan.The effect of hypoxic pretreatment of bone marrow mesenchymal stem cells on IL-12 after cerebral ischemia-reperfusion injury in mice[J].Journal of Tianjin Medical University,2024,30(03):522.[doi:10.20135/j.issn.1006-8147.2024.06.0522]

备注/Memo

备注/Memo:

基金资助 国家重点研发计划（2022YFC3601900）；天津市自然科学基金重点项目（22JCZDJC00340）；天津市卫生健康科技项目（TJWJ2022QN053）
作者简介: 周丽芸（1999-），女，硕士在读，研究方向：骨质疏松；
通信作者：马剑雄，E-mail：yjslwtg@126.com。

常用功能

更新日期/Last Update: 2025-06-01