[1]耿卢婧,孙智欣,李俞辰,等.温度对过氧化氢抑制前成骨细胞MC3T3-E1细胞增殖和成骨分化的影响[J].新乡医学院学报,2024,(2):109-114.[doi:10.7683/xxyxyxb.2024.02.002]
　GENG Lujing,SUN Zhixin,LI Yuchen,et al.Effect of temperature on the inhibitory effect induced by hydrogen peroxide on cell proliferation and osteogenic differentiation in preosteoblast MC3T3-E1 cells[J].Journal of Xinxiang Medical University,2024,(2):109-114.[doi:10.7683/xxyxyxb.2024.02.002]
点击复制

温度对过氧化氢抑制前成骨细胞MC3T3-E1细胞增殖和成骨分化的影响
分享到：

《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
期数:
2024年2
页码:
109-114
栏目:
基础研究
出版日期:
2024-02-05

文章信息/Info

Title:
Effect of temperature on the inhibitory effect induced by hydrogen peroxide on cell proliferation and osteogenic differentiation in preosteoblast MC3T3-E1 cells
作者:
耿卢婧; 孙智欣; 李俞辰; 张瑜; 史培培
（新乡医学院生命科学技术学院，河南　新乡　453003）
Author(s):
GENG Lujing; SUN Zhixin; LI Yuchen; ZHANG Yu; SHI Peipei
(College of Life Sciences and Technology,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
关键词:
孵育温度; 氧化损伤; 成骨细胞; MC3T3-E1细胞; 细胞增殖; 成骨分化
Keywords:
incubation temperature; oxidative damage; preosteoblast; MC3T3-E1 cell; cell proliferation; osteogenic differentiation
分类号:
R329.2
DOI:
10.7683/xxyxyxb.2024.02.002
文献标志码:
A
摘要:
目的　探讨温度对过氧化氢(H2O2)抑制前成骨细胞增殖和成骨分化的影响。
方法　取对数生长期MC3T3-E1细胞，随机分为0、450、500、550、600、650 μmol·L-1H2O2干预组，分别给予0、450、500、550、600、650 μmol·L-1H2O2溶液干预2 h。另取对数生长期MC3T3-E1细胞，随机分为对照组、模型组、低温组和高温组。对照组细胞置于37 ℃、含体积分数5%CO2培养箱中孵育24 h；模型组细胞置于37 ℃、含体积分数5%CO2培养箱中孵育24 h，并给予H2O2刺激2 h；低温组细胞置于32 ℃、含体积分数5%CO2培养箱中孵育24 h，并给予H2O2刺激2 h；高温组细胞置于40 ℃、含体积分数5%CO2培养箱中孵育24 h，并给予H2O2刺激2 h。采用细胞计数试剂盒-8检测各组细胞增殖能力，实时荧光定量聚合酶链反应法检测细胞中Runt相关转录因子2（RUNX2）、骨桥蛋白（OPN）和骨钙素（OC）mRNA表达水平，Western blot法检测 MC3T3-E1 细胞中RUNX2、OPN和OC蛋白表达水平。
结果　0、450、500 μmol·L-1H2O2干预组细胞增殖率比较差异无统计学意义（P>0.05）；550、600、650 μmol·L-1 H2O2干预组细胞增殖率显著低于0、450、500 μmol·L-1H2O2干预组，且随H2O2浓度增加细胞增殖率显著降低（P<0.05）。为保证后续实验有足够的细胞，选择H2O2的干预浓度为 550 μmol·L-1。模型组和低温组细胞增殖率显著低于对照组和高温组，低温组细胞增殖率显著低于模型组（P<0.05）；对照组与高温组细胞增殖率比较差异无统计学意义（P>0.05）。模型组和高温组细胞中RUNX2 mRNA相对表达量显著高于对照组和低温组，低温组细胞中RUNX2 mRNA相对表达量显著低于对照组（P<0.05）；模型组与高温组细胞中RUNX2 mRNA相对表达量比较差异无统计学意义（P>0.05）。模型组、低温组和高温组细胞中OPN mRNA相对表达量显著高于对照组，低温组和高温组细胞中OPN mRNA相对表达量显著高于模型组，低温组细胞中OPN mRNA相对表达量显著高于高温组（P<0.05）。模型组、低温组和高温组细胞中OC mRNA相对表达量显著高于对照组，低温组和高温组细胞中OC mRNA相对表达量显著高于模型组（P<0.05）；低温组与高温组细胞中OC mRNA相对表达量比较差异无统计学意义（P>0.05）。模型组、低温组和高温组细胞中RUNX2、OPN和OC蛋白相对表达量显著低于对照组（P<0.05）。低温组细胞中RUNX2、OPN蛋白相对表达量显著低于模型组和高温组，OC蛋白相对表达量显著低于高温组（P<0.05）；低温组与模型组细胞中OC蛋白相对表达量比差异无统计学意义（P>0.05）。高温组细胞中RUNX2、OPN和OC蛋白相对表达量显著高于模型组（P<0.05）。
结论　H2O2可抑制MC3T3-E1细胞增殖和成骨分化，低温可增强H2O2对MC3T3-E1细胞增殖和成骨分化的抑制作用，高温可缓解H2O2对细胞增殖和成骨分化的抑制作用。RUNX2、OPN和OC蛋白可能在温度调控细胞增殖和成骨分化的过程中发挥重要作用。
Abstract:
Objective　To investigate the effect of temperature on cell proliferation and osteogenic differentiation inhibition of preosteoblast induced by hydrogen peroxide(H2O2).
Methods　The MC3T3-E1 cells in the logarithmic phase were randomly divided into 0,450,500,550,600,650 μmol·L-1 H2O2 intervention groups and incubated with 0,450,500,550,600,650 μmol·L-1 H2O2 for 2 h,respectively.Other MC3T3-E1 cells in the logarithmic phase were selected and randomly divided into the control group,model group,low-temperature group,and high-temperature group.Cells in the control group were cultured in an incubator with 5% CO2 for 24 h at 37 ℃;cells in the model group were incubated with H2O2 for 2 h and cultured in an incubator with 5% CO2 for 24 h at 37 ℃;cells in the low-temperature group were incubated with H2O2 for 2 h and cultured in an incubator with 5% CO2 for 24 h at 32 ℃;cells in the high-temperature group were incubated with H2O2 for 2 h and cultured in an incubator with 5% CO2 for 24 h at 40 ℃.The cell proliferation in all groups was detected by cell counting kit-8.The expression levels of Runt-related transcription factor 2 (RUNX2),osteopontin (OPN) and osteocalcin (OC) mRNA were detected by real-time fluorescence quantitave polymerase chain reaction;and the expression levels of RUNX2,OPN and OC protein were detected by Western blot.
Results　There was no statistically significant difference in cell proliferation among the 0,450 and 500 μmol·L-1 H2O2 intervention groups (P>0.05);the cell proliferation rate in the 550,600 and 650 μmol·L-1 H2O2 intervention groups was significantly lower than that in the 0,450 and 500 μmol·L-1 H2O2 intervention groups,showing a significant decrease in cell proliferation with the increase of H2O2 concentrations (P<0.05).In order to ensure that there were enough cells to perform the following experiments,550 μmol·L-1 H2O2 was chosen.The cell proliferation rate in the model group and the low-temperature group was significantly lower than that in the control group and high-temperature group (P<0.05);there was no significant difference in the cell proliferation rate between the control group and high-temperature group (P>0.05).The relative expression of RUNX2 mRNA in the model group and high-temperature group were significantly higher than that in the control group and low-temperature group (P<0.05);the relative expression of RUNX2 mRNA in the low-temperature group was significantly lower than that in the control group (P<0.05);there was no significant difference in the relative expression of RUNX2 mRNA between the model group and high-temperature group(P>0.05).The relative expression of OPN mRNA in the model group,low-temperature group and high-temperature group was significantly higher than that in the control group (P<0.05);the relative expression of OPN mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group (P<0.05);the relative expression of OPN mRNA in the low-temperature group was significantly higher than that in the high-temperature group (P<0.05).The relative expression of OC mRNA in the model group,low-temperature group and high-temperature group was significantly than that in the control group(P<0.05);the relative expression of OC mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group (P<0.05);there was no significant difference in the relative expression of OC mRNA between the low-temperature group and high-temperature group (P>0.05).The relative expressions of RUNX2,OPN and OC protein the model group,low-temperature group and high-temperature group were significantly lower than those in the control group(P<0.05);the relative expressions of RUNX2 and OPN protein in the low-temperature group were significantly lower than those in the model group and high-temperature group (P<0.05);the relative expression of OC protein was significantly lower than that in the high-temperature group (P<0.05);and there was no siqnificantly difference in the relatiwe experesson of OC protein between the low-temperature group and model group (P>0.05);the relative expressions of RUNX2,OPN and OC protein in the high-temperature group were significantly higher than those in the model group (P<0.05).
Conclusion　The inhibitory effects of H2O2 on cell proliferation and osteogenic differentiation are observed in MC3T3-E1 cells;low-temperature incubation can enhance the inhibition of H2O2 on cell proliferation and osteogenic differentiation in MC3T3-E1 cells,while high-temperature incubation can relieve its inhibitory effect on cell proliferation and osteogenic differentiation.RUNX2,OPN and OC protein might play an important role in cell proliferation and osteogenic differentiation mediated by temperature.

参考文献/References:

［1］　CUI Z,FENG H,MENG X,et al.Age-specific 1-year mortality rates after hip fracture based on the populations in mainland China between the years 2000 and 2018:a systematic analysis［J］.Arch Osteoporos,2019,14(1):55.
［2］　KHOSLA S,FARR J N,TCHKONIA T,et al.The role of cellular senescence in ageing and endocrine disease［J］.Nat Rev Endocrinol,2020,16(5):263-275.
［3］　LI R,BOER C G,OEI L,et al.The gut microbiome:a new frontier in musculoskeletal research［J］.Curr Osteoporos Rep,2021,19(3):347-357.
［4］　孙钰,杨利学,王官林，等.从肠道微生态探讨“肝-肠相通”理论在骨质疏松症防治中的应用［J］.中医学报,2022,37(6):1148-1152.
SUN Y,YANG L X,WANG G L,et al.Exploring application of "Liver-Intestine Connectivity" theory in prevention and treatment of osteoporosis from perspective of intestinal microecology［J］.Acta Chin Med,2022,37(6):1148-1152.
［5］　LI X,XU J,DAI B,et al.Targeting autophagy in osteoporosis:from pathophysiology to potential therapy［J］.Ageing Res Rev,2020,62:101098.
［6］　祝震亚,童蕾,陆燕群.miR-181a调控PINK1/Parkin通路对骨质疏松大鼠破骨细胞线粒体自噬的影响［J］.解放军医学杂志,2022,47(6):569-578.
ZHU Z Y,TONG L,LU YQ.Effect of miR-181a regulating PINK1/Parkin pathway on mitochondrial autophagy of osteoclasts in osteoporotic rats［J］.Med J Chin PLA,2022,47(6):569-578.
［7］　MA H,WANG X,ZHANG W,et al.Melatonin suppresses ferroptosis induced by high glucose via activation of the Nrf2/HO-1 signaling pathway in type 2 diabetic osteoporosis［J］.Oxid Med Cell Longev,2020,2020:9067610.
［8］　曹娟,李玲玲,姚瑶,等.氧化应激相关细胞衰老指标与骨关节炎的相关性探讨［J］.中华全科医学,2023,21(3):396-400.
CAO J,LI L L,YAO Y,et al.Correlation between oxidative stress related cell senescence indicators and osteoarthritis［J］.Chin J Gen Pract,2023,21(3):396-400.
［9］　KIMBALL J S,JOHNSON J P,CARLSON D A.Oxidative stress and osteoporosis［J］.J Bone Joint Surg Am,2021,103(15):1451-1461.
［10］　谢英毫.高温环境下人体年龄对热生理与行为能力的影响［D］.苏州:苏州大学,2021.
XIE Y H.Effect of human age on thermophysiological response and behavior ability in a high temperature environment［D］.Suzhou:Soochow University,2021.
［11］　WANG H,LGEN M,TRAJKOVSKI M.Importance of temperature on immuno-metabolic regulation and cancer progression［J］.FEBS J,2022,Epub ahead of print.
［12］　HUUS K E,LEY R E.Blowing hot and cold:body temperature and the microbiome［J］.mSystems,2021,6(5):e0070721.
［13］　HYLANDER B L,GORDON C J,REPASKY E A.Manipulation of ambient housing temperature to study the impact of chronic stress on immunity and cancer in mice［J］.J Immunol,2019,202(3):631-636.
［14］　LEE H J,ALIRZAYEVA H,KOYUNCU S,et al.Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes［J］.Nat Aging,2023,3(5):546-566.
［15］　中国研究型医院学会神经再生与修复专业委员会心脏重症脑保护学组,中国研究型医院学会神经再生与修复专业委员会神经重症护理与康复学组.亚低温脑保护中国专家共识［J］.中华危重病急救医学,2020,32(4):385-391.
CEREBRAL PROTECTION IN CARDIAC INTENSIVE CARE GROUP,NEURAL REGENERATION AND REPAIR COMMITTEE,CHINESE RESEARCH HOSPITAL ASSOCIATION,NEURAL INTENSIVE NURSING AND REHABILITATION GROUP,NEURAL REGENERATION AND REPAIR COMMITTEE,CHINESE RESEARCH HOSPITAL ASSOCIATION.Chinese consensus for mild hypothermia brain protection［J］.Chin Crit Care Med,2020,32(4):385-391.
［16］　YUAN X,ZHANG J,MA T T,et al.Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia［J］.Biochem Biophys Res Commun,2021,534:240-247.
［17］　LI S,CHIEN S,BRANEMARK P I.Heat shock-induced necrosis and apoptosis in osteoblasts［J］.J Orthop Res,1999,17(6):891-899.
［18］　YANG Y,SUN Y,MAO W W,et al.Oxidative stress induces downregulation of TP53INP2 and suppresses osteogenic differentia-tion of BMSCs during osteoporosis through the autophagy degradation pathway［J］.Free Radic Biol Med,2021,166:226-237.
［19］　阮建伟.抑制microRNA-29a促使Gabl蛋白上调保护OB-6人成骨细胞免受H2O2破坏的机制研究［D］.苏州:苏州大学,2018.
RUAN J W.MicroRNA-29a inhibition induces Gab1 upregulation to protect OB-6 human osteoblasts from hydrogen peroxide［D］.Suzhou:Soochow University,2018.
［20］　卢茜.硒对H2O2诱导血管平滑肌细胞向成骨细胞分化作用的影响及机理［D］.武汉:华中科技大学,2011.
LU Q.Effect of selenium on H2O2-induced osteoblastic differentiation of vascular smooth muscle cells and the related mechanisms［D］.Wuhan:Huazhong University of Science and Technology,2011 .
［21］　黄灿华.应激医学与人类健康［J］.四川大学学报(医学版),2021,52(1):1-4.
HUANG C H.Stress medicine and human health［J］.J Sichuan Univ(Med Sci),2021,52(1):1-4.
［22］　PREUBNER M,SMITH H L,HUGHES D,et al.ASO targeting RBM3 temperature-controlled poison exon splicing prevents neurodegeneration in vivo［J］.EMBO Mol Med,2023,15(5):e17157.
［23］　FU R,HUANG Z,LI H,et al.A hemidesmosome-to-cytoplasm translocation of small heat shock proteins provides immediate protection against heat stress［J］.Cell Rep,2020,33(8):108410.
［24］　杨彤涛,甘璐,周勇.热作用下成骨细胞HSP70 mRNA的表达变化及其对破骨细胞增殖的影响［J］.现代生物医学进展,2010,10(19):3639-3642.
YANG T T,GAN L,ZHOU Y.Effect of thermal stimulation on expression of HSP70 mRNA in osteoblasts & its medium′s effect on proliferation of osteoclasts in mice［J］.Progr Mod Biomed,2010,10(19):3639-3642.
［25］　ZENG H,HUANG J,REN J,et al.Spatially resolved single-cell translatomics at molecular resolution［J］.Science,2023,380(6652):eadd3067.
［26］　DE SOUSA ABREU R,PENALVA L O,MARCOTTE E M,et al.Global signatures of protein and mRNA expression levels［J］.Mol Biosyst,2009,5(12):1512-1526.

相似文献/References:

[1]卢　娜,李　晓,刘冬玲,等.线粒体DNA氧化损伤在动脉粥样硬化发生、发展中的作用研究进展[J].新乡医学院学报,2021,38(3):201.[doi:10.7683/xxyxyxb.2021.03.001]
[2]赵颖丹,张天嵩,马　骏,等.细颗粒物2.5在肾小管上皮细胞氧化损伤中的作用及其调控机制[J].新乡医学院学报,2020,37(12):1118.[doi:10.7683/xxyxyxb.2020.12.004]
　ZHAO Yingdan,ZHANG Tiansong,MA Jun,et al.Role of particulate matter 2.5 in oxidative damage of renal tubular epithelial cells and its regulatory mechanism[J].Journal of Xinxiang Medical University,2020,37(2):1118.[doi:10.7683/xxyxyxb.2020.12.004]
[3]程　莹,张利彬,丁延峰,等.P2X7受体阻断剂亮蓝G对氧化应激所致红细胞氧化损伤的保护机制[J].新乡医学院学报,2016,33(6):462.[doi:10.7683/xxyxyxb.2016.06.004]
　CHENG Ying,ZHANG Li-bin,DING Yan-feng,et al.Protection mechanism of P2X7 receptor antagonist brilliant blue G to oxidative damage of erythrocyte[J].Journal of Xinxiang Medical University,2016,33(2):462.[doi:10.7683/xxyxyxb.2016.06.004]

更新日期/Last Update: 2024-02-05