[1]刘明明,陈翀,许海燕,等.加味六味地黄汤对巨首相关蛋白-1基因的影响[J].中国中医骨伤科杂志,2024,32(07):11-17.[doi:10.20085/j.cnki.issn1005-0205.240702 ]
　LIU Mingming,CHEN Chong,XU Haiyan,et al.Effect of Modified Liuwei Dihuang Decoction on Cytosine Hydroxymethylation of Dickkopf-Related Protein 1 Gene in BMSCs under High Glucose Stress[J].Chinese Journal of Traditional Medical Traumatology & Orthopedics,2024,32(07):11-17.[doi:10.20085/j.cnki.issn1005-0205.240702 ]
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加味六味地黄汤对巨首相关蛋白-1基因的影响()
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《中国中医骨伤科杂志》[ISSN:1005-0205/CN:42-1340/R]

卷:
第32卷
期数:
2024年07期
页码:
11-17
栏目:
实验研究
出版日期:
2024-07-15

文章信息/Info

Title:
Effect of Modified Liuwei Dihuang Decoction on Cytosine Hydroxymethylation of Dickkopf-Related Protein 1 Gene in BMSCs under High Glucose Stress
文章编号:
1005-0205(2024)07-0011-07
作者:
刘明明1; 陈翀2; 许海燕2; 杨伏猛1; 吕南宁1; 李洪叶1△
1徐州医科大学连云港临床学院(江苏 连云港,222003)
2徐州医科大学
Author(s):
LIU Mingming1; CHEN Chong2; XU Haiyan2; YANG Fumeng1LÜ Nanning1; LI Hongye1△
1Lianyungang Clinical College of Xuzhou Medical University,Lianyungang 222003,Jiangsu China; 2Xuzhou Medical University,Xuzhou 221004,Jiangsu China.
关键词:
骨髓间充质干细胞;  加味六味地黄汤;  巨首相关蛋白-1;  胞嘧啶羟甲基化;  糖尿病性骨质疏松症
Keywords:
bone marrow mesenchymal stem cells;  modified Liuwei Dihuang decoction;  dickkopf-related protein 1;  cytosine hydroxymethylation;  diabetic osteoporosis
分类号:
R-33
DOI:
10.20085/j.cnki.issn1005-0205.240702
文献标志码:
A
摘要:
目的:探讨加味六味地黄汤在骨髓间充质干细胞(BMSCs)高糖应激下巨首相关蛋白-1(DKK1)基因胞嘧啶羟甲基化的作用机制。方法:将雄性SD大鼠随机分为空白组(等量生理盐水)和加味六味地黄汤干预组(24.255 g/kg)灌胃干预,制备含药血清。加味六味地黄汤含药血清干预骨髓间充质干细胞48 h,通过qRT-PCR和ELISA测定高糖应激下DKK1基因及蛋白的表达; 用双荧光素酶报告基因检测高糖应激下DKK1基因启动子活性; 5-hmC和5-mC分析加味六味地黄汤含药血清对高糖应激下DKK1基因启动子区的CpG甲基化和羟甲基化水平及Abct和Pgk1基因启动子区的羟甲基化水平; 通过TETs活性及qRT-PCR测定加味六味地黄汤含药血清对TETs活性及TETs基因表达水平的影响。结果:与0%含药血清相比,20%加味六味地黄汤含药血清可以显著抑制高糖状态下DKK1 mRNA表达水平,差异有统计学意义(P<0.01); 同时显著减少培养上清中DKK1的浓度,差异有统计学意义(P<0.01); 抑制了高糖诱导的DKK1启动子活性,差异有统计学意义(P<0.01)。5-hmC和5-mC分析结果显示20%加味六味地黄汤含药血清降低了DKK1启动子区羟甲基化水平,差异有统计学意义(P<0.05); 同时提高了该区域甲基化水平,差异有统计学意义(P<0.01); 加味六味地黄汤含药血清对TETs的活性没有显著影响,差异无统计学意义(P>0.05),提高了TET2基因表达水平,差异有统计学意义(P<0.05)。结论:加味六味地黄汤选择性抑制DKK1基因胞嘧啶羟甲基化,减轻高糖应激诱发的小鼠骨髓间充质干细胞DKK1表达。
Abstract:
Objective:To investigate the effect of modified Liuwei Dihuang decoction on cytosine hydroxymethylation of dickkopf-related protein 1(DKK1)gene in bone marrow mesenchymal stem cells(BMSCs)under high glucose stress.Methods:Male SD rats were randomly divided into blank group(equivalent normal saline)and intervention group treated with a modified Liuwei Dihuang decoction(24.255 g/kg)by gavage to prepare medicated serum.BMSCs were treated with 20% serum containing modified Liuwei Dihuang decoction for 48 h,and the relative protein and gene expression levels of DKK1 were analyzed by qRT-PCR and ELISA tests under high-glucose environments.Dual luciferase reporter gene assay was employed to evaluate the the DKK1 promoter activity.The 5-hmC and 5-mC was used to analyze the methylation level of the DKK1 gene and hydroxymethylation level of CpG island of the DKK1,Abct and Pgk1 genes under high glucose stress.TETs activity and qRT-PCR were used to detected the TET(ten-eleven translocation)activity of TETs and expression level of TETs.Results:Compared with 0% modified Liuwei Dihuang decoction containing serum,20% modified Liuwei Dihuang decoction containing serum significantly inhibited the expression level of DKK1 mRNA under high glucose conditions(P<0.01),while significantly reduced the concentration of DKK1(P<0.01).Additionally,high-glucose-induced DKK1 promoter activity overexpression was attenuated(P<0.01).The results of 5-hmC and 5-mC analysis showed that 20% modified Liuwei Dihuang decoction containing serum reduced the hydroxymethylation level in the DKK1 promoter region(P<0.05),while increased the methylation level in this region(P<0.01).The 20% modified Liuwei Dihuang decoction containing serum had no significant effect on the activity of TETs(P>0.05).However,it can increase the expression level of TET2 gene(P<0.05).Conclusion:Modified Liuwei Dihuang decoction selectively inhibits cytosine hydroxymethylation of DKK1 gene and alleviates the expression of DKK1 in high-glucose-induced BMSCs.

参考文献/References:

[1] 刘明明,马勇,许海燕,等.加味六味地黄汤治疗肝肾亏虚型糖尿病性骨质疏松症的临床疗效[J].中国中医骨伤科杂志,2022,30(3):12-16.
[2] YUAN S,ZHANG C,WANG B.Neohesperidin promotes the proliferation and osteogenic differentiation of BMSCs via BMP2-Wnt/β-catenin pathway[J].Cell Cycle,2022,21(2):187-201.
[3] HE S C,GUAN Y,WU Y C,et al.DEC1 deficiency results in accelerated osteopenia through enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3β signaling[J].Metabolism,2021,118:154730.
[4] VANOVA M M,DAO J,KASACI N,et al.Wnt signaling pathway inhibitors,sclerostin and DKK-1,correlate with pain and bone pathology in patients with Gaucher disease[J].Front Endocrinol(Lausanne),2022,13:1029130.
[5] FAN M,SHI H,YAO H,et al.Glutamate regulates gliosis of BMSCs to promote ENS regeneration through α-KG and H3K9/H3K27 demethylation[J].Stem Cell Res Ther,2022,13(1):255.
[6] QIN W,SCICLUNA B P,VAN DER POLL T.The role of host cell DNA methylation in the immune response to bacterial infection[J].Front Immunol,2021,12:696280.
[7] SHEN H X,YANG E S,CONRY M,et al.Predictive biomarkers for immune checkpoint blockade and opportunities for combination therapies[J].Genes Dis,2019,6(3):232-246.
[8] 王建勇,黄诗诗,金婉君,等.DNA甲基化和羟甲基化在帕金森病发病机制中的研究进展[J].浙江医学,2023,45(3):315-318.
[9] TAIRA A,PALIN K,ANNA K K,et al.Vitamin C boosts DNA demethylation in TET2 germline mutation carriers[J].Clin Epigenetics,2023,15(1):7.
[10] KAASINEN E,KUISMIN O,RAJAMÄKI K,et al.Impact of constitutional TET2 haploinsufficiency on molecular and clinical phenotype in humans[J].Nat Commun,2019,10(1):1252.
[11] LI R,ZHENG C,WANG Q,et al.Identification of an immunogenic DKK1 long peptide for immunotherapy of human multiple myeloma[J].Haematologica,2021,106(3):838-846.
[12] 王童瑶,李思源,李军,等.绝经后女性2型糖尿病合并骨质疏松与DKK1基因多态性关系的研究[J].糖尿病新世界,2022,25(7):10-12.
[13] JANECZEK A A,TARE R S,SCARPA E,et al.Transient canonical Wnt stimulation enriches human bone marrow mononuclear cell isolates for osteoprogenitors[J].Stem Cells,2016,34(2):418-430.
[14] SUN T,YAN Z,CAI J,et al.Effects of mechanical vibration on cell morphology,proliferation,apoptosis,and cytokine expression/secretion in osteocyte-like MLO-Y4 cells exposed to high glucose[J].Cell Biol Int,2020,44(1):216-228.
[15] HE C,ZHANG H Y,ZHANG Y X,et al.Cytosine methylations in the promoter regions of genes involved in the cellular oxidation equilibrium pathways affect rice heat tolerance[J].BMC Genomics,2020,21(1):560.
[16] AIVALIOTI M M,BARTHOLDY B A,PRADHAN K,et al.PU.1-dependent enhancer inhibition separates TET2-deficient hematopoiesis from malignant transformation[J].Blood Cancer Discov,2022,3(5):444-467.

备注/Memo

备注/Memo:
基金项目:江苏省卫生健康委医学科研项目(H2023049)
连云港市第六期“521高层次人才培养工程”科研项目(LYG06521202159)
连云港市中医药科技发展计划(重点项目)(ZD202210)
连云港市“十四五”花果山科教强医工程(连卫科教〔2021〕1号)
△通信作者 E-mail:li19816269587@163.com
更新日期/Last Update: 2024-07-05