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Effect of Jieduquyuziyin prescription-treated rat serum on MeCP2 gene expression in Jurkat T cells

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Abstract

How genomic DNA methylation and methyl CpG-binding protein 2 (MeCP2) gene expression affect the pathogenesis of systemic lupus erythematosus (SLE) remains poorly understood. Traditional Chinese medicine has a unique effect in the treatment of SLE patients. This study aimed to investigate the effect of Jieduquyuziyin prescription (JP)-treated rat serum on the gene expression of MeCP2 in Jurkat T cells and its role in the pathogenesis of SLE. Jurkat T cells were harvested, and drug-containing serum was prepared. The ferulic acid and paeoniflorin content in the drug-containing serum were determined by liquid chromatography-mass spectrometry (LC-MS/MS). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were used to screen the optimal concentration of drug-containing serum. The DNA methylation level in Jurkat T cells was detected with a Methylamp™ Total DNA Methylation Kit. The methylation status of the MeCP2 promoter region was detected using bisulfite modification and methylation-specific PCR (MSP). Real-time PCR was used to measure MeCP2 mRNA expression. Western blotting and flow cytometry were done to detect MeCP2 protein expression in Jurkat cell nuclei. Paeoniflorin and ferulic acid were detected in the drug-containing serum of JP-treated rats. The results showed that cell growth was affected in the high serum-containing drug group. The experimental results showed that JP and prednisone acetate increased the level of genomic DNA methylation and MeCP2 gene promoter region methylation in Jurkat cells. MeCP2 mRNA and protein levels were also increased in the JP and prednisone acetate groups. Furthermore, flow cytometry revealed that the expression of MeCP2 protein in Jurkat T cell nuclei was higher in the drug group than the blank control group, and these results were consistent with the western blot analysis results. Our study found that there is a negative correlation between drug-containing serum and cell survival rate. JP upregulated the levels of DNA methylation, MeCP2 mRNA and protein as effectively as prednisone acetate and thus may activate the MeCP2 gene by increasing the methylation level, thereby inhibiting the pathogenesis of SLE. Therefore, JP may potentially be used to treat SLE patients. The Jurkat T lymphocyte in vitro experiments provided a foundation to study the effects of JP on the lupus mouse CD4+ T cell methylation mechanism and to further explore the pathogenesis of SLE.

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Abbreviations

JP:

Jieduquyuziyin prescription

MeCP2:

methyl CpG-binding protein 2

SLE:

systemic lupus erythematosus

TCM:

traditional Chinese medicine

SD:

Sprague–Dawley

PBS:

phosphate-buffered saline

FBS:

fetal bovine serum

LC-MS/MS:

liquid chromatography technique coupled with tandem mass spectrometry

HPLC:

high-performance liquid chromatography

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

MTS:

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

PBMCs:

peripheral blood mononuclear cells

ATCC:

American Type Culture Collection

PFA:

paraformaldehyde

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Acknowledgments

The authors would like to thank Dr. Li Rongqun, Dr. Pan Xiaoping, Dr. Hou Xiaoli, and Professor Liu Wenhong for their suggestions regarding the study.

Funding

This study was supported by the Natural Science Foundation of Zhejiang Province (no. LY15H270007) and the National Natural Science Foundation of China (no. 81673863).

Author information

Author notes

  1. Rongqun Li, Aiwen Zhuang, and Jiawei Ma contributed equally to this work.

Authors and Affiliations

  1. School of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, China

    Rongqun Li, Jiawei Ma, Lina Ji & Wenhong Liu

  2. Institute of TCM Literature and Information, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, China

    Aiwen Zhuang

  3. Chinese Medicine Hospital Shangyu Shaoxing, Shangyu, China

    Jiawei Ma

  4. Center of Analysis and Testing, Zhejiang Chinese Medical University, Hangzhou, China

    Xiaoli Hou & Xiaoping Pan

  5. Zhejiang Traditional Chinese Medical Hospital, Hangzhou, China

    Hongbo Chen

Authors
  1. Rongqun Li
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  8. Wenhong Liu
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Contributions

Conceived and designed the experiments: RL, AZ, JM, WL. Performed the experiments: RL, AW, JW, LN. Analyzed the data: RL, AW, JW. Contributed reagents/materials/analysis tools: XH, HC, XP. Write the paper: RL, AZ, JM. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Wenhong Liu.

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The authors declare that they have no competing interests.

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Not applicable.

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Studies involving animals

The animal experiments were approved by the Laboratory Animal Research Center and Use Committee of Zhejiang Chinese Medical University, Hangzhou, China.

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Editor: Tetsuji Okamoto

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Li, R., Zhuang, A., Ma, J. et al. Effect of Jieduquyuziyin prescription-treated rat serum on MeCP2 gene expression in Jurkat T cells. In Vitro Cell.Dev.Biol.-Animal 54, 692–704 (2018). https://doi.org/10.1007/s11626-018-0295-x

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