Role and Mechanism of Metformin in Oligodendrocyte Precursor Cell Differentiation

doi:10.13523/j.cb.2103013

China Biotechnology

2021, Vol. 41

Issue (9): 1-9 DOI: 10.13523/j.cb.2103013

Role and Mechanism of Metformin in Oligodendrocyte Precursor Cell Differentiation

TAN Pei-lin^1,²,ZHANG Ying²,ZHANG Jun¹,GAO Xiao¹,WANG Shu-kun²,HOU Lin¹,YUAN Zeng-qiang^1,^2,^**(

)

1 School of Basic Medicine, Qingdao 266071, China
2 Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Beijing 100850, China

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Abstract

Objective:To demonstrate the role of metformin in oligodendrocyte precursor cell (OPC) differentiation and preliminarily discuss the molecular mechanism.Methods:OPC was directly isolated and purified by immune adsorption from the brain and identified using immunofluorescence. Firstly, the concentration of metformin was decided through cell viability assay. Then, the effects of metformin on OPC-differentiation related positive cells, the mRNA or protein level were analyzed by immunofluorescence, flow cytometry, qRT-PCR, and western blot.Results:High purity of primary cells were obtained. CCK8 assay showed that there is no significant toxicity of metformin (<100 μmol/L) on cell viability. Moreover, the significant increasement of PDGFRα⁺OLIG2⁺ and MBP⁺ cells, up-regulation levels of Mag, Olig2, Mbp and Sox10 mRNA and OLIG2, MBP protein were detected in OPC after metformin treatment. Mechanically, compared to the control group, RAS, p-MEK and p-ERK proteins were significantly increased after metformin treatment for 5min in Oli-neu cells and OPC. Conclusion:Metformin promotes the differentiation of oligodendrocyte precursor cells through the RAS-MEK-ERK signaling pathway.

Key words： Metformin Oligodendrocyte precursor cells(OPCs) Demyelinating diseases

Received: 09 March 2021 Published: 30 September 2021

ZTFLH:

Q819

Corresponding Authors: Zeng-qiang YUAN E-mail: zyuan620@yahoo.com

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	Pei-lin TAN
	Ying ZHANG
	Jun ZHANG
	Xiao GAO
	Shu-kun WANG
	Lin HOU
	Zeng-qiang YUAN

Cite this article:

TAN Pei-lin,ZHANG Ying,ZHANG Jun,GAO Xiao,WANG Shu-kun,HOU Lin,YUAN Zeng-qiang. Role and Mechanism of Metformin in Oligodendrocyte Precursor Cell Differentiation. China Biotechnology, 2021, 41(9): 1-9.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103013 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I9/1

Table 1 qRT-PCR primers

Fig.1 Isolation and cultivation of oligodendrocyte precursor cells (a) Flowchart showing the isolation and cultivation of OPC in vitro (b) Immunofluorescence labeling of PDGFRα (red) and OLIG2 (green) in cultured cells

Fig.2 Effect of metformin on cell viability and protein expression (a) Western blot analysis of MAG, ALDH1L using indicated antibodies in Oli-neu cells after metformin (0, 100 μmol/L, 300 μmol/L, 900 μmol/L) treatment for 48 h (b) Western blot analysis of OLIG2, MBP, ALDH1L1, GFAP in OPC after metformin treatment for 48 h (c) CCK8 assay showed relative cell viability in OPC after metformin treatment

Fig.3 Metformin modulates OPC differentiation (a) Immunofluorescence labeling of PDGFRα (green) and OLIG2 (red) in cells after 100 μmol/L metformin treatment for 48 h (b) Quantification of the percentage of PDGFRα⁺OLIG2⁺ cells *** P< 0.001 (c),(d) Flow cytometry analysis of PDGFRα in living cells after 100 μmol/L metformin treatment for 48 h (e) Quantification of the percentage of PDGFRα⁺ cells *** P<0.001 (f) Immunostainings of MBP (green) in cells treated with metformin for 48 h (g) Quantification of MBP⁺ cells *** P<0.001 (h) The mRNA levels of Mag, Mbp, Olig2, Sox10 and Gfap in primary cells after 100 μmol/L metformin treatment n=4 each group, * P< 0.05, ** P<0.01 (i) Western blot analysis of protein levels (OLIG2, MBP, ALDH1L1, GFAP) in OPCs treated with metformin for 48 h

Fig.4 The mechanism of metformin modulating OPC differentiation (a) Immunoblotting analysis of RAS, p-MEK, MEK, p-ERK, ERK protein levels in Oli-neu cells after metformin treatment for 0, 5, 10 and 30 min (b) Immunoblotting analysis of the protein expression in OPC after metformin treatment for 5 min (c) Quantification of protein expression * P<0.05, *** P<0.001 (d) The model depicts the role and mechanism of metformin in oligodendrocyte precursor cell differentiation

Fig.5 Effect of metformin on oligodendrocytes Western blot analysis of p-FoxO3a, FoxO3a expression in cells after 100μmol/L metformin treatment for 0, 5, 10 and 30 min


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