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TLR4 activation inhibits the proliferation and osteogenic differentiation of skeletal muscle stem cells by downregulating LGI1

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Abstract

Skeletal muscle stem cells (SMSCs) are vital to the growth, maintenance, and repair of the muscles; emerging evidence has indicated that Toll-like receptor 4 (TLR4) can potentially regulate muscle regeneration. In present study, in vitro and in vivo experiments were performed to explore the correlation of TLR4 with leucine-rich glioma-inactivated 1 (LGI1) as well as their effects on the proliferation and osteogenesis potential of SMSCs. In order to examine the regulatory mechanisms of TLR4 and LGI1 in SMSCs, the obtained cells were treated with lipopolysaccharide (LPS, used as an activator of TLR4) of different concentration at different time points as well as the siRNA against LGI1. Subsequently, a series of detection was undertaken in order to measure the proliferation and differentiation potential of SMSCs, which involved detection of the related factors, cell activity, and the sphere-forming capability. Following LPS treatment, the increased TLR4 expression and reduced LGI1 expression were observed. Consequently, we also discovered that Erk signaling pathway was inactivated and cell proliferation and osteogenesis capabilities declined, presented by the downregulation of related factors such as cyclin B1 and runt-related transcription factor 2. Moreover, the cell activity and sphere-formation performance of SMSCs were also declined. These results were also validated in rats with cecal ligation and perforation-induced rat models with sepsis. In conclusion, the present study reveals a regulatory mechanism in SMSCs whereby LGI1 expression is reduced by TLR4, thus impeding cell proliferation and osteogenesis, highlighting TLR4 as a potential therapeutic target against many diseases related to SMSCs.

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Data availability

Data are available upon reasonable request.

Abbreviations

TLR4:

Toll-like receptor 4

LGI1:

Leucine-rich glioma-inactivated 1

Erk:

Extracellular regulated protein kinases

CCNB1:

Cyclin B1

CCND1:

Cyclin D1

ALP:

Alkaline phosphatase

BGP:

Biliary glycoprotein

RUNX2:

Runt-related transcription factor 2

MuRF1:

Muscle RING-finger 1

MAFbx:

Muscle atrophy F-box

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

SMSCs:

Skeletal muscle stem cells

LPS:

Lipopolysaccharide

PBS:

Phosphate-buffered saline

CLP:

Cecal ligation and perforation

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

RT-qPCR:

Reverse transcription quantitative polymerase chain reaction

NC:

Negative control

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

OD:

Optical density

ELISA:

Enzyme-linked immunosorbent assay

DEPC:

Diethyl pyrocarbonate

PI:

Propidium iodide

C5a:

Complement 5a

IL-6:

Interleukin 6

HE:

Hematoxylin and eosin

ANOVA:

One-way analysis of variance

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Author information

Authors and Affiliations

  1. Department of Orthopedics, Renmin Hospital of Wuhan University, No. 99, Zhangzhidong Street, Wuchang District, Wuhan, 430060, Hubei Province, People’s Republic of China

    Haiying Tao & Hai Tao

  2. General Department, Zhongnan Hospital of Wuhan University, Wuhan, 430071, People’s Republic of China

    Xiaoyan Tang

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  1. Haiying Tao
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Contributions

Haiying Tao and Hai Tao designed the study. Xiaoyan Tang and Hai Tao collated the data, carried out data analyses, and produced the initial draft of the manuscript. Haiying Tao and Xiaoyan Tang contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Corresponding author

Correspondence to Hai Tao.

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Ethics approval

Animal use and experimental procedures were carried out with the approval of the Experimental Animal Ethics Committee of Renmin Hospital of Wuhan University (approval number: 202011-A-035). Animal operating procedures were in line with the US National Institutes of Health (NIH) laboratory animal care and usage guidelines.

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Not applicable.

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The authors declare no competing interests.

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Haiying Tao and Xiaoyan Tang are co-first authors.

Key points

• LPS-induced TLR4 activation inhibits the LGI1 expression in SMSCs.

• TLR4 activation reduces SMSC proliferation-related gene expression.

• TLR4 activation hinders the osteogenic differentiation of SMSCs.

• TLR4 activation inhibits the sphere formation ability of SMSCs.

• This study provides a potential therapeutic target against diseases related to SMSCs.

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Tao, H., Tang, X. & Tao, H. TLR4 activation inhibits the proliferation and osteogenic differentiation of skeletal muscle stem cells by downregulating LGI1. J Physiol Biochem 78, 667–678 (2022). https://doi.org/10.1007/s13105-022-00888-3

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  • DOI: https://doi.org/10.1007/s13105-022-00888-3

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