Endocrinol Metab.  2020 Sep;35(3):647-655. 10.3803/EnM.2020.658.

Irisin Regulates the Functions of Hepatic Stellate Cells

• Dong HN
• Park SY
• Le CT
• Choi DH
• Cho EH

• KMID: 2508015
• DOI: http://doi.org/10.3803/EnM.2020.658

Abstract

Background
Hepatic stellate cells (HSCs) are known to play a fundamental role in the progression of liver fibrosis. Once HSCs are activated, they are involved in proliferation, migration, and contractility which are characteristics of liver fibrogenesis. Recent studies have shown that irisin, a myokine secreted during physical exercise, has a protective effect in various metabolic diseases, especially in renal fibrosis. However, whether irisin is involved in HSC activation and other processes associated with liver fibrosis has not yet been investigated. In this study, we reveal the role of irisin in HSC activation as well as in proliferation, migration, and contractile properties of HSCs in vitro.
Methods
LX-2 cells, immortalized human HSCs, were treated with transforming growth factor beta 1 (TGF-β1), a core regulator of HSC fibrosis, with or without irisin, and markers of the aforementioned processes were analyzed. Further, an inflammatory response was stimulated with TGF-β1 and lipopolysaccharide (LPS) in combination with irisin and the expression of cytokines was measured.
Results
Recombinant irisin significantly suppressed the expression of TGF-β1-stimulated fibrosis markers including alpha-smooth muscle actin and collagen type 1 alpha 1 and prevented the TGF-β1-induced proliferation, migration, and contractility of LX-2 cells. Additionally, irisin ameliorated the production of interleukin-6 (IL-6) and IL-1β induced by TGF-β1 and LPS treatments.
Conclusion
These findings suggested that irisin potently improved the progression of hepatic fibrosis by regulating HSC activation, proliferation, migration, contractility, and HSC-mediated production of inflammatory cytokine.

Keyword

Hepatic stellate cells; Irisin; Lipopolysaccharides; Liver cirrhosis; Transforming growth factor beta1

Figure

• 

  Fig. 1 Irisin abolishes hepatic stellate cell activation and migration. (A) LX-2 cells were stimulated with transforming growth factor beta 1 (TGF-β1; 6 ng/mL) or TGF-β1 plus irisin (100 nM) for 24 hours. Western blotting was conducted to measure the level of fibrogenesis proteins, alpha-smooth muscle actin (α-SMA) and collagen type 1 alpha 1 (COL1A1). Band intensities were measured using ImageJ software (National Institutes of Health) and are plotted to the right of the membrane images. Protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B) Wound healing migration assay was used to investigate the effect of irisin (100 nM) on the migration of LX-2 cells following 6 hours of co-treatment with TGF-β1 (6 ng/mL). aP<0.001; bP<0.01 vs. control; cP<0.05 vs. TGF-β1 only treatment (mean±standard error of the mean, n=3).

• 

  Fig. 2 Irisin reduces the contractility of activated hepatic stellate cells. LX-2 cells were treated with transforming growth factor beta 1 (TGF-β1; 6 ng/mL) or TGF-β1 plus irisin (100 nM) for 24 hours. (A) Cell contraction assay was used to test contraction capability, (B) Western blotting was used to analyze the ratio of the different forms of the contractile marker, phosphorylated myosin light chain 2 (p-MLC 2) and total myosin light chain 2 (t-MLC 2) in LX-2 lysates. Band intensities were measured using ImageJ software (National Institutes of Health) and are plotted to the right of the membrane images. Protein levels were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). aP<0.01 vs. control; bP<0.01 vs. TGF-β1 only treatment (mean±standard error of the mean, n=3); cP<0.05 vs. control.

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  Fig. 3 Irisin inhibits proliferation of activated hepatic stellate cell but is not involved in the apoptotic pathway. LX-2 cells were treated with transforming growth factor beta 1 (TGF-β1; 6 ng/mL) or TGF-β1 plus irisin (100 nM) for 24 hours. (A) Cell proliferation was measured using EZ-Cytox assay kit (DoGen). The optical density (OD) was measured at 450 nm. (B) Western blotting was used to analyze the apoptotic markers, and poly (ADP-ribose) polymerase (PARP) and cleaved-PARP (cl-PARP). Band intensities were measured using ImageJ software (National Institutes of Health) and are plotted to the right of the membrane images. Protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). aP<0.01 vs. control; bP<0.01 vs. TGF-β1 only treatment (mean±standard error of the mean [SEM], n=6); cP<0.001 vs. control (mean±SEM, n=7).

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  Fig. 4 Irisin attenuates transforming growth factor beta 1 (TGF-β1)- and lipopolysaccharide (LPS)-induced inflammatory cytokine expression in hepatic stellate cells. LX-2 cells were treated with (A) TGF-β1 (6 ng/mL) or (B) LPS (100 ng/mL) with recombinant of irisin (100 nM) for 24 hours. Western blotting was used to detect the expression of inflammatory cytokines, interleukin 6 (IL-6) and IL-1β in the cell lysates. Band intensities were measured using ImageJ software (National Institutes of Health) and are plotted to the right of the membrane images. Protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Enzyme-linked immunosorbent assay (ELISA) was used to measure secretion of IL-6 in the cell culture medium. aP<0.05 vs. control; bP<0.05 vs. TGF-β1 only treatment (mean± standard error of the mean [SEM], n=7 for IL-6 and n=5 for IL-1β); cP<0.001 vs. control; dP<0.05 vs. TGF-β1 only treatment (mean±SEM, n=3); eP<0.01 vs. control; fP<0.05 vs. LPS only treatment (mean±SEM, n=3 for IL-1β, n=4 for IL-6); gP<0.05 vs. LPS only treatment (mean±SEM, n=5).

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