Endocrinol Metab.  2024 Dec;39(6):908-920. 10.3803/EnM.2024.1984.

Irisin Attenuates Hepatic Stellate Cell Activation and Liver Fibrosis in Bile Duct Ligation Mice Model and Improves Mitochondrial Dysfunction

• Lai TL
• Park SY
• Nguyen G
• Pham PTM
• Kang SM
• Hong J
• Lee JH
• Im SS
• Choi DH
• Cho EH

• KMID: 2562719
• DOI: http://doi.org/10.3803/EnM.2024.1984

Abstract

Background
Liver fibrosis is a common outcome of chronic liver disease and is primarily driven by hepatic stellate cell (HSC) activation. Irisin, a myokine released during physical exercise, is beneficial for metabolic disorders and mitochondrial dysfunction. This study aimed to explore the effects of irisin on liver fibrosis in HSCs, a bile duct ligation (BDL) mouse model, and the associated mitochondrial dysfunction.
Methods
In vitro experiments utilized LX-2 cells, a human HSC line, stimulated with transforming growth factor-β1 (TGF-β1), a major regulator of HSC fibrosis, with or without irisin. Mitochondrial function was assessed using mitochondrial fission markers, transmission electron microscopy, mitochondrial membrane potential, and adenosine triphosphate (ATP) production. In vivo, liver fibrosis was induced in mice via BDL, followed by daily intraperitoneal injections of irisin (100 μg/kg/day) for 10 days.
Results
In vitro, irisin mitigated HSC activation and reduced reactive oxygen species associated with the TGF-β1/Smad signaling pathway. Irisin restored TGF-β1-induced increases in fission markers (Fis1, p-DRP1) and reversed the decreased expression of TFAM and SIRT3. Additionally, irisin restored mitochondrial membrane potential and ATP production lowered by TGF-β1 treatment. In vivo, irisin ameliorated the elevated liver-to-body weight ratio induced by BDL and alleviated liver fibrosis, as evidenced by Masson’s trichrome staining. Irisin also improved mitochondrial dysfunction induced by BDL surgery.
Conclusion
Irisin effectively attenuated HSC activation, ameliorated liver fibrosis in BDL mice, and improved associated mitochondrial dysfunction. These findings highlight the therapeutic potential of irisin for the treatment of liver fibrosis.

Keyword

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

Figure

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  Fig. 1. Irisin suppressed transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cells (HSCs) activation. LX-2 cells were treated with 5 ng/mL TGF-β1 and co-treated with 10 or 20 nM irisin for 24 hours. (A) Western blot analysis of α-smooth muscle actin (α-SMA) and collagen type 1 expression in LX-2 cells. (B) Western blot analysis of p-SMAD2, SMAD2, p-SMAD3, and SMAD3 expression in LX-2 cells. The quantitation of band intensities in Western blot images was calculated using ImageJ (National Institute of Health). The protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. All data are presented as mean±standard error of mean (n≥3). COL1A1, collagen type I alpha 1 chain. aP<0.01 vs. control; bP<0.05, cP<0.01 vs. TGF-β1.

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  Fig. 2. Irisin alleviated transforming growth factor-β1 (TGF-β1)-induced reactive oxygen species (ROS) and malondialdehyde (MDA) production in hepatic stellate cells (HSCs). LX-2 cells were treated with 5 ng/mL TGF-β1 and co-treated with 10 or 20 nM irisin for 24 hours. (A) Cellular ROS by dichlorodihydrofluorescein diacetate (DCFDA) assay. The graph showing fluorescence intensity of immunostaining was quantified using ImageJ (National Institute of Health). (B) MDA level. (C) Western blot analysis of the expression of superoxide dismutase 2 (SOD2) acetylation on lysine 68 (acetyl superoxide dismutase2-K68 [AcSOD2-K68]) in LX-2 cells. The protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. All data are presented as mean±standard error of mean (n ≥ 3). aP<0.05, bP<0.01 vs. control; cP<0.05, dP<0.01 vs. TGF-β1.

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  Fig. 3. Irisin attenuated transforming growth factor-β1 (TGF-β1)-induced mitochondrial fission in hepatic stellate cells (HSCs). LX-2 cells were treated with 5 ng/mL TGF-β1 and co-treated with 10 or 20 nM irisin for 24 hours. (A) Mitochondrial fission morphology was detected using transmission electron microscopy. Black arrows indicate mitochondria. (B) Western blot analysis of fission protein 1 (Fis1), phosphodynamin-related protein 1 (p-DRP1), phospho-mitochondrial fission factor (p-MFF), total DRP1, total MFF, optic atrophy 1 (OPA1), mitofusin 1 (MFN1), and mitofusin 2 (MFN2) expression in LX-2 cells. The quantitation of band intensities in Western blot images was calculated using ImageJ (National Institute of Health). The protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. All data are presented as mean±standard error of mean (n≥3). aP<0.05, bP<0.01 vs. control.

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  Fig. 4. Irisin alleviated transforming growth factor-β1 (TGF-β1)-induced mitochondrial dysfunction in hepatic stellate cells (HSCs). LX-2 cells were treated with 5 ng/mL TGF-β1 and co-treated with 10 or 20 nM irisin for 24 hours. (A) Western blot analysis of sirtuin 3 (SIRT3) and mitochondrial transcription factor A (TFAM) expression in LX-2 cells. (B) Mitochondrial membrane potential (ΔψM) was assessed using the JC-1 probe assay. The fluorescence intensity of immunostaining was quantified using ImageJ (National Institute of Health). (C) Adenosine triphosphate (ATP) production level in various groups of in vitro experiments. (D) Western blot analysis of succinate dehydrogenase subunit A (SDHA) expression in LX-2 cells. The quantitation of band intensities in Western blot images was calculated using ImageJ. The protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. All data are presented mean±standard error of mean (n≥3). (E) Seahorse analysis of extracellular acidification rate (ECAR), and glycolysis, glycolytic capacity and glycolytic reserve with 5 ng/mL TGF-β1 and co-treated with irisin (10 or 20 nM). Analysis of densitometry was performed and present data as the mean±standard error values of three independent experiments. OD, optical density; 2-DG, 2-deoxy-D-glucose. aP<0.05, bP<0.01 vs. control; cP<0.05, dP<0.01 vs. TGF-β1.

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  Fig. 5. Irisin improved bile duct ligation (BDL)-induced liver injury and liver fibrosis in mice. Mice underwent BDL to induce liver fibrosis. (A) Morphology of representative mice livers from the sham, BDL, and BDL+I groups. (B) The ratio of liver weight-to-body weight, serum alanine transaminase (ALT), and aspartate transaminase (AST) levels. (C) Sections of liver were stained with hematoxylin and eosin (H&E) staining; Masson’s trichrome. The slides are magnified 10× and 40×. (D) Western blot analysis of α-smooth muscle actin (α-SMA), collagen type 1, p-SMAD2, and SMAD2 expression in liver from the sham, BDL, and BDL+I groups. The protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. All data are presented as mean±standard error of mean (n≥3). COL1A1, collagen type I alpha 1 chain. aP<0.05, bP<0.01 vs. sham group; cP<0.05 vs. BDL group.

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  Fig. 6. Irisin recovered oxidative stress and mitochondrial dysfunction in bile duct ligation (BDL) mice. Irisin reversed the oxidative stress in the liver, as illustrated by the markers of oxidative stress. (A) The protein expression of acetyl superoxide dismutase 2-K68 (AcSOD2-K68) and (B) malondialdehyde (MDA) level. (C) Western blot analysis of fission protein 1 (Fis1), phospho-dynamin-related protein 1 (p-DRP1), phospho-mitochondrial fission factor (p-MFF), sirtuin 3 (SIRT3), mitochondrial transcription factor A (TFAM), and succinate dehydrogenase subunit A (SDHA) expression in liver. The quantitation of band intensities in Western blot images was calculated using ImageJ (National Institute of Health). The protein levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. All data are presented as mean±standard error of mean (n≥3). SOD2, superoxide dismutase 2; NS, no significance. aP<0.05, bP<0.01 vs. sham group; cP<0.05, dP<0.01 vs. BDL group.

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