Endocrinol Metab.  2023 Aug;38(4):395-405. 10.3803/EnM.2023.1661.

Phloretin Ameliorates Succinate-Induced Liver Fibrosis by Regulating Hepatic Stellate Cells

• Le CT
• Nguyen G
• Park SY
• Dong HN
• Cho YK
• Lee JH
• Im SS
• Choi DH
• Cho EH

• KMID: 2545272
• DOI: http://doi.org/10.3803/EnM.2023.1661

Abstract

Background
Hepatic stellate cells (HSCs) are the major cells which play a pivotal role in liver fibrosis. During injury, extracellular stimulators can induce HSCs transdifferentiated into active form. Phloretin showed its ability to protect the liver from injury, so in this research we would like to investigate the effect of phloretin on succinate-induced HSCs activation in vitro and liver fibrosis in vivo study.
Methods
In in vitro, succinate was used to induce HSCs activation, and then the effect of phloretin on activated HSCs was examined. In in vivo, succinate was used to generated liver fibrosis in mouse and phloretin co-treated to check its protection on the liver.
Results
Phloretin can reduce the increase of fibrogenic markers and inhibits the proliferation, migration, and contraction caused by succinate in in vitro experiments. Moreover, an upregulation of proteins associated with aerobic glycolysis occurred during the activation of HSCs, which was attenuated by phloretin treatment. In in vivo experiments, intraperitoneal injection of phloretin decreased expression of fibrotic and glycolytic markers in the livers of mice with sodium succinate diet-induced liver fibrosis. These results suggest that aerobic glycolysis plays critical role in activation of HSCs and succinate can induce liver fibrosis in mice, whereas phloretin has therapeutic potential for treating hepatic fibrosis.
Conclusion
Intraperitoneal injection of phloretin attenuated succinate-induced hepatic fibrosis and alleviates the succinate-induced HSCs activation.

Keyword

Hepatic stellate cells; Phloretin; Succinate; Liver cirrhosis; Aerobic glycolysis

Figure

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  Fig. 1. Phloretin reduced the upregulation in fibrogenic markers expression, inhibited cell proliferation and increased apoptosis of hepatic stellate cells (HSCs) induced by succinate in HSCs. LX-2 cells were treated with succinate (1,600 μM) and phloretin (50 or 100 μM) for 24 hours. (A) Real-time polymerase chain reaction to check mRNA expression of α-smooth muscle actin (α-SMA), collagen type I, transforming growth factor β1 (TGFβ1). (B) Western blot analysis of α-SMA, collagen type I, TGFβ1 were detected using specific antibodies. (C) Measure cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assessment and (D) Western blot analysis of cleaved poly-adenosine diphosphate-ribose polymerase (PARP), and PARP. Analysis of densitometry was performed and present data as the mean±standard error values of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Statistical significance: aP<0.05, bP<0.001 vs. the control group; cP<0.05, dP<0.01 vs. succinate.

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  Fig. 2. Phloretin inhibited succinate-induced migration of hepatic stellate cells (HSCs). LX-2 cells were exposure with succinate (1,600 μM) for 16 hours and phloretin (100 μM) before measure distance of migration by microscopy with 4× of magnification. (A) LX-2 cells were treated with succinate (1,600 μM) and phloretin (100 μM) for 16 hours. (B) LX-2 cells were treated with succinate (1,600 μM) and phloretin (100 μM) for 24 hours and measure collagen gel area. (C) Western blot analysis of tissue inhibitor of metalloproteases 1 (TIMP-1), phosphorylation myosin light chain 2 (p-MLC2), and MLC2 were detected using specific antibodies. Analysis of densitometry was performed and present data as the mean mean±standard error values of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Statistical significance: aP<0.05, bP<0.01, and cP<0.001 vs. the control group; dP<0.05, eP<0.01, and fP<0.001 vs. succinate.

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  Fig. 3. Phloretin reduced succinate-induced aerobic glycolysis in activated hepatic stellate cells (HSCs). LX-2 cells were treated with succinate (1,600 μM) and phloretin (50 or 100 μM) for 24 hours. (A) Real-time polymerase chain reaction to check mRNA expression of glucose transporter 1 (GLUT-1) and lactate dehydrogenase (LDH). (B) Western blot analysis of GLUT-1, hexokinase II (HK II), and lactate dehydrogenase A (LDHA) were detected using specific antibodies. (C) Western blot analysis of phosphorylated adenosine monophosphate protein kinase α (p-AMPKα) and AMPKα were detected using specific antibodies. (D) Seahorse analysis of extracellular acidification rate (ECAR), and glycolysis, glycolytic capacity and glycolytic reserve with succinate and phloretin (50 or 100 μM). Analysis of densitometry was performed and present data as the mean±standard error values of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OD, optical density; 2-DG, 2-deoxy-D-glucose. Statistical significance: aP<0.05, bP<0.01, and cP<0.001 vs. the control group; dP<0.05, eP<0.01, and fP<0.001 vs. succinate.

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  Fig. 4. Phloretin administration improved liver fibrosis induced by a sodium succinate diet in mice. (A) Serum alanine transaminase (ALT) levels and hepatic triglyceride levels. (B) Hematoxylin and eosin (H&E) stain and Masson’s trichrome stain show the effect of phloretin intraperitoneal (10 mg/kg every other day) on sodium succinate 2% containing diet-induced liver fibrosis in mice. H&E staining 40×, Masson’s Trichrome 20×. (C) Liver from control group, sodium succinate 2% containing diet-feeding mice, and intraperitoneal phloretin group (10 mg/kg every other day) were used to analyze expression of α-smooth muscle actin (α-SMA) by Western blot. Analysis of densitometry was performed and present data as the mean±standard error values of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Statistical significance: aP<0.05, bP<0.01, and cP<0.001 vs. the control group; dP<0.05, eP<0.01, and fP<0.001 vs. the succinate group.

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  Fig. 5. Administration of phloretin decreased glycolytic markers in the livers of mice with sodium succinate diet-induced liver fibrosis. (A) Hepatic lactate levels and serum lactate levels. (B) Liver from control group, sodium succinate 2% containing diet-feeding mice, and intraperitoneal phloretin group (10 mg/kg every other day) were used to analyze expression of glucose transporter 1 (GLUT-1), lactate dehydrogenase A (LDHA) by Western blot. Analysis of densitometry was performed and present data as the mean±standard error values of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Statistical significance: aP<0.05, bP<0.01 vs. the control group; cP<0.05, dP<0.01 vs. the succinate group.

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