TRPM8 deficiency attenuates liver fibrosis through S100A9-HNF4α signaling

Abstract

Background: Liver fibrosis represent a major global health care burden. Data emerging from recent advances suggest TRPM8, a member of the transient receptor potential (TRP) family of ion channels, plays an essential role in various chronic inflammatory diseases. However, its role in liver fibrosis remains unknown. Herein, we assessed the potential effect of TRPM8 in liver fibrosis.

Methods: The effect of TRPM8 was evaluated using specimens obtained from classic murine models of liver fibrosis, namely wild-type (WT) and TRPM8^-/- (KO) fibrotic mice after carbon tetrachloride (CCl₄) or bile duct ligation (BDL) treatment. The role of TRPM8 was systematically evaluated using specimens obtained from the aforementioned animal models after various in vivo and in vitro experiments.

Results: Clinicopathological analysis showed that TRPM8 expression was upregulated in tissue samples from cirrhosis patients and fibrotic mice. TRPM8 deficiency not only attenuated inflammation and fibrosis progression in mice but also helped to alleviate symptoms of cholangiopathies. Moreover, reduction in S100A9 and increase in HNF4α expressions were observed in liver of CCl₄- and BDL- treated TRPM8^-/- mice. A strong regulatory linkage between S100A9 and HNF4α was also noticed in L02 cells that underwent siRNA-mediated S100A9 knockdown and S100A9 overexpressing plasmid transfection. Lastly, the alleviative effect of a selective TRPM8 antagonist was confirmed in vivo.

Conclusions: These findings suggest TRPM8 deficiency may exert protective effects against inflammation, cholangiopathies, and fibrosis through S100A9-HNF4α signaling. M8-B might be a promising therapeutic candidate for liver fibrosis.

Keywords: ECM; HNF4α; Inflammation; Liver fibrosis; S100A9; TRPM8.

Fig. 1

TRPM8 is highly expressed in the livers of cirrhotic patients and fibrotic mice. A Representative images of immunohistochemical staining of TRPM8 in healthy control and cirrhotic human liver tissues, and its statistical summary. Scale bars, 100 μm. B Cirrhotic patients were divided into three different categories according to their etiologies: chronic hepatitis B (CHB), alcohol-related liver disease (ALD), and autoimmune hepatitis (AIH). C, D Immunoblotting analysis of hepatic TRPM8 expression in CCl₄- and BDL-induced liver fibrosis model (n = 3 per group). The results are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Liver fibrosis is attenuated in TRPM8^−/− mice after CCl₄ treatment. A Representative histology of H&E, Sirius Red, Masson’s trichrome, and IHC staining for α-SMA and COL1A1 in the liver of WT and TRPM8^−/− mice induced by CCl₄ (n = 5 per group). Positive staining areas were quantified by by Image J software. Scale bars, 100 μm. B Liver function was assessed by measuring the serum levels of ALT and AST in mice (n = 5 per group). C Immunoblotting analyses of α-SMA and COL1A1 expression in the mouse liver (n = 3 per group). D Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR (n = 5 per group). The results are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

TRPM8 deficiency alleviates BDL-induced liver fibrosis in mice. A Representative images of H&E, Sirius Red, Masson’s trichrome, and IHC staining for α-SMA and COL1A1 in the liver of WT and TRPM8^−/− mice operated with BDL (n = 5 per group). Positive staining areas were quantified by Image J software. Scale bars, 100 μm. B Liver function was assessed by measuring the serum levels of ALT and AST in mice (n = 5 per group). C Immunoblotting analyses of α-SMA and COL1A1 expression in the mouse liver (n = 3 per group). D Hepatic mRNA levels of fibrogenic genes were measured by qRT-PCR (n = 5 per group). The results are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

TRPM8 deficiency attenuates bile canalicular abnormalities, dysregulation of bile transporters, ductular reaction and liver inflammation in fibrotic liver. A Electron microscopy images show bile canaliculi (arrows) in liver sections obtained from CCl₄-treated WT and TRPM8^−/− mice. B, C Hepatic mRNAs of bile acid transport genes were measured by qRT-PCR in liver specimens obtained from CCl₄- or BDL-treated WT and TRPM8^−/− mice (n = 5 per group). D, F Histology of CK19 and F4/80 IHC staining in liver sections obtained from CCl₄- or BDL-treated WT and TRPM8^−/− mice. Positive staining areas were quantified by Image J software. Scale bars, 100 μm. E, G Hepatic mRNAs of IL-6, IL-1β, TNFα, and MCP-1 were measured by qRT-PCR in liver specimens obtained from CCl₄- or BDL-treated WT and TRPM8^−/− mice (n = 5 per group). The results are expressed as mean ± SD. *P < 0.05, **P < 0.01

Fig. 5

TRPM8 regulates the expression of S100A9 and HNF4α. A Volcano plot and heatmap presentation of significantly up- and downregulated genes in liver specimens obtained from CCl₄-treated TRPM8^−/− mice compared to WT (n = 3 per group). B Serum S100A9 expressions in liver cirrhosis patients and healthy donors. C, E Histology of S100A9 and HNF4α IHC staining in liver sections obtained from CCl₄- or BDL-treated WT and TRPM8^−/− mice (n = 5 per group). Scale bars, 50 μm. D, F Expressions of S100A9 and HNF4α were detected by immunoblotting using liver specimens obtained from CCl₄- or BDL-treated WT and TRPM8^−/− mice (n = 3 per group). G Immunoblotting assays of TRPM8, S100A9, and HNF4α in primary hepatocytes from WT and TRPM8^−/− mice. H Immunoblotting assays of TRPM8, S100A9, and HNF4α in L02 cells transfected with TRPM8 siRNA after 48 h. I, J Immunoblotting assays of S100A9 and HNF4α in L02 cells transfected with S100A9 siRNA or plasmid after 48 h. The results are expressed as mean ± SD. **P < 0.01

Fig. 6

TRPM8 inhibitor mitigates liver fibrosis in CCl₄-treated mice. A H&E, Sirius Red, Masson’s trichrome, and IHC staining for α-SMA and COL1A1 in liver sections of CCl₄-treated mice (n = 5 per group). Image J was used to quantify positively stained areas. Scale bars, 100 μm. B Serum levels of ALT and AST were measured in mice (n = 5 per group). C Expressions of α-SMA and COL1A1 were detected by immunoblotting (n = 3 per group). D Hepatic mRNAs of fibrogenic genes were measured by qRT-PCR assays in mice treated with M8-B or DMSO after CCl₄ induction (n = 5 per group). The results are expressed as mean ± SD. *P < 0.05, **P < 0.01