The CYP4/20-HETE/GPR75 axis in the progression metabolic dysfunction-associated steatosis liver disease (MASLD) to chronic liver disease

Abstract

Introduction: Metabolic-dysfunction-associated steatosis liver disease (MASLD) is a progressive liver disease from simple steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Chronic liver diseases (CLDs) can lead to portal hypertension, which is a major cause of complications of cirrhosis. CLDs cause structural alterations across the liver through increased contents of extracellular matrix (ECM), driving dysfunction of liver sinusoidal endothelial cells (LSECs) alongside hepatic stellate cells (HSCs) and activated resident or infiltrating immune cells. Bioactive arachidonic metabolites have diverse roles in the progression of MASLD. Both secreted levels of 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acid (EET) are elevated in patients with liver cirrhosis.

Methods: CLD samples were evaluated for changes in free fatty acids (FFA), cholesterol, bilirubin, bile acid, reactive oxygen species (ROD), lipid peroxidation, myeloperoxidase activity and hydroxyproline levels to evaluate the degrees of liver damage and fibrosis. To address the role of the CYP4/20-HETE/GPR75 axis, we measured the amount and the synthesis of 20-HETE in patients with CLD, specifically during the progression of MASLD. Additionally, we evaluated gene expression and protein levels of GPR75, a high-affinity receptor for 20-HETE across CLD patient samples.

Results: We observed an increase in 20-HETE levels and synthesis during the progression of MASLD. Increased synthesis of 20-HETE correlated with the expression of CYP4A11 genes but not CYP4F2. These results were confirmed by increased P4504A11 protein levels and decreased P4504F2 protein levels during the development and progression of MASLD. The gene expression and protein levels of GPR75, the major receptor for 20-HETE, increased in the progression of MASLD. Interestingly, the CYP4A11 and GPR75 mRNA levels increased in steatohepatitis but dramatically dropped in cirrhosis and then increased in patients with HCC. Also, protein levels of P4504A11 and GPR75 mirrored their mRNA levels.

Discussion: These results indicate that the CYP4A11 and subsequent GPR75 genes are coordinately regulated in the progression of MASLD and may have multiple roles, including 20-HETE activation of peroxisome proliferator-activated receptor α (PPARα) in steatosis and GPR75 in CLD through either increased cell proliferation or vasoconstriction in portal hypertension during cirrhosis. The abrupt reduction in CYP4A11 and GPR75 in patients with cirrhosis may also be due to increased 20-HETE, serving as a feedback mechanism via GPR75, leading to reduced CYP4A11 and GPR75 gene expression. This work illustrates key correlations associated with the CYP4/20-HETE/GPR75 axis and the progression of liver disease in humans.

Keywords: 20-HETE; CYP4A11; GPR75; MASLD; chronic liver disease.

FIGURE 1

Graphic view on the role of CYP4 arachidonic acid ω-hydroxylase and the 20-HETE receptor GPR75 in chronic liver disease (CLD). The liver is in different stages of CLD, and various factors are shown, from the accumulation of lipid droplets in steatosis to fibrosis, cirrhosis, and hepatocellular carcinoma, with the significant complication of portal hypertension seen in the progression of MASLD. Metabolism of arachidonic acid (AA) by CYP4A/4F ω-hydroxylase to 20-HETE, which activates the GPR75 receptor, eliciting different unknown responses by liver Kupffer cells, hepatocytes, endothelial cells, bile canaliculi cells, and activation of stellate cells in hepatic fibrosis. Non-selective β-blockers (NSBB) inhibit vasoconstriction of portal vein vasoconstriction and portal hypertension.

FIGURE 2

Biochemical markers in twenty-eight liver samples at different stages of MASLD progression (A) Free fatty acid (FFA) levels at various stages of liver diseases. (B) Hepatic Cholesterol levels, (C) The concentration of liver bilirubin, (D) Total bile acid amount at different stages of MASLD, (E) Reactive oxygen species (ROS) in the liver, (F) The level of lipid peroxidation product, (G) The activity of myeloperoxidase in MASLD progression, (H) The degree of liver fibrosis determined by hydroxyproline levels. n = 28 per group, 140 total patient samples. Statistically significant changes relative to control normal livers are indicated by *p < 0.05.

FIGURE 3

20-HETE levels and its synthesis rates in MASLD (A) The level of 20-HETE in hepatic tissues at different stages of MASLD, (B) the synthesis rates of 20-HETE in liver microsomes at different stages of MASLD n = 28 per group, 140 total patient samples. Statistical significance compared to normal control liver *p < 0.05.

FIGURE 4

The level of CYP4A11 mRNA and protein in MASLD. (A) Changes in CYP4A11 mRNA as mRNA molecules per nanogram cDNA, (B) The level of P4504A11 protein, (C) Western immunoblot of P4504A11 protein at different stages of MASLD, (D) Total protein stain of proteins separated in a 4% stacked polyacrylamide gel at different stages of MASLD n = 28 per group, 14 representative immunoblot samples per group. Statistical significance compared to control liver *p < 0.05.

FIGURE 5

The amount of CYP4A22 mRNA and protein in the progression of MASLD (A) CYP4A22 mRNA levels as mRNA molecules per nanogram cDNA, (B) P4504A22 protein level at different stages of MASLD, (C) Western immunoblot of P4504A22 protein in different stages of MASLD n = 28 per group, 14 representative immunoblot samples per group. Statistical significance compared to control liver *p < 0.05.

FIGURE 6

The amount of CYP4F2 mRNA and protein at different stages of MASLD (A) CYP4F2 mRNA expressed as mRNA molecules per nanogram cDNA, (B) Amount of P4504F2 protein at different stages of MASLD, (C) Immunoblot analysis of P4504F2 protein at different stages of MASLD n = 28 per group, 14 representative immunoblot samples per group. Statistical significance compared to control liver *p < 0.05.

FIGURE 7

The Regulation of GPR75 mRNA and protein in MASLD. (A) GRP75 mRNA expressed as mRNA molecules per nanogram cDNA, (B) The Protein levels of GPR75, (C) Western immunoblot analysis of GPR75 protein at different stages of MASLD, (D) Level of β-actin protein at different stages of MASLD n = 28 per group, 14 representative immunoblot samples per group. Statistical significance compared to normal liver *p < 0.05.