Nitroxoline mitigates hepatic steatosis by enhancing cholesterol efflux and promoting bile acid synthesis through LRH-1 signaling

Abstract

Background: Metabolic associated fatty liver disease (MAFLD) has emerged as the most common chronic liver disease worldwide. However, effective pharmacological treatments remain limited. Dysregulated lipid metabolism and impaired bile acid synthesis are recognized as key contributors to the pathogenesis of MAFLD. This study aimed to investigate the therapeutic potential and underlying mechanisms of nitroxoline (Nit), an antimicrobial agent identified through drug repurposing, in ameliorating hepatic steatosis.

Methods: Nit was administered to high-fat diet (HFD)-fed low-density lipoprotein receptor knockout (Ldlr^⁻/⁻) mice to assess hepatic steatosis, aortic atherosclerosis, serum lipid levels, and bile acid metabolism comprehensively. In vitro, Huh-7 cells were used to examine Nit-mediated regulation of lipid metabolism-related genes. RNA sequencing (RNA-seq) and pharmacologic inhibition studies were conducted to elucidate the underlying molecular mechanisms.

Results: Nit treatment significantly reduced liver weight without affecting body weight in HFD-fed Ldlr⁻/⁻ mice. Serum total cholesterol, low-density lipoprotein (LDL)-cholesterol, and triglyceride levels were markedly decreased. Mechanistically, Nit enhanced the expression of ATP-binding cassette subfamily G5 (ABCG5) and G8 (ABCG8) transporters, along with cholesterol 7α-hydroxylase (CYP7A1), thereby promoting cholesterol efflux into bile and bile acid synthesis. In Huh-7 cells, Nit induced ABCG5, ABCG8 and CYP7A1 expression in a dose-dependent manner. Furthermore, RNA-Seq analysis revealed liver receptor homolog-1 (LRH-1) as a potential transcriptional regulator related to Nit. Notably, pretreatment with the LRH-1 inhibitor, ML-180 abolished Nit-induced upregulation of ABCG5, ABCG8 and CYP7A1, suggesting that Nit may alleviate hepatic lipid accumulation primarily through LRH-1 activation.

Conclusions: This study identifies Nit as a promising pharmacological candidate for MAFLD by modulating cholesterol metabolism and bile acid synthesis through LRH-1-mediated activation. These findings not only advance the understanding of metabolic liver disease pathogenesis but also support the development of innovative and accessible therapeutic strategies by leveraging existing compounds to improve health outcomes.

Keywords: Bile acids biosynthesis; Fatty liver; Lipid metabolism; Nitroxoline.

Fig. 1

Nitroxoline reduces HFD-induced hepatic steatosis. Eight-week-old Ldlr^−/− mice were fed with a high-fat diet for 12 weeks and then treated with 20 mg/kg Nit or vehicle (DMSO) from the fourth to the 12th week. A Body weight, live weight, GPT and GOT levels of mouse serum (n = 3). B En face ORO staining results of aortic plaques in the vehicle group or nitroxoline group were shown (n = 3). C H&E and ORO staining of the Liver. Scale bar: 100 μm. D TCHO, TG, LDL-C, HDL-C levels of mouse serum. TCHO, TG and Bile acids in mouse (E) liver and (F) feces. Data was presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, **** P < 0.001, compared with the control group by Student’s t-test

Fig. 2

Nitroxoline regulates ABCG8, ABCG5 and CYP7A1 expression in Ldlr^−/− mice fed with a HFD. A The mRNA levels of ABCA1, ABCG1, ABCG5, ABCG8, SR-B1, NPC1L1, CPT1A and CYP7A1 of mouse liver tissues were assessed by qPCR analysis (n = 3). B The protein levels of ABCG5, ABCG8, CYP7A1 and GAPDH were determined by western blot analysis (n = 3). Data was presented as mean ± SD of three independent experiments. *P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001, compared with the vehicle group by Student’s t-test

Fig. 3

Nitroxoline enhances ABCG5, ABCG8 and CYP7A1 expression in vitro. Huh7 cells incubated with LPDS (5%) were treated with Nit (5 μM) and the solvent vehicle for 24 h. A The mRNA levels of ABCA1, ABCG1, ABCG5, ABCG8, SR-B1, NPC1L1, CPT1A and CYP7A1 were assessed by qPCR analysis (n = 3). Huh-7 cells incubated with LPDS (5%) were treated with various doses of Nit or solvent vehicle for 24 h. B The expression of ABCG5, ABCG8 and CYP7A1 at the mRNA levels was determined by qPCR. C The protein levels of ABCG5, ABCG8, CYP7A1 and GADPH were determined by western blot analysis (n = 3). Data was presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.005, compared with the vehicle group by Student’s t-test

Fig. 4

Nitroxoline preserved the protein expression of ABCG8 and CYP7A1. Huh-7 cells were incubated with Nit (5 μM) and the solvent vehicle and treated with for Act. D (5 μM) indicated period. A The expression of ABCG5, ABCG8 and CYP7A1 at the mRNA levels was determined by qPCR analysis. B Huh-7 cells were incubated with Nit (5 μM) and the solvent vehicle and treated with for CHX (5 μM) indicated period. Data were presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, compared with the vehicle group by Student’s t-test

Fig. 5

Nitroxoline modulated ABCG5, ABCG8 and CYP7A1 expression through LRH-1 signaling. A The heatmap plot shows the differential expression of RNA-seq analysis in Nit-treated and untreated Huh-7 cells. The values of three independent experiments were transformed into z scores for comparison. The color scale indicates high (red), average (white), and low (blue) values. B Huh-7 cells were transfected with 200 ng LRH-1 response element reporter plasmid and then, pretreated with/without LRH-1 inhibitor, ML-180 (5 μM), followed by Nit treatment. Promoter activities were detected using the Dual-Luciferase Reporter Assay System. C Huh 7 cells were incubated with LPDS (5%) were pre-treated with/without ML-180 (5 μM), then incubated with the solvent vehicle and/or Nit (5 μM) for 24 h. The mRNA levels of LRH-1, CYP7A1, ABCG5 and ABCG8 were assessed by qPCR analysis (n = 3). D The protein levels of ABCG5, ABCG8, CYP7A1 were determined by western blotting. GAPDH served as a loading control. E The mRNA and (F) protein level of LRH-1 of mouse liver tissues were assessed by qPCR analysis (n = 3). Data was presented as mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.005, compared with the vehicle group by Student’s t-test