NXT629 Ameliorates Cholesterol Gallstones in Mice Model by Improving Lipid Metabolism Disorder and Cholesterol Homeostasis Through Inhibiting the GPAM Pathway

Abstract

Background: NXT629, a PPAR-alpha antagonist, exerts widespread effects in many diseases; however, its function and relevant mechanism in cholesterol gallstones (CG) remain largely unknown.

Methods: Male C57BL/6 J mice were fed a regular diet or lithogenic diet (LD), followed by treatment with intraperitoneal injection of NXT629. H&E staining was performed to analyze hepatic pathological changes, and Oil red O staining was conducted to detect lipid accumulation. Concentrations of total cholesterol (TC), triglyceride (TG), phospholipids (PL), total bile acids (TBA), and cholesterol saturation index (CSI) in both bile and serum were analyzed using commercially available kits. The mRNA expressions of ABCG5/8, CYP7A1, CYP7B1, PPAR-α, and ABCB11 in mouse liver tissues were measured by qRT-PCR assay. Overexpression of glycerol-3-phosphate acyltransferase mitochondrial (GPAM) was constructed to investigate the molecular mechanism of NXT629 in CG.

Results: NXT629 could prevent the formation of cholesterol gallstones (CG) and improve lipid metabolic disorders in mice fed a lithogenic diet (LD). Treatment with NXT629 significantly reduced the levels of ABCG5, ABCG8, and ABCB11, while increasing the levels of CYP7A1 and CYP7B1 in the LD group. Additionally, NXT629 treatment downregulated GPAM expression in hepatic tissue from LD-fed mice. Overexpression of GPAM partially counteracted the beneficial effects of NXT629 on CG formation, lipid metabolic disorders, and lipid-related gene expressions.

Conclusion: NXT629 can inhibit CG formation, improve lipid metabolism disorders and cholesterol homeostasis by inhibiting GPAM expression, suggesting that NXT629 may serve as a potential therapeutic strategy for cholesterol stones prevention and treatment.

Keywords: Cholesterol gallstones; GPAM; Lipid metabolism disorder; NXT629; PPAR-α.

Fig. 1

NXT629 prevented lithogenic diet-induced cholesterol gallstone formation in mice model. a Appearance of gallbladder in mice from control, LD, and LD + NXT629 groups. b The formation rate of gallstones. c Liver weight. d Body weight gain. e The ratio of liver weight/body weight. f Representative images of H&E staining and Oil red O staining of hepatic tissues from different groups. * P < 0.05, **P < 0. 01, ***P < 0. 001

Fig. 2

NXT629 ameliorated lithogenic diet-induced lipid metabolic disorders in mice model. a–d The levels of TC, TG, PL and TBA in mice bile from different groups. e The value of CSI in mice bile was assessed based on the Carey table. f–i The levels of TC, TG, LDL-C, and HDL-C in mice serum. * P < 0.05, **P < 0. 01, ***P < 0. 001

Fig. 3

NXT629 altered levels of lipid-related genes in hepatic tissues of lithogenic diet-fed mice. a–f The mRNA levels of ABCG5, ABCG8, CYP7A1, CYP7B1, PPAR-α, and ABCB11 in hepatic tissues from different groups were detected by qRT-PCR. * P < 0.05, **P < 0. 01, ***P < 0. 001

Fig. 4

NXT629 prevented cholesterol gallstone formation by mediating GPAM pathway in mice model. a–c The mRNA and protein levels of GPAM in hepatic tissues from different groups were determined by qRT-PCR and western blot. d Appearance of gallbladder in mice from different groups. e The formation rate of gallstones. f Liver weight. g Body weight gain. h The value of liver weight/body weight. i Representative images of H&E staining and Oil red O staining of hepatic tissues. * P < 0.05, **P < 0. 01, ***P < 0. 001

Fig. 5

NXT629 weakened lithogenic diet-induced lipid metabolic disorders by repressing GPAM pathway. a–d The levels of TC, TG, PL and TBA in mice bile. e The value of CSI in mice bile was assessed based on the Carey table. f–i) The levels of TC, TG, LDL-C and HDL-C in mice serum. * P < 0.05, **P < 0. 01, ***P < 0. 001

Fig. 6

NXT629 mediated lipid-related gene expression by GPAM pathway. a–f Relative mRNA levels of ABCG5, ABCG8, CYP7A1, CYP7B1, PPAR-α, and ABCB11 in hepatic tissues from different groups. * P < 0.05, **P < 0. 01, ***P < 0. 001