Butein Alleviates Non-Alcoholic Steatohepatitis in Leptin-Deficient Mice by Modulating the PDE4/cAMP/p-CREB Pathway

Abstract

Purpose: Non-alcoholic steatohepatitis (NASH) is a prevalent liver disease characterized by steatosis, inflammation, and liver injury. Despite its increasing incidence, effective treatments are limited. Butein, a flavonoid with anti-cancer, anti-inflammatory, and antioxidant properties, has not been thoroughly studied for its potential therapeutic effects in NASH. This study aimed to evaluate the effects of butein in NASH using both in vivo and in vitro experimental models, with emphasis on elucidating the underlying molecular signaling mechanisms.

Methods: The leptin-deficient (ob/ob) mouse model of NASH, induced by the Gubra amylase NASH (GAN) diet, was employed to assess the therapeutic effects and mechanistic pathways of butein treatment. In vitro investigations utilized palmitic acid-induced HepG2 human hepatocellular carcinoma cells and LX-2 hepatic stellate cells to explore butein's impact on oxidative stress, inflammatory responses, and fibrotic processes.

Results: Butein treatment resulted in significant amelioration of glucolipid metabolism dysregulation, hepatic inflammation, and liver fibrosis in the mouse model, potentially mediated through modulation of the PDE4/cAMP/p-CREB signaling pathway. In in vitro experimental models, butein effectively attenuated lipid-induced oxidative stress in HepG2 cells and reduced inflammatory and fibrotic responses in LX-2 cells, demonstrating consistent protective effects across both experimental models.

Conclusion: These findings establish the protective effects of butein against NASH progression through PDE4/cAMP/p-CREB pathway modulation, supporting its potential as a therapeutic candidate for NASH treatment pending further clinical validation.

Keywords: fibrosis; inflammation; lipid metabolism; non-alcoholic fatty liver disease.

Graphical abstract

Figure 1

Butein mitigates GAN diet-induced obesity and insulin resistance without impacting food intake in GAN ob/ob mice. (A) Depiction of the external morphological characteristics of mice at 46 days. (B) Growth curve illustrating body weight changes over time. (C) Representation of caloric intake (Kcal/day). (D–E) The plasma glucose profile following a 2 g/kg glucose oral challenge in mice and the mean area under the curve (AUC) measured between 0 and 120 minutes post-glucose loading. (F–H) Fasting glucose concentration, fasting insulin concentration, homeostatic model assessment for insulin resistance (HOMA-IR) index. Data are presented as mean ± SEM (n=7 mice/group). Statistical significance is indicated by * for p < 0.05 and ** for p < 0.01 compared to the vehicle group. Comparison between groups is shown by p-values or asterisks.

Figure 2

Butein decreased hepatic lipid accumulation and oxidative stress in GAN ob/ob mice. (A) Representative images of the liver. (B) Liver weight at necropsy (g). (C) Liver total cholesterol (TC) (µmol/g tissue). (D) Liver triglycerides (TG) (µmol/g tissue). (E) Liver malondialdehyde (MDA) (nmol/g tissue). (F) Liver glutathione (GSH) (µmol/g tissue). The data are expressed as mean ± SEM for 7 mice per group. *p<0.05 and ***p<0.001 compared to the vehicle group.

Figure 3

Butein mitigates steatohepatitis, inflammation and fibrosis in GAN ob/ob mice. Histological liver sections stained with hematoxylin-eosin (H&E), Oil Red O, and Sirius Red across normal control, vehicle, and butein-treated mice. H&E staining (top row) displays cellular morphology; Oil Red O (middle row) visualizes lipid content; Sirius Red (bottom row) detects collagen. Scale bars = 100 µm.

Figure 4

Continued.

Figure 4

Serum biochemical parameters in GAN ob/ob mice. (A) Triglycerides (TG), (B) total cholesterol (TC), (C) high-density lipoprotein cholesterol (HDL-C), (D) low-density lipoprotein cholesterol (LDL-C), (E) lactic dehydrogenase (LDH), (F) free fatty acids (FFA) (G), alanine aminotransferase (ALT), (H) aspartate aminotransferase (AST), (I) alkaline phosphatase (ALP) levels at baseline 0 day, 21 days, and 46 days post-treatment with normal control, vehicle, and butein. Data represent mean ± SEM; *p<0.05 **p < 0.01 and ***p < 0.001 compared to the vehicle group. Comparison between groups is shown by p-values or asterisks.

Figure 5

Butein modulates PDE4/cAMP/p-CREB pathways in GAN ob/ob mice. (A) mRNA expression changes in phosphodiesterase 4D (PDE4D) and phosphodiesterase 4B (PDE4B). (B) Levels of cyclic adenosine monophosphate (cAMP) in liver tissues. (C) Western blot analysis of phosphorylated cyclic AMP response element binding protein (p-CREB) relative to total CREB and GAPDH as a loading control. (D) Densitometry analysis of p-CREB normalized to total CREB. (E) mRNA levels of pro-inflammatory cytokines TNF-α and IL-6, and anti-inflammatory cytokine IL-10. (F) mRNA expression of fibrosis-related genes transforming growth factor-β (TGF-β), tissue inhibitor of metalloproteinases-1 (TIMP-1), and collagen type 1 alpha 1 chain (COL1A1). Data represent mean ± SEM. Statistical significance is denoted as *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the vehicle group. Comparison between groups is shown by p-values or asterisks.

Figure 6

Cellular steatosis and hepatocyte injury markers after butein treatment in palmitic acid (PA)-induced HepG2 cells. (A) Representative images of Oil Red O-stained HepG2 cells under control conditions, with PA induction, and PA induction followed by butein treatment. Butein mitigates lipid accumulation as visualized by decreased Oil Red O staining. (B–D) Enzymatic activity of ALT, AST, and TG within hepatocytes, respectively, indicating reduced hepatocellular injury and steatosis upon butein treatment. (E–G) Measurements of TC, LDH, and SOD in HepG2 cells, where butein demonstrates protective effects against PA-induced oxidative stress. Data represent mean ± SEM. Statistical significance is denoted as **p < 0.01, ***p < 0.001 compared to the PA group.

Figure 7

Gene expression analysis in PA-treated LX-2 cells with butein administration. (A) mRNA expression levels of inflammatory cytokines cells (TNF-α, IL-1β, IL-6) show a dose-dependent decrease in expression with butein treatment. (B) Gene expression of fibrosis markers (TGF-β, COL1A1, α-SMA, TIMP-1, MMP-9) indicates that butein modulates the fibrogenic response in a dose-responsive manner, affirming its anti-inflammatory and antifibrotic properties. Data represent mean ± SEM. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001 compared to the PA group. Comparison between groups is shown by p-values or asterisks.