Metabolic Dysfunction-Associated Steatohepatitis （MASH）

ALMS1 spontaneous mutant mice progressively developed obesity and metabolic abnormalities with age, as shown by increased body weight, fat percentage, food intake, plasma cholesterol, HDL-C, LDL-C, and postprandial blood glucose, together with impaired glucose tolerance. These findings support this model for studies of obesity-associated glucose and lipid metabolic disorders.

(A) Body weight changes. Compared with B6 control mice, ALMS1 spontaneous mutant mice showed progressive body weight gain with age, indicating spontaneous development of obesity; (B) Fat percentage. ALMS1 spontaneous mutant mice showed markedly higher fat percentage than B6 control mice at 8, 12, and 16 weeks of age, suggesting increased adiposity; (C) Food intake. Food intake was generally higher in ALMS1 spontaneous mutant mice than in B6 control mice, suggesting that increased food consumption may contribute to the obese phenotype; (D) Plasma total cholesterol levels. Plasma Chol levels increased with age and were significantly higher in ALMS1 spontaneous mutant mice than in B6 controls; (E) Plasma HDL-C levels. HDL-C levels were elevated in ALMS1 spontaneous mutant mice, indicating altered lipoprotein metabolism; (F) Plasma LDL-C levels. LDL-C levels were increased in ALMS1 spontaneous mutant mice compared with B6 controls, suggesting marked dyslipidemia; (G) Plasma triglyceride levels. TG levels showed an age-dependent decreasing trend in ALMS1 spontaneous mutant mice, reflecting dynamic changes in lipid metabolism; (H) Blood glucose curve during the glucose tolerance test. ALMS1 spontaneous mutant mice showed delayed glucose clearance after glucose challenge, indicating impaired glucose tolerance; (I) Area under the curve from 0 to 120 min. AUC was significantly increased in ALMS1 spontaneous mutant mice, further confirming impaired glucose tolerance; (J) Postprandial blood glucose levels. ALMS1 spontaneous mutant mice showed significantly higher postprandial blood glucose at multiple ages, indicating persistent glucose metabolic dysfunction. Black circles indicate B6 male mice, and red triangles indicate B6-Alms1-del male spontaneous mutant mice. Data are presented as mean ± SEM. Statistical significance is indicated in the figure: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

 ALMS1 spontaneous mutant mice progressively developed MASH-like liver pathology with age, characterized by increased hepatic steatosis, hepatocellular ballooning, lobular inflammation, and collagen deposition. The elevated NAS and fibrosis scores indicate that this model spontaneously develops obesity-associated steatohepatitis and liver fibrosis.

Figure 2. Characterization of MASH-like liver pathology in ALMS1 spontaneous mutant mice. (A) Representative liver H&E staining. The upper row shows B6J control mice, and the lower row shows ALMS1 spontaneous mutant mice. Compared with B6J controls, ALMS1 mutant mice exhibited more prominent hepatic steatosis, hepatocellular ballooning, and lobular inflammation, indicating spontaneous development of steatohepatitis-like liver injury. Yellow arrows indicate hepatocellular ballooning, red arrows indicate inflammatory foci, and green arrows indicate steatotic areas; (B) Quantification of NAFLD Activity Score (NAS). NAS was increased in ALMS1 spontaneous mutant mice with disease progression, suggesting aggravated steatosis, ballooning, and inflammatory activity; (C) Representative liver Sirius Red staining. The upper row shows B6J control mice, and the lower row shows ALMS1 spontaneous mutant mice. Increased collagen deposition and fibrotic changes were observed in ALMS1 mutant mice. Black arrows indicate collagen-positive fibrotic areas; (D) Quantification of fibrosis score. Fibrosis score was increased in ALMS1 spontaneous mutant mice, indicating that this model develops liver fibrosis together with spontaneous obesity and metabolic dysfunction. Data are presented as mean ± SEM. Statistical significance is indicated in the figure: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

Efruxifermin and Tirzepatide reduced weight gain and food intake and improved glucose/lipid metabolic parameters in B6-Alms1-del mice, with Tirzepatide showing stronger effects on body weight and lipid profiles.

Figure 1. Effects of Efruxifermin and Tirzepatide on body weight, food intake, and glycemic/lipid parameters in B6-Alms1-del mice.

(A) Body weight changes (B) Food intake (C) Cumulative food intake curves (D) Endpoint fasting blood glucose levels (E) Endpoint serum triglyceride (TG) levels (F) Endpoint serum total cholesterol (Chol) levels,  (G) Endpoint serum HDL-C levels. (H) Endpoint serum LDL-C levels. n=6~8, Data presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 by one-way ANOVA.

Compared to the control group, both Efruxifermin and Tirzepatide reduced selected liver injury- and hepatic lipid-related endpoints. Tirzepatide showed a more pronounced decreasing trend in hepatic TG/Chol accumulation, while Efruxifermin also improved serum transaminases and hepatic lipid abnormalities. Gross liver images further showed improved liver appearance after treatment.

Figure 2. Effects of Efruxifermin and Tirzepatide on liver injury and hepatic lipid endpoints in B6-Alms1-del mice.

(A) Endpoint serum ALT levels (B) Endpoint serum AST levels (C) Quantitative analysis of hepatic TG levels (D) Quantitative analysis of hepatic Chol levels (E) Representative gross liver images from each group, showing changes in liver size, color, and steatosis-related appearance.  n=6~8, Data presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 by one-way ANOVA.

Tirzepatide and Efruxifermin markedly improved liver pathology in B6-Alms1-del mice, reducing steatosis, inflammation, ballooning, collagen deposition, NAS, and fibrosis scores.

Figure 3．Tirzepatide and Efruxifermin improve liver histopathology and fibrosis in B6-Alms1-del mice.

(A) Representative liver H&E staining images from each group. Compared with B6J controls, B6-Alms1-del mice treated with PBS showed marked hepatic steatosis, ballooning, and inflammatory cell infiltration, which were alleviated after Tirzepatide or Efruxifermin treatment. (B) NAFLD activity score, including steatosis, inflammation, and ballooning components, used to evaluate overall liver disease activity. (C) Representative liver Sirius Red staining images showing collagen deposition and fibrosis. Increased collagen accumulation was observed in B6-Alms1-del + PBS mice, while treatment reduced fibrosis-related staining signals. (D) Quantitative fibrosis score. Tirzepatide and Efruxifermin both reduced fibrosis scores in B6-Alms1-del mice, indicating attenuation of model-associated liver fibrosis. n=6~8, Data presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 by one-way ANOVA.

Long-term GAN diet induced progressive liver enlargement and steatosis-like changes in B6 mice, as shown by increased liver weight, elevated liver index, and pale enlarged liver morphology.

Figure 1. GAN diet induces liver enlargement and steatosis-like phenotypes in B6 mice.

(A) Body weight changes in B6 mice during GAN diet feeding. B) Quantitative analysis of liver weight at different induction time points. Compared with the control diet group (B6+CD), the GAN diet group (B6+GAN) showed markedly increased liver weight at 24, 36, and 48 weeks. (C) Quantitative analysis of liver index at different induction time points. GAN diet feeding significantly increased liver index, indicating increased hepatic burden. (D) Representative gross liver images from each group at different induction time points. Compared with B6+CD mice, B6+GAN mice showed enlarged and paler livers, suggesting pronounced hepatic steatosis-like changes and liver enlargement after long-term GAN diet induction.  n=5~10, Data presented as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 by 2-Way ANOVA.

GAN diet feeding induced persistent dyslipidemia, liver injury, and hepatic lipid accumulation in B6 mice, as shown by increased plasma T-CHO, HDL-C, LDL-C, ALT, AST, and hepatic T-CHO/TG levels, together with reduced plasma TG. These findings indicate that long-term GAN feeding robustly establishes MASH-associated metabolic and hepatic injury phenotypes.

Figure 2. GAN diet induces dyslipidemia, liver injury, and hepatic lipid accumulation in B6 mice.

(A) Plasma total cholesterol (T-CHO) levels. Compared with B6+CD mice, B6+GAN mice showed significantly elevated T-CHO levels at 24, 36, and 48 weeks; (B) Plasma triglyceride (TG) levels. Plasma TG levels were generally lower in B6+GAN mice than in B6+CD mice, with significant differences observed at 24 and 48 weeks; (C) Plasma high-density lipoprotein cholesterol (HDL-C) levels. B6+GAN mice showed significantly increased HDL-C levels at all time points, indicating altered circulating lipoprotein metabolism; (D) Plasma low-density lipoprotein cholesterol (LDL-C) levels. LDL-C levels were significantly higher in B6+GAN mice at 24, 36, and 48 weeks; (E) Serum ALT levels. ALT was significantly elevated in B6+GAN mice, suggesting GAN diet-induced hepatocellular injury; (F) Serum AST levels. AST levels were increased in B6+GAN mice at 24, 36, and 48 weeks, further indicating liver injury; (G) Hepatic total cholesterol (Liver T-CHO) content. B6+GAN mice showed markedly increased hepatic T-CHO levels, indicating hepatic cholesterol accumulation; (H) Hepatic triglyceride (Liver TG) content. Hepatic TG levels were significantly increased in B6+GAN mice at all time points, suggesting pronounced hepatic lipid deposition induced by GAN diet feeding. Black bars indicate the B6+CD group, and green bars indicate the B6+GAN group. Data are presented as mean ± SEM. Statistical significance is indicated in the figure: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

 Long-term GAN diet feeding induced progressive MASH-like liver injury in B6 mice, demonstrates hepatic steatosis, inflammation, ballooning, collagen deposition, and increased fibrosis-related parameters.

Figure 3. Long-term GAN diet feeding induces hepatic steatosis, inflammatory injury, and fibrosis in B6 mice.

(A) Representative liver H&E staining images from each group at different induction time points. Compared with control diet-fed mice, GAN diet-fed mice showed marked hepatic lipid vacuoles, inflammatory cell infiltration, and hepatocellular injury. Colored arrows indicate representative pathological lesions. (B) Quantitative histological scoring of liver lesions, including steatosis, inflammation, and ballooning-related scores. (C) Representative liver Sirius Red staining images from each group at different induction time points, showing collagen deposition and fibrosis development. Arrows indicate representative collagen-positive regions. (D) Quantitative analysis of liver fibrosis-related parameters. Long-term GAN diet feeding induced progressive liver pathological injury in B6 mice, characterized by hepatic steatosis, inflammatory infiltration, tissue architectural abnormalities, and increased collagen deposition supporting the stable development of MASH-associated steatohepatitis and fibrosis phenotypes in this model.  n=5~10. Data presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001 by T-test.

In the GAN diet-induced MASH model, different treatments showed distinct pharmacodynamic profiles. Tirzepatide and Semaglutide markedly reduced body weight and body weight change rate, accompanied by reduced cumulative food intake, whereas Efruxifermin showed a body weight-lowering trend without reducing food intake. Multiple treatments improved liver injury markers, plasma cholesterol, and hepatic lipid accumulation, with Tirzepatide and Semaglutide showing clear benefits in body weight control and hepatic lipid reduction.

Figure 1:Pharmacodynamic evaluation of metabolic therapies in the GAN diet-induced MASH model.

(A) Body weight changes during the dosing period. GAN vehicle-treated mice maintained high body weight, whereas Tirzepatide, Semaglutide, and Lanifibranor treatment resulted in marked body weight reduction; (B) Body weight change rate relative to Day 0. GAN vehicle-treated mice maintained a higher body weight change rate, while Tirzepatide and Semaglutide induced sustained reductions. Efruxifermin also showed a decreasing trend; (C) Daily food intake. Food intake varied among treatment groups. Tirzepatide- and Semaglutide-treated mice showed relatively lower food intake, whereas Efruxifermin-treated mice showed higher food intake, suggesting different mechanisms underlying body weight regulation; (D) Cumulative food intake. Tirzepatide and Semaglutide showed the lowest cumulative food intake, indicating that their weight-lowering effects were associated with food intake suppression; (E) Serum ALT levels. GAN vehicle-treated mice showed elevated ALT levels, while Tirzepatide, Semaglutide, and Efruxifermin treatment generally reduced ALT, suggesting improved hepatocellular injury; (F) Serum AST levels. Compared with GAN vehicle-treated mice, AST levels were lower in the Tirzepatide, Semaglutide, and Efruxifermin groups, further indicating reduced liver injury; (G) Plasma total cholesterol (T-CHO) levels. GAN vehicle-treated mice showed increased plasma T-CHO, while most treatments reduced T-CHO, indicating improved circulating cholesterol metabolism; (H) Plasma triglyceride (TG) levels. Plasma TG was lower in GAN vehicle-treated mice than in CD controls, and different treatments showed variable regulatory effects on plasma TG; (I) Hepatic total cholesterol (T-CHO) content. GAN vehicle-treated mice showed markedly increased hepatic T-CHO, whereas all treatments reduced hepatic cholesterol accumulation to varying degrees; (J) Hepatic triglyceride (TG) content. GAN vehicle-treated mice showed significantly increased hepatic TG, while Lanifibranor, Tirzepatide, Efruxifermin, Resmetirom, and Semaglutide reduced hepatic TG to different extents, indicating improved hepatic lipid accumulation.  Data are presented as mean ± SEM. Statistical significance is indicated in the figure: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

In the GAN diet-induced MASH model, vehicle-treated mice developed marked hepatic steatosis, hepatocellular ballooning, lobular inflammation, and fibrosis, accompanied by increased NAFLD Activity Score (NAS) and fibrosis score. Pharmacological treatments improved liver histological activity to varying degrees, while fibrosis improvement was relatively limited, supporting the utility of this model for evaluating MASH-related liver injury and anti-MASH efficacy.

Figure 2. Effects of metabolic therapies on liver histopathology in the GAN diet-induced MASH model.

(A) Representative liver H&E staining. G1 CD vehicle-treated mice showed largely normal liver architecture, whereas G2 GAN vehicle-treated mice exhibited marked hepatic steatosis, hepatocellular ballooning, and lobular inflammation. Treatment with Lanifibranor, Tirzepatide, Efruxifermin, Resmetirom, or Semaglutide improved steatosis- and inflammation-related histological lesions to different extents. Yellow arrows indicate hepatocellular ballooning, and red arrows indicate inflammatory foci; (B) Quantification of NAFLD Activity Score (NAS), including steatosis, ballooning, and lobular inflammation. GAN vehicle-treated mice showed a marked increase in NAS, while treatment groups showed variable reductions in NAS, indicating improved histological disease activity; (C) Representative liver Sirius Red staining. GAN vehicle-treated mice showed increased collagen deposition and fibrosis, while treated mice still displayed varying degrees of collagen deposition, suggesting limited fibrosis reversal after 12 weeks of intervention. Black and blue arrows indicate collagen-positive fibrotic areas; (D) Quantification of fibrosis score. GAN vehicle-treated mice showed significantly increased fibrosis score compared with CD controls, and fibrosis scores remained relatively high across treatment groups.  Data are presented as mean ± SEM. Statistical significance is indicated in the figure: ****, p < 0.0001.

Drug treatment improved metabolic abnormalities in the HFD + CCl4-induced MASH model, reducing body weight gain and food intake while improving glucose and lipid-related parameters compared with the control group.

Figure 1 .Effects of different treatments on body weight, food intake, liver function, and hepatic lipid parameters.

(A) Body weight dynamics and body weight change. The left panel shows body weight changes during the study period, and the right panel shows endpoint body weight change (B) Food intake and cumulative food intake. The left panel shows food intake changes during the study period, and the right panel shows cumulative food intake (C) Serum liver function markers. The left panel shows endpoint serum AST levels, and the right panel shows serum ALT levels, (D) Hepatic lipid content. The left panel shows endpoint hepatic total cholesterol (CHOL) levels, and the right panel shows hepatic triglyceride (TG) levels. Different treatments showed distinct effects on body weight control, food intake, serum transaminase levels, and hepatic lipid accumulation. Some treatments reduced body weight gain and cumulative food intake and improved liver-related endpoints, including AST, ALT, hepatic CHOL, and hepatic TG, suggesting beneficial effects on metabolic abnormalities and liver injury in study mice. Data presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001.

In the HFD + CCl4-induced MASH model, DIO vehicle mice developed severe steatosis, inflammation, ballooning, and fibrosis. Lanifibranor, Tirzepatide, Efruxifermin, and Resmetirom reduced NAFLD Activity Score (NAS) to varying degrees, while Lanifibranor and Resmetirom showed clearer anti-fibrotic effects.

Figure 2 Effects of different treatments on liver histopathological injury and fibrosis.

(A) Representative liver H&E staining images from each group, used to evaluate hepatic steatosis, inflammatory cell infiltration, and hepatocyte ballooning. Colored arrows indicate representative pathological lesions. (B) Quantitative analysis of liver NAS score, reflecting the overall severity of hepatic steatosis, inflammation, and ballooning. (C) Representative liver Sirius Red staining images from each group, showing collagen deposition and hepatic fibrosis. Arrows and circled areas indicate representative fibrotic or collagen-positive regions. (D) Quantitative fibrosis score, used to assess treatment effects on model-associated liver fibrosis progression. HFD + CCl4 group showed marked hepatic steatosis, inflammatory infiltration, hepatocyte ballooning, and increased collagen deposition, indicating the development of a MASH-like liver pathology. After treatment, NAS scores and fibrosis scores were reduced to varying degrees, suggesting that the interventions alleviated liver histopathological injury and fibrosis progression in model mice. Data presented as mean ± SEM, *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001.