DIO-B6-M Mouse

Figure 1. Weight changes and blood glucose measurements in DIO-B6-M mice (n_ND=10, n_HFD=20). Male C57BL/6NCya mice, aged 4 weeks, were fed a 60% high-fat diet after a 1-week adaptation period. Body weight was periodically monitored, and random blood glucose levels were measured after 25 weeks of high-fat feeding. The results reveal that compared to the normal diet group (ND), the 60% high-fat diet group (HFD) displayed significant weight gain, with an average body weight of approximately 50g after 25 weeks of high-fat feeding. Additionally, random blood glucose levels were significantly higher in the HFD group compared to the ND group at 25 weeks (Data presented as Mean±SD; ***p<0.001).

Figure 2. Body composition in Lean and DIO mice. Body composition analysis was performed during diet treatments using a Conscious Small Animal Body Composition Analyzer to assess the fat mass and lean mass of each mouse (n=4).

Figure 3. Flow cytometry analysis of immune cells (T cells, B cells, NK cells, and macrophages) in peripheral blood of normal diet (ND) and 60% high-fat diet (HFD) fed mice after 25 weeks (n=10). The results indicate that DIO-B6-M mice exhibit a significant increase in NK cells and macrophages after 25 weeks of high-fat feeding, suggesting an enhanced inflammatory response (Mouse weight range for analysis: 50-55g; Data presented as Mean±SD; *p<0.05, **p<0.01).

Figure 4. Blood lipid and liver function indicators in normal diet (ND) and 60% high-fat diet (HFD) fed mice after 25 weeks of dietary induction* (n_ND=10, n_HFD=20). The results show that After 25 weeks, DIO-B6-M mice exhibited significant elevations in T-CHO, HDL-C, LDL-C, AST, and ALT levels compared to the ND group (Data presented as Mean±SD; **p<0.01, **p<0.001). *TG: Triglycerides; T-CHO: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase.

Figure 5. The efficacy of Semaglutide in reducing body weight and blood glucose in DIO-B6-M mice, as well as improving glucose tolerance. A: After 12 weeks of a 60% high-fat diet, DIO-B6-M mice received subcutaneous injections of 30 nmol/kg Semaglutide twice weekly (30-90 minutes before the dark cycle). Control groups (B6N+ND and B6N+HFD) received equivalent doses of a control solvent. Weight, food intake, and blood glucose levels were monitored regularly. B~E: Semaglutide treatment significantly reduced body weight, blood glucose, and food intake in DIO-B6-M mice (n=6). F~G: Semaglutide significantly improved glucose tolerance in DIO-B6-M mice, with the area under the IPGTT curve (AUC) comparable to the B6N+ND group and substantially lower than the B6N+HFD group (n=6). H: At the experimental endpoint, DIO-B6-M mice treated with Semaglutide had a leaner body type compared to the B6N+HFD group, with minimal differences compared to the B6N+ND group. *Semaglutide is an anti-obesity drug with a relatively long half-life ^[1].

Figure 6. Body Weight and Body Composition Analysis. Body weight was measured every two days. Semaglutide significantly reduced body weight and fat mass in mice. Data are presented as mean ± SEM, n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the normal diet control group, #P < 0.05 and ##P < 0.001 compared to the high-fat diet + CCl4 group, using one-way ANOVA.

Figure 7. Liver Weight and Serum Lipid Analysis. Liver weight was measured at the study endpoint, and the Semaglutide group showed a significant reduction in liver weight. After treatment, Semaglutide group significantly alleviated the hyperlipidemia phenotype induced by HFD. Data are presented as mean ± SEM, n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the normal diet control group, #P < 0.05 and ###P < 0.01 compared to the high-fat diet + CCl4 group, using one-way ANOVA.

Figure 8. H&E Staining and Histopathological Analysis. Liver tissues were collected at the study endpoint for histopathological analysis. Compared to the DIO model group, Semaglutide treatment reduced ballooning degeneration and steatosis. Based on composite scoring, the Semaglutide group showed a significant reduction in the NAFLD Activity Score (NAS) compared to the DIO and DIO + CCl4 groups. Yellow arrows indicate steatosis, blue arrows indicate hepatocellular ballooning, red arrows indicate lymphocyte infiltration in various liver regions, green arrows indicate congestion in central and portal veins, and brown arrows indicate occasional fibroblast proliferation. Data are presented as mean ± SEM, n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the normal diet control group; #P < 0.05 and ###P < 0.01 compared to the high-fat diet + CCl4 group, using one-way ANOVA.

Figure 9. Sirius Red Staining and Pathological Analysis. Sirius Red staining revealed that the CCl4 model group exhibited significantly increased fibrosis compared to the DIO model group. Semaglutide significantly reduced CCl4-induced liver fibrosis. Black arrows indicate increased collagen fiber proliferation around the central veins in liver tissue. Data are presented as mean ± SEM, n = 5. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the normal diet control group; #P < 0.05 and ###P < 0.01 compared to the high-fat diet + CCl4 group, using one-way ANOVA.