A total of eighteen male SPF ICR mice aged 5 - 6 weeks were used in this study. The sorghum grains were processed and milled into flour prior to the experiment. The experimental diets were formulated according to the AIN-93G guidelines (Table 1).

Table 1. Composition of experimental diets (g/kg).

Assay kits for total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Reagents and antibodies, including RIPA lysis buffer, BCA protein assay kit, ECL substrate, and specific primary (IL-6, FASN, GAPDH) and secondary (Goat Anti-Rabbit IgG) antibodies, were obtained from Beyotime Biotechnology (Shanghai, China). The instruments used included an electronic balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.), an Alpha chemiluminescence gel imaging system (Purseen Biotechnology (Shanghai) Co., Ltd.), an optical microscope and RM2125 rotary microtome (Leica, Germany), and a multifunctional microplate reader (Thermo Scientific, USA).

2.2.1. Experimental Grouping and Animal Feeding

All the experimental animals were housed in SPF animal facility maintained at (22 ± 2)˚C with a 12 h light/dark cycle, with ad libitum access to food and water. Following a 1-week acclimatization period, the mice were randomly divided into two groups using a random number table: the normal control (NC) group (n = 6), fed a normal diet, and the High-fat diet (HFD) group (n = 12), fed a High-fat diet. After successful model establishment (defined as the HFD group exhibiting a mean body weight ≥ 20% greater than the NC group), the HFD-fed mice were randomly reassigned to two groups (n = 6 per group): the RGS group, which received the RGS-based diet, and the HFD group, which continued on the high-fat diet. The intervention period lasted 7 weeks. All animal experiments received approval from the Experimental Animal Ethics Committee of Guilin Medical College (approval number: GLMC202005013).

2.2.2. Sample Collection and Preparation

At the end of the experiment, mice were fasted for 12 - 16 h. Body weight was measured, and whole blood (1 - 2 mL) was collected via orbital venous plexus puncture and centrifuged to separate serum. The supernatant was stored at −80˚C until analysis. Following euthanasia, the liver was rapidly excised and weighed. Approximately 0.5 g of liver tissue was fixed in 10% neutral formalin for histopathological analysis. The remaining liver tissue, bilateral abdominal adipose tissue, and epididymal adipose tissue were snap-frozen and stored at −80˚C for subsequent biochemical and molecular analysis.

2.2.3. Histopathological Analysis of Liver Tissue

Liver tissue samples were fixed in 10% neutral formalin, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections were cut at 4 μm thickness, stained with hematoxylin and eosin (HE), and mounted with coverslips. The morphological structure of hepatocytes was examined under a light microscope.

2.2.4. Determination of Liver Index and Fat Index

The liver was gently blotted dry with filter paper and immediately weighed. The liver index was calculated according to Equation (1):

Abdominal and epididymal adipose tissues were carefully dissected, with adhering blood vessels and non-adipose tissues removed. The tissues were then blotted dry with filter paper and weighed. The fat index was calculated using Equation (2):

2.2.5. Determination of Serum and Hepatic Biochemical Indices

The levels of TC, TG, LDL-C, and HDL-C in mouse serum and liver tissue were measured using commercially available assay kits. All procedures were performed strictly according to the manufacturer’s instructions.

2.2.6. Western Blot Analysis of IL-6 and FASN in Liver Tissue

Liver tissues were homogenized on ice in RIPA lysis buffer supplemented with protease inhibitors. The homogenate was centrifuged at 3000 × g for 17 min at 4˚C. The supernatant was collected for subsequent analysis. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane using the wet transfer method. After transfer, the membrane was blocked with 5% skimmed milk for 2 h at room temperature. Following three washes with TBST, the membranes were incubated overnight at 4˚C with primary antibodies against FASN (1:5000) and IL-6 (1:7500). After washing with TBST, the membranes were incubated with the secondary antibody (1:10,000) for 1 h at room temperature. GAPDH was used as the internal loading control. Protein bands were visualized using an ECL chemiluminescent substrate, and grayscale analysis was performed to quantify the relative expression levels of the target proteins.

Data were processed and analyzed using SPSS version 25.0 (IBM, USA). The data met the assumptions of normality and homogeneity of variance. Results are expressed as mean ± SD. Comparisons among multiple groups were performed using one-way ANOVA, followed by pairwise comparisons using the least significant difference (LSD) test. A P-value < 0.05 was considered statistically significant. Graphs were prepared using GraphPad Prism version 9.5.

The changes in body weight throughout the experiment are shown in Figure 1Figure 1. Food intake was not recorded during the experimental period. During the 9 weeks modeling period, the body weight of mice in the NC group showed a gradual physiological increase and subsequently stabilized, whereas the body weight of mice in the HFD group increased progressively. By the end of the modeling period, the mean body weight of the HFD group was 8.70 g higher than that of the NC group, confirming the successful establishment of the obesity model. Before the dietary intervention, there was no statistically significant difference in body weight between the HFD (37.93 g) and RGS (38.38 g) groups (P > 0.05). Therefore, the grouping did not affect the results of subsequent experiments. Following the 7-week dietary intervention, the body weight of mice in the RGS group was significantly lower than that of the HFD group, with a reduction of 2.86 g (P < 0.01). The results indicate that RGS significantly suppressed high-fat diet-induced body weight gain in mice, thereby effectively controlling body weight and preventing further obesity progression.

Figure 1. Body weight of mice throughout the experimental period. HFD: High-fat diet group; RGS: Redglutinous sorghum group; NC: Normal control group. Compared with the High-fat diet group, *P< 0.05; **P < 0.01.

The effects of RGS on liver weight, liver index, fat weight, and fat index are shown in Figure 2. Compared with the NC group, both liver weight and liver index were significantly increased in the HFD group (P< 0.05), with liver weight increasing by 0.40 g (Figure 2(A) and Figure 2(B)). Following RGS intervention, liver weight and liver index were significantly reduced compared to the HFD group (P < 0.05), with liver weight decreasing by 0.18 g. These results suggest that long-term high-energy intake impairs lipid metabolism, promotes hepatic lipid deposition, aggravates metabolic burden, and induces liver damage, all of which can be effectively alleviated by RGS intervention.

The fat weight and fat index of the mice are shown in Figure 2(C) and Figure 2(D). Both fat weight and fat index were significantly higher in the HFD group than in the NC group (P < 0.05), with fat weight increasing by 0.97 g. Compared with the HFD group, fat weight and fat index were significantly reduced in the RGS group (P < 0.05), with fat weight decreasing by 0.46 g. These results indicate that a high-fat diet can promote fat accumulation in mice, leading to an increase in body weight and body fat percentage, whereas RGS intervention can effectively inhibit fat accumulation and reduce body fat levels.

Figure 2. Effects of RGS on liver weight, liver index, fat weight, and fat index in mice. (A) Liver weight; (B) Liver index; (C) Fat weight; (D) Fat index. HFD: High-fat diet group; RGS: Red glutinous sorghum group; NC: Normal control group. Different letters indicate significant differences (P < 0.05).

The results of liver histopathological examination are shown in Figure 3Figure 3. In the NC group, hepatocytes were tightly and regularly arranged with intact morphology, and no obvious lipid vacuoles or inflammatory infiltration were observed. In the HFD group, hepatocytes were disorganized, some cells exhibited irregular morphology and prominent lipid vacuoles were clearly visible. In contrast, hepatocytes in the RGS group were orderly arranged with relatively tight intercellular junctions and essentially intact morphology; no obvious inflammatory response was observed, and only a few lipid vacuoles were present. These results indicate that RGS intervention can effectively alleviate high-fat diet-induced hepatic lipid accumulation and structural damage.

Figure 3. Liver HE staining sections of mice (400× magnification). HFD: High-fat diet group; RGS: Red glutinous sorghum group; NC: Normal control group. Arrows indicate lipid vacuoles.

The serum and hepatic biochemical parameters are shown in Figure 4Figure 4. Compared with the NC group, the serum and hepatic levels of TC, TG, and LDL-C in the HFD group were significantly increased (P < 0.05), while the HDL-C level was significantly decreased (P < 0.05), indicating that long-term high-fat diet feeding induces dyslipidemia and hepatic lipid deposition, ultimately leading to lipid metabolism disorders. Compared with the HFD group, the serum and hepatic levels of TC, TG, and LDL-C in the RGS group were significantly decreased (P < 0.05), while HDL-C level was significantly increased (P < 0.05). These results demonstrate that RGS effectively suppresses high-fat diet-induced elevation of lipid levels and ameliorates abnormal lipid metabolism.

Figure 4. Effects of RGS on serum and hepatic biochemical parameters in mice. (A-D) Serum levels of TC, TG, HDL-C, and LDL-C; (E-H) Hepatic levels of TC, TG, HDL-C, and LDL-C. HFD: High-fat diet group; RGS: Red glutinous sorghumgroup; NC: Normal control group. Different letters indicate significant differences (P < 0.05).

The protein expression levels of IL-6 and FASN in liver tissue are shown in Figure 5Figure 5. Compared with the NC group, the protein expression levels of both IL-6 and FASN were significantly upregulated in the HFD group (P < 0.05). Following RGS intervention, the expression of both IL-6 and FASN proteins was significantly reduced compared with the HFD group (P < 0.05). These results suggest that RGS

Figure 5. Effects of RGS on IL-6 and FASN protein expression in liver tissue. A: Representative protein bands of IL-6 and FASN; B: Relative expression level of IL-6 protein; C: Relative expression level of FASN protein. HFD: High-fat diet group; RGS: Red glutinous sorghum group; NC: Normal control group. Different letters indicate significant differences (P < 0.05).

may exert its lipid-lowering effects partly through the suppression of hepatic inflammatory signaling and de novo lipogenesis.