Animal Model Screening for Hyperlipidemic ICR Mice

Abstract

This study aimed to establish a hyperlipidemia model in ICR mice using a homemade high-fat diet. It further investigated hyperlipidemia-related indicators in control and model mice at various feeding durations to determine the optimal time frame for successful model establishment. Sixteen male ICR mice were introduced at intervals of 3 weeks, starting from weeks 0, 3, 6, 9, and 12. The control group was fed a standard diet, while the model group received a homemade high-fat diet to induce hyperlipidemia. Blood lipid related indices were detected at 15 weeks. The liver, scapular fat, abdominal fat, and epididymal fat were harvested to calculate the organ index. The contents of T-CHO, TG, and TBA in the liver were measured. HE staining was used to observe pathological changes in liver tissue and white adipose tissue, while Oil Red O staining was used to observe lipid droplets in liver tissue. The mRNA and protein expression of SREBP-2, insig1, HMGCR, LXRα, ABCA1, and CYP7A1 in the liver were detected by RT-qPCR and Western Blot. In the model group, blood lipid levels significantly increased by the 9th week, aligning with pathological changes indicative of hyperlipidemia. The mRNA and protein expression levels of SREBP-2, Insig-1, HMGCR, LXRα, ABCA1, and CYP7A1 were markedly elevated at 9 weeks and remained relatively stable thereafter. This study provides a reliable reference for determining the optimal establishment time of hyperlipidemia models and for in vivo hyperlipidemia animal experiments.

Keywords: HMGCR; cholesterol reverse transport; cholesterol synthesis; hyperlipidemia; model screening.

Figure 1

Mouse body weight and food intake. (A) shows the body weight of mice in each week, (B) shows the average food intake of mice in each week. Data of eight mice in each group were expressed as mean ± SD, * p < 0.05, ** p < 0.01.).

Figure 2

Mouse liver organ index, scapular fat organ index, abdominal fat organ index, and epididymal fat organ index. (A) shows the mouse liver organ index. (B) shows the mouse scapular fat organ index. (C) shows the mouse abdominal fat organ index. (D) shows the mouse epididymal fat organ index. Data of eight mice in each group are expressed as mean ± SD, * p < 0.05, ** p < 0.01.

Figure 3

Serum levels of ALT, AST, GLU, CHO, TG, HDL, and LDL in each group of ICR mice. (A) shows the serum levels of CHO in mice. (B) shows the TG levels in serum of mice. (C) shows the HDL levels in mouse serum. (D) shows the LDL content in mouse serum. (E) shows the serum AST in mice. (F) shows the serum ALT in mice. (G) shows the serum GLU content in mice. Data from eight mice per group are expressed as mean ± SD. * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01, compared with the control group corresponding to the number of weeks, compared with the model group at week 9.

Figure 4

Total cholesterol (T-CHO), triglyceride (TG), and total bile acids (TBA) content in mouse liver. (A) shows the T-CHO content in mouse liver. (B) shows the TG content in mouse liver. (C) shows the TBA content in mouse liver. Data from 8 mice per group are expressed as mean ± SD. * p < 0.05, ** p < 0.01, compared with the model group of corresponding weeks.

Figure 5

HE staining results of mouse liver tissue (200×).

Figure 6

HE staining results of mouse epididymal fat (200×).

Figure 7

Results of Oil Red O staining of mouse liver tissue (400×).

Figure 8

The HMGCR content in mouse livers. Data from eight mice per group are expressed as mean ± SD. * p < 0.05, compared with the model group of corresponding weeks.

Figure 9

Liver LXRα immunofluorescence plots of mice in each group at 6, 9, and 12 weeks. (Right) shows the results of LXRα immunofluorescence intensity analysis in each group. ** p < 0.01, compared with the model group of corresponding weeks.

Figure 10

Immunofluorescence plots of mouse liver CYP7A1 in each group at 6, 9, and 12 weeks. (Right) shows the results of LXRα immunofluorescence intensity analysis in each group. ** p < 0.01, compared with the model group of corresponding weeks.

Figure 11

SREBP-2, Insig-1, HMGCR, LXR, ABCA1, and CYP7A1 gene expression in mouse livers. (A) is Insig-1 mRNA expression, (B) is SREBP2 mRNA expression, (C) is HMGCR mRNA expression, (D) is LXRα mRNA expression, (E) is ABCA1 mRNAexpression, and (F) is CYP7A1 mRNA expression. * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01, compared with the control group of the corresponding weeks and compared with the model group at week 9.

Figure 12

Insig1, SREBP-2, HMGCR, LXRα, ABCA1, and CYP7A1 protein expression in mouse livers. (A) is the Insig1, SREBP-2, and HMGCR protein wb plot; (B) is the LXRα, ABCA1, and CYP7A1 protein wb plot; (C) is the Insig1 grayscale analysis value; (D) is the SREBP-2grayscale analysis values; (E) is the HMGCR grayscale analysis values; (F) is the LXRα grayscale analysis values; (G) is the ABCA1 grayscale analysis values; (H) is the CYP7A1 grayscale analysis values. * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01, compared with the control group of the corresponding weeks and compared with the model group of week 9.

Figure 13

SREBP2-mediated negative feedback pathway and gp78-mediated ubiquitination pathway of HMGCR.

Figure 14

Cholesterol efflux and bile acid conversion process (by Figdraw).

Figure 15

Flowchart of the experimental methodology (by Figdraw).