3.1 Changes in food and water intake, body mass and liver wet weight levels between each group
3.1.1 Liver appearance in mice from each group
Grossly, the livers from mice in the Con group were dark red and soft. The livers from mice in the HFHC group showed different degrees of yellow at different time points and looked full and blunt; the sections were greasy, and the texture became tough. At 30 weeks, some livers from HFHC mice had yellowish white focal fat deposition in their livers (Fig. 1A).
3.1.2 Changes in average food intake, water intake and liver wet weight
No mice died during the experiment. The body mass of the mice in each group increased gradually over time (Fig. 1B). At 20 weeks, the body mass and liver wet weight in the HFHC group were significantly increased (P<0.01). Compared with those of the Con group mice at 30w, the body mass and liver wet weight levels of the HFHC group mice at 30w were significantly higher (P<0.01). Compared with those the HFHC group mice at 20w, the body mass and liver wet weight levels of the HFHC group mice at 30w were significantly higher (P<0.05, P<0.05) (Fig. 1C). The average food and water intake levels gradually increased over time, with no significant differences observed between the HFHC and Con groups at each time point (P>0.05) (Fig. 1D-F).
3.2 Enzymology and glucose metabolism status in each group
3.2.1 Changes in ALT and AST activities in each group
At 20 weeks, the serum ALT and AST activities in the HFHC group mice were significantly elevated (P<0.01), and these activities increased progressively over time. At 30 weeks, the serum ALT and AST activities in the HFHC group mice were significantly higher than those in the Con group mice (P<0.01) at 30w and the HFHC group mice at 20w (P<0.01, P<0.01) (Fig. 2A and B).
3.2.2 Changes in FBG, FINS and HOMA-IR in each group
Compared with the Con group mice at 20w, the FBG and FINS levels in the HFHC group mice at 20w were significantly higher (P<0.05, P<0.01), and these levels increased progressively over time. At 30 weeks, the FBG and FINS levels in the HFHC group mice were elevated even more compared with those in Con group mice (P<0.05, P<0.01). Compared with the Con group mice at the same time points, the HOMA-IR indices in the HFHC group mice were significantly higher (P<0.01, P<0.05) (Fig. 2C-E).
3.3 Liver steatosis and inflammation status of the mice in each group
3.3.1 Changes in liver TG content
The TG liver content increased gradually over time in both mouse groups. At 20 weeks, the TG liver content in the HFHC group was significantly elevated compared with that in the Con group (P<0.01). Similarly, at 30 weeks, the TG liver content in the HFHC group was significantly elevated compared with that in the Con group (P<0.01). Additionally, the TG content in the HFHC group at 30w was significantly higher than that at 20w (P<0.05) (Fig. 3C).
3.3.2 H&E staining
Under a light microscope, H&E staining of the liver tissue showed that hepatic steatosis affected the entire liver lobule, with substantial inflammatory cell infiltration and scattered necrosis. The HFHC mice at 30w showed ballooning degeneration of hepatocytes and inflammatory foci in the hepatic lobule. The NAFLD activity scores (NAS) ranged from 4-8, among which 80% were higher than 5, meeting the NASH diagnosis criteria, and 40% were close to 8 (Fig. 3A and D).
3.3.3 Oil red O staining
Oil red O staining showed that both macrovesicular and microvesicular steatosis were present in the liver tissues from mice in the HFHC group at 20w, ranging from 33 to 66%. The area, density and intensity of Oil red O staining in liver tissue from the HFHC group increased gradually over time. At 30 weeks, severe macrovesicular and microvesicular steatosis was present in the liver tissues from the HFHC group, ranging from 66 to 99% (Fig. 3B).
3.4 Collagen deposition and hepatic stellate cell (HSC) activation status in liver tissue from mice in each group
3.4.1 HYP content
The liver HYP content in the HFHC group increased gradually over time. Compared with that in the Con group at 20w, the liver HYP content in the HFHC group mice at 20w was significantly increased (P<0.05). Similarly, the liver HYP content in the HFHC group at 30w was significantly elevated compared with that in the Con group at 30w (P<0.01) and the HFHC group at 20w (P<0.05) (Fig. 4A).
3.4.2 Sirius red staining
Under a light microscope, Sirius red staining of the liver tissue showed that at 20 weeks, fibrosis in the perisinusoidal space was present in a star shape in the HFHC mouse livers, and the fibrosis stage ranged from F1-F2, with the majority (60%) at stage F2. At 30 weeks, there was substantial perisinusoidal fibrosis in the liver, the presence of cage-like structures and massive fibrous connective tissue hyperplasia, with some bridging formations. Semiquantitative analysis showed that at 30 weeks, the collagen level in the HFHC group was significantly higher than that in the Con group (P<0.01), and more than half of the fibrosis was at the F2-F3 stage (Fig. 4B-D).
3.4.3 Changes in the Col-I, α-SMA and TGF-β1 protein expression levels in liver tissues from mice in each group
Western blot analysis showed that the Col-I, α-SMA and TGF-β1 protein expression levels in the HFHC group livers at 20w and 30w were significantly higher than those in Con group livers at 0w, and the expression levels increased over time (P<0.01, P<0.01, P<0.05) (Fig. 5A).
3.4.4 IHC of Col-1 and α-SMA in the mouse livers in each group
This α-SMA-positive staining was significantly enhanced in the fibrotic liver septum in the HFHC group at 20w and 30w and was mainly located in the thick and dense fibrotic septum. The positive staining area increased over time. Similarly, Col-I positive staining was present only in the blood vessel walls in the Con group at 0w. This Col-I positive staining was significantly enhanced in the fibrotic liver septum in the HFHC group at 20w and 30w and was mainly located in the perisinusoidal hepatocyte membranes and fibrotic septum. The positive staining area increased over time (Fig. 5B).
3.4.5 Col-I, Col-4, α-SMA, TGF-β1 and Smad3 mRNA expression levels in mice in each group
Compared with those in the Con group livers at 0w, the Col-I, TGF-β1 and α-SMA mRNA expression levels in the HFHC group livers at 20w were significantly increased (P<0.05, P<0.05 and P<0.01). At 30 weeks, the Col-I, Col-4, α-SMA and TGF-β1 mRNA levels in the HFHC group livers were significantly increased (P<0.01), and there was a trend toward increased Smad3 mRNA levels, although the difference was not significant (Fig. 5C-G).
3.5 Transcriptome analysis of differential gene expression between the HFHC group and Con group
3.5.1 Model group and normal group differential gene clustering analysis heat map
The genes differentially expressed between the normal group and model group were selected (P<0.05), and a heat map analysis was performed via cluster analysis, as shown in the following figure. The three samples in the normal group and the model group were naturally clustered, and the expression levels of the genes were similar. However, the difference in gene expression between the two groups was significant (Fig. 6A).
3.5.2 Differentially expressed genes between the model group and normal group
There were 151 differentially expressed genes with Log2FC≥2 and P<0.05 between the model group and the normal group, including 104 upregulated genes (Table 1) and 47 downregulated genes (Table 2).
3.5.3 Signal-regulated pathways associated with the genes differentially expressed between the model group and the normal group
The 151 differentially expressed genes between the model group and the normal group with a fold change ≥2 according to Log2FC values were analyzed via KEGG PATHWAY analysis using R script. When the corrected P value (P adjust) was <0.05, the KEGG PATHWAY function was considered to show significant enrichment. The results showed that the above 151 differentially expressed genes were involved in 41 signaling pathways, suggesting that the 41 signaling pathways are significant in the high-trans fatty acid, high-glucose diet-induced NAFLD model (Fig. 6B, Table 3).