Our study showed that NASH mice induced by a high-fat, high-sugar diet with trans-fat as the main fat for 25 weeks with an increased ratio of cholesterol and bile salts exhibited advanced liver inflammation, glucose and lipid metabolism disorders, and fibrosis. After the HFHCC diet model, significant changes were observed in the levels of innate immune cells and their released cytokines in the liver of mice, while the related changes in adaptive immune cells were not obvious. This model is more inclined to trigger an innate immune response than an adaptive immune response.
The liver TG content was 2.5-folds higher in the HFHCC mice at 25 weeks, and serum ALT and AST activities were significantly increased. Liver pathological analysis showed that the HFHCC diet induced hepatocytes in mice with different degrees of balloon-like changes after 25 weeks. The steatotic hepatocytes were swollen in round or oval shapes, and there are large amounts of fat vacuils within the hepatocytes. Macrophages could be observed in the hepatic sinuses, and neutrophil and monocyte infiltrates could be seen within the lobules of the liver. The mean NAS score was 4.25 points. In terms of fibrosis, col-1 positivity and α-sma positivity were significantly increased in mouse liver tissue, suggesting significant stellate cell activation and hepatic collagen deposition. Hepatic fibrosis was staged as F1-2. In terms of glucose metabolism, compared with the normal group, the glucose, insulin, and insulin resistance indices of the HFHCC model mice were significantly increased, showing obvious glucose metabolism disorder, which is very similar to the characteristics of glucose metabolism disorder in human patients with NASH.
C57BL/6J mice fed a conventional high-fat diet (60% energy from fat) for 10 weeks exhibited obesity and disturbances in glucose and lipid metabolism, developed steatosis and inflammation at 16 weeks, and showed mild fibrosis at 50 weeks[11]. C57BL/6 mice fed a high-fat, high-sugar diet for 30 weeks showed NASH characteristics, such as ballooning, glucose and lipid metabolism disorders, liver damage, and inflammation, but the degree of fibrosis was very mild[7]. Compared with conventional high-fat high-sugar diet models, the HFHCC diet induces more pronounced and quicker pathological features of NASH. Tu et al[12]. also used a high-fat and high-cholesterol bile salt diet to induce NASH, in which 37.1% of the energy came from fat, the cholesterol content was 1.25%, and the sodium cholate content was 0.5%. The pathological results showed that the diet was modeled, and post-mice showed steatosis and inflammation but very low levels of fibrosis. On the basis of Tu’s study, our study increased cholesterol content (2%) and added high-sugar drinking water to further accelerate NASH progression, inducing more pronounced glucose and lipid metabolism disorders, and the appearance of fiber change. Similar to our study, Mells et al. found that C57BL/6J mice fed a high-trans-fat, high-sucrose, and high-cholesterol diet not only developed steatosis, lobular inflammation, and hepatocyte ballooning but also exhibited fibrosis. Together, our experiments demonstrated that the HFHCC diet could replicate the NASH model with similar metabolic and histological features in humans[13].
However, it was worth noting that although the HFHCC diet-treated mice showed significantly increased liver weight and liver body ratio compared to control diet-treated mice, yet mice in the HFHCC group exhibited significant decreased body weight by approximately 9% at 25 weeks compared to that in the Control group. A possible reason is that bile salts can participate in the metabolism of bile acids and promote the metabolism and absorption of cholesterol, activate G protein-coupled receptor 5, induce the activity of type 2 deiodinase, promote the conversion of tetraiodothyronine to triiodothyronine, and finally promote the energy expenditure, resulting in weight loss[14]. In conclusion, this model could simulate the entire disease course of human NASH patients, while exhibiting the characteristics of NASH fibrosis, showing characteristics of patients with NASH, especially non-obese patients with NASH liver fibrosis.
To further analyze the immune disorder state of this model, flow cytometry and immunohistochemistry were used to measure the proportion of immune cells in the liver, and multiplex bead immunoassay and luminex technology was used to detect the protein levels of related cytokines in liver tissue. Our findings supported that the HFHCC diet activated the liver immune response, compared with the control group, the proportions of innate immune cells such as KCs, neutrophils, and DCs were evidently increased in the model group, while the proportion of adaptive immunity-related cells, only CD3 + T cells, was higher in the model group. There were no differences in the proportions of B cells, CD4 + T cells, and CD8 + T cells between the two groups. The protein levels of pro-inflammatory cytokines, chemokines, and colony-stimulating factors released by innate immune-related cells in the liver of HFHCC mice were significantly increased, whereas the changes in inflammatory factors released by adaptive immune cells were not obvious. These results suggest that the HFHCC diet is more likely to activate innate immunity in mice.
There is mounting evidence that innate immunity plays an important role in the pathogenesis of NASH. Liver innate immune cells include KCs, neutrophils, DC, and NK[8]. KC cells are the first line of defense in the liver. When the HFHCC diet induces disorders of glucose and lipid metabolism, lipotoxicity caused by excessive accumulation of lipids in hepatocytes activates macrophages, leading to TNF-α, IL-6, and IL-1β, which further promotes liver steatosis, inflammation, and liver fibrosis[15]. In this study, In this study, immunohistochemical results showed that the HFHFF diet induced the activation of KCs. At the same time, the pro-inflammatory cytokines (TNF-α, IL-1α, IL-1β, and IL-6) and chemokines (including CXCL1, CCL2, CCL11), and the levels of colony-stimulating factor recruitment (GM-CSF, G-CSF) released by KCs were also significantly increased. Neutrophils are another major effector of the innate immunity. The accumulation of metabolites induces the recruitment of neutrophils in the peripheral blood to the liver. Neutrophils activate macrophages, which increases the secretion of adhesion molecules and recruits neutrophils. More white blood cells enter the liver, triggering an inflammatory response. This study showed that neutrophils were significantly recruited in the liver tissue of mice in the model group, and at the same time, the levels of inflammatory factors IL1-α and G-CSF released by neutrophils were significantly increased. The role of DCs as antigen-presenting cells in NASH is complex and controversial, as hepatic DCs play a pro-inflammatory role in thioacetamide diet-induced liver fibrosis[16,17]. In the MCD diet, the reduction in hepatic DCs induces the production of inflammatory factors such as IL-6 and TNF-α, and the decrease in hepatic DCs is a type of damage to the liver[18,19]. Our study found that the number of DCs in the model group was higher than that in the normal group, and the levels of IL-10 and IL-12, two cytokines released by DCs, were significantly increased. The differences in the experimental results of the different studies may be related to the NASH modeling method. NKT cells express the surface markers CD3 of T cells and the surface markers of NK cells. These are the bridges between innate and adaptive immunity. Once recognized by antigens, they can rapidly secrete pro-inflammatory or anti-inflammatory inflammatory factors such as IFNγ and IL-4. Michael et al[20–22]. found increased numbers of NKT cells in the liver and peripheral blood of patients with NASH, NAFLD, or obesity. BhattacharjeeJ[23] found increased numbers of NKT cells in the livers of NASH mice. In our experiment, it was also observed that the proportion of NKT cells in the livers of NASH mice, and the levels of related inflammatory factors such as IFNγ were significantly increased.
T cell-mediated adaptive immunity can cause inflammation and fibrosis in NASH through cytotoxicity, cytokines, and other proinflammatory and profibrotic mediators[24]. CD3 is a surface marker for mature T lymphocytes. T cells are divided into two categories, αβ T cells (such as CD4 and CD8) and γδ T cells, according to different TCRs[25]. This study showed that, compared with the normal group, the proportion of CD3 + T cells in the HFHCC group was significantly higher than that in the control group, while no significant difference was found in CD4 + T cells and CD8 + T cells. IL5, IL13, and IL17A are mainly secreted by adaptive immune cells, such as Th2, Th12, and CD4 + T cells, and these cytokines did not change significantly after modeling. Moreover, there were no significant differences in the number of B cells. Overall, it is hypothesized that the HFHCC diet has a weaker effect on adaptive immunity than on innate immunity. However, a deeper understanding of the mechanism requires further verification and research.
This study had some potential limitations. Inconsistent with the disease characteristics of most patients with NASH, the weight of the model mice was lower than that of the normal group, which was mainly related to the involvement of bile salts in bile acid metabolism affecting energy metabolism; on the other hand, this model also provided a reference for the study of non-obese NASH. In addition, due to the limitation of the number of experimental channels for flow analysis, the study only used immunohistochemistry for the analysis of liver macrophages, and lacked the results of flow analysis; in terms of adaptive immunity, Th1, Th2, Th17, and Treg have not been further detected and analyzed, which is also the direction for further research in the later stage.
In conclusion, we established an HFHCC diet-induced NASH model that is stable and reproducible, showing the emergence of NASH pathological features such as inflammation, steatosis, and fibrosis. This model is likely to trigger innate immunity. This could serve as a suitable experimental model for drug testing and for understanding the pathogenesis of innate immunity in NASH.