NAFLD is currently the most frequently occurring of all chronic liver diseases, with a yearly rise in morbidity and mortality (1). Despite the abundance of national and foreign studies and the deepened understanding on NAFLD, its pathogenesis is still unclear, and the treatment options are very limited (1, 2). Thus, at this stage, the establishment of animal models is still necessary in the research on NAFLD pathogenesis and the discovery of potential novel therapeutic targets. Various approaches have now been used for the establishment of animal NAFLD models, which can be divided into two major categories; the first method is based on the establishment of animal NAFLD models through diet induction; the second is realized by specific gene knockout through genetic modification (16–19).
The animal model of diet induction has been often used at home and abroad, which can simulate the natural formation process of human NAFLD, with a high success rate, low mortality rate, and good repeatability (20–24). The most commonly applied diet models are choline-deficient, L-amino acid‐defined (CDAA) diet, high-cholesterol diet (HCD), MCD, high-sugar diet, etc. Unlike other diet models, MCD can lead to insulin resistance and a significant weight loss, which is inconsistent with the human metabolic model. However, MCD diet can well simulate the pathological characteristics of NASH, and the time for its implementation is short (approximately 2–4 weeks). Therefore, MCD diet has been most extensively investigated and applied in the development of NAFLD models (20–24). Therefore, our mouse NAFLD model was developed using an MCD diet, which was simple and rapid. We assumed that the establishment of this model was closer to the natural course of the disease in patients with NAFLD. Here, we successfully set up a NAFLD mice model and found that a four-week probiotics treatment reduced the liver function damage caused by MCD diet and attenuated liver and intestinal pathological injuries.
The gut and the liver are closely linked by their tight anatomical and functional relationships, also collectively known as the “gut–liver axis” (25, 26). Accumulating recent evidence from many human and animal model studies has shown that gut microbiota dysfunction is conducive to the development and progression of NAFLD (27). The increase in intestinal permeability was found to cause injuries in the intestinal mucosal barrier, leading to the overproduction of bacteria in the intestinal cavity, destruction of the intestinal microenvironment, and the production of a large number of toxic metabolites and endotoxins (26). On the one hand, the endotoxins entering the blood circulation not only directly damage the intestinal mucosal epithelial cells, but also induce intestinal microvasculature contractions and intestinal tissue ischemia and hypoxia, leading to excessive ROS production. Meanwhile, the increase in the intestinal permeability augments endotoxin absorption, which is also a huge burden on the liver (28, 29). On the other hand, because of the presence of the intestine-liver axis, the endotoxins entering the blood circulation also act directly on the liver, causing its inflammation by activating Toll-like receptors (29–31).
A recent clinical trial found that probiotics alleviated intestinal microecological and metabolic disorders in patients with NAFLD (32). ALT and AST concentrations are considered important markers for liver injury evaluation. Our results showed significantly increased serum AST and ALT levels in the model group and pathologically damaged liver tissue. After four weeks of probiotics treatment, the elevated serum ALT, AST, TC, and TG levels, induced by the MCD diet, were alleviated and liver steatosis improved, demonstrating the beneficial effects of probiotics on the liver.
Lipid peroxidation and oxidative stress are vital pathogenetic mechanisms of NAFLD, and thus their suppression may serve as an effective method for NAFLD prevention or intervention (33). Studies have shown that excessive free fatty acids (FFAS) in the liver cells induce lipid peroxidation during NAFLD, which considerably increases the production of ROS (33, 34). On the one hand, the produced ROS promote the release of TNF-α, which triggers inflammation through the NF-κB signaling pathways and aggravates the inflammatory response of the liver. On the other hand, ROS can participate in the JNK signaling pathway as the second messenger, eventually activating the JNK signaling pathway and inducing hepatocyte apoptosis (34, 35). To elucidate the potential mechanism of probiotics action in NAFLD remedy, we analyzed the expression of ROS/JNK signaling pathway-related factors.
JNK is well recognized as an important signaling involved in stress response and apoptosis, which is activated by oxidative stress, DNA damage and UV exposure, and subsequently regulates the downstream targets expression, such as that of Bax and Caspase-3 (35, 36). Bax is a pro-apoptotic gene, and Caspase-3 is critically involved in apoptosis (37). In this study, we established that probiotics exerted an important part in antioxidant stress and apoptosis alleviation. The large amount of ROS, produced due to action of different stimuli, activates the JNK pathway through a JNK-specific kinase, JNKK. Then, JNK phosphorylation enhances the activity of the transcription factor complex AP-1, resulting in upregulated expression of pro-apoptotic proteins, such as p53, Bax, and TNF, but inhibited expression of apoptotic proteins such as Bcl-2. Subsequently, the overexpressed pro-apoptotic proteins apply to and accelerate the release of cytochrome C from the mitochondria into cytoplasm. Next, cytochrome C binds to caspase-9, and that complex then interacts with caspase-3. Finally, activated caspase-3 binds to apoptotic substrates and causes apoptosis (37–39). In a previous study, the inhibition of JNK phosphorylation reduced liver fat deposition and improved liver function and inflammatory response in animals with MCD-induced NAFLD (40). To further confirm the anti-inflammatory effect of probiotics on MCD-induced oxidant stress and apoptosis, we investigated the changes in JNK downstream signaling. Our results showed that the expression levels of ROS, P-JNK, Bax, and caspase-3 were significantly increased in the model group; the oxidative stress response was enhanced and liver cell apoptosis increased. Moreover, we found that probiotics diminished ROS production, inhibited JNK phosphorylation, significantly suppressed the expression of Bax and Caspase-3 in the downstream JNK signaling pathway, and further impeded cell apoptosis, which alleviated NAFLD. These results indicate that probiotics can be utilized as a therapeutic target for the treatment of NAFLD by regulating the ROS/JNK signaling pathway. This study provides a potential target for NAFLD and additional evidence for the significance of the clinical use of probiotics in the treatment of NAFLD.
However, this study has some limitations. First of all, it was focused on the expression of pro-apoptotic genes in the JNK signaling pathway, but did not examine the importance of the expression of anti-apoptotic genes and related proteins. Second, the test results of a single sex of mice and a single NAFLD modeling method may be controversial to some extent, and this study needs to be supplemented and verified in the later stage by combining different genders of mice and different NAFLD modeling methods.Finally, we only analyzed the intestinal barrier function and permeability, but did not analyze and evaluate the changes of gut microbiota in mice. If the above limitations had been overcome, our experimental results would have been more reliable. Multiple mechanisms of probiotics action can be exerted in the treatment of NAFLD, but only one of them was investigated in this study. Therefore, future studies should be performed for further clarification of the potential mechanisms of probiotics action in the treatment of NAFLD.