Role and Mechanisms of Probiotics in Regulating the ROS/JNK Signaling Pathway in the Pathogenesis of Nonalcoholic Fatty Liver Disease.

This study was aimed to investigate the impact of probiotics on regulating the ROS/JNK signaling pathway their underlying mechanism of action in the treatment of nonalcoholic fatty liver disease. For this purpose, male C57BL/6 mice were randomly divided into three groups: control, probiotics, and model groups. Methionine and choline deciency (MCD) diets were fed for four weeks to establish a NAFLD mouse model. Serum levels of ALT, AST, TC, and TG were detected. Moreover, the pathological changes of the liver and ileum tissues were observed by hematoxylin and eosin (H&E) staining, and the content of reactive oxygen species (ROS) in liver tissues was determined. In addition, the levels of D-lactic acid and plasma and small intestine diamine oxidase were measured to evaluate the effects of probiotics on the intestinal tract of NAFLD mice. The expression levels of p-JNK, Bax, and Caspase-3 were established to analyze the regulatory mechanism of probiotics on the JNK signaling pathway. We found that probiotics improve liver function, repair intestinal barrier and signicantly suppressed oxidative stress, JNK phosphorylation. Moreover, the application of probiotics regulated the expression of signaling pathway-related proteins and promoted the intestinal barrier function repair and decreased intestinal permeability. The data above suggest that probiotics alleviate NAFLD, whose mechanism might be associated with the regulation of ROS/JNK signaling pathway and the suppression of oxidative stress and apoptosis.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic progressive liver disease all over the world, with a global incidence of approximately 25% (1,2). It is a complex disease whose pathogenesis remains unclear. An earlier study showed that NAFLD is closely associated with metabolic abnormalities, oxidative stress, in ammatory response, apoptosis, and other factors (3). To date, clinical guidelines indicate that dietary interventions and lifestyle changes are the major factors involved in the treatment of NAFLD and its complications while no speci c drugs are available for NAFLD therapy (4). Therefore, it is critically important to investigate and discover novel therapeutic targets and approaches.
C-Jun N-terminal kinase (JNK) is one of the most important pathways in the mitogen-activated protein kinase (MAPK) family that is mainly involved in stress response and apoptosis, among other physiological processes (5). The accumulation of reactive oxygen species (ROS) induces apoptotic signal transduction and activates apoptotic proteins, such as caspase-3 and Bax, causing apoptosis (6,7). The bodily production of ROS is induced through the action of various factors causing oxidative stress, leading to activation of apoptosis signal-regulating kinase 1 (ASK1). In turn, JNK phosphorylation is promoted, and phosphorylated JNK further activate and promote the overexpression of the apoptosis proteins Bax, cysteinyl aspartate-speci c proteinase-3 (caspase-3), resulting in liver cell apoptosis, which can lead to liver degeneration and necrosis ( Fig. 1) (6,8,9).
Probiotics are a group of bioactive microorganisms that are bene cial to the host and improve its microecological balance and a favorable impact on the status and function of the intestinal tract (10,11).
Probiotics may improve the intestinal epithelial barrier function and reduce intestinal permeability, in ammation, and oxidative stress by regulating the abundance and diversity of the natural intestinal ora; thus, it can be applied in the treatment of NAFLD, but their speci c mechanism of action has not yet been clari ed (12). Moreover, the e cacy of probiotics in metabolic disease treatment remains controversial, and further studies are needed to assess their safety and signi cance in that respect.
Hence, the purpose of this investigation was to elucidate the role and mechanism of probiotics in regulating ROS/JNK signaling pathway in the treatment of NAFLD, which would provide novel insights, further evidence, and reference for the clinical use of probiotics in the treatment of NAFLD.

Materials And Methods
Lactobacillus plantarum N3117 isolation and culture methods Lactobacillus plantarum N3117 was isolated from Inner Mongolia traditional fermented milk and stored in the central laboratory of A liated Hospital of Southwest Medical University,was cultured at 37℃ for 24 h in de Man, Rogosa, and Sharpe (MRS) broth.Bacteria were collected from MRS broth cultures by centrifugation (6000g for 10 min at 4℃).Afterwards, phosphate buffered saline (PBS) was used to wash the particles twice and live bacteria used as oral probiotic supplements were suspended in sterile saline.Each bacterial suspension was given an adjusted oral dose of 1.0 x 108 colony-forming units

NAFLD model
All C57BL/6 mice were fed normal diet and water ad libitum for a week at room temperature of 18°C-22°C, humidity of 50%, and day-night cycle of 12 h. After another week of new environment acclimation, C57BL/6 mice (n = 18) were randomly divided into three groups (n = 6 in each group): control, probiotic, and model group. In the model and probiotic groups, mice were fed MCD diet, whereas in the control group, MCS diet was applied for four weeks(The duration of probiotics treatment was determined by preliminary studies). Then, a mouse was randomly selected from each group and killed by Cervical dislocation. H&E staining of the liver tissue was used to con rm the successful establishment of the model. In the probiotic group, mice received probiotics gavage once a day, whereas the control and the model groups were given the same amount of saline gavage once a day for four weeks. After the drug intervention was complete, the mice were fasted for 12 h, and their weight was measured. Next, using pentobarbital sodium for intraperitoneal injection anesthesia, blood was collected from the heart, followed by settlement for 1 h and centrifugation for 10 min at 2000 g. After centrifugation, we collected the supernatant and preserved it at -80°C for analyses of the levels of lipids, transaminases, and related parameters. Liver and ileum tissues were dissected and weighed. Then, part of them was xed in 4% formaldehyde and sent to the Pathology Department for H&E staining and immunohistochemical analysis. The remaining liver and ileum tissues were frozen at -80°C in a refrigerator for further Western blot and PCR analyses.

Biochemical analysis
After the mice were killed, blood samples were taken from the heart. Then, the blood samples were centrifuged at 5000 rpm for 20 min for supernatant serum extraction. The levels of ALT, AST, TG, TC, D-Lac, DAO, and ROS were determined by ALT, AST, TG, TC, D-Lac, DAO, and ROS assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

Western blot
Liver and ileum tissues were cleaned with precooled PBS buffer 2-3 times to purify the blood. Next, the appropriate volume of total cell protein extraction reagent was placed into an ice bath (protease inhibitor was added a few minutes before use), where the homogenate was hatched lasted for 30 min. The homogenate was centrifuged at 4°C and 13,000 g for 5 min, followed by supernatant collection. Further, the concentration of the supernatant protein was measured by a BCA protein concentration assay kit (Wuhan Aspen Biotechnology Co., Ltd., Wuhan, China). The right quantity of protein loading buffer was added to the protein samples, and they were boiled in a water bath at 95°C-100°C for 5 min and transferred onto PVDF membranes (Millipore Co., Ltd., Shanghai, China). Next, the con ning solution was removed, and the primary antibody was added (Table I) and left undisturbed overnight at 4°C. The secondary antibody (Table II) was then added, followed by incubation at room temperature for 30 min. The fresh ECL mixture was next added to the protein side of the membrane for its exposition in the in the dark.

Real-time quantitative PCR
Total mRNA was extracted from the liver employing a TRIpure RNA Extraction Reagent kit (ELK Biotechnology Co., Ltd., Wuhan, China). The rst-strand of cDNA of Bax and Caspase-3 were synthesized by the EntiLink 1st Strand cDNA Synthesis Kit (ELK Biotechnology, Co., Ltd., Wuhan, China).The primers sequences were shown in Table III. The real-time quantitative uorescence PCR was completed on a StepOne real-time PCR instrument of Life Technologies, and each sample was detected by employing the EnTurbo SYBR Green PCR SuperMix kit (ELK Biotechnology, Co., Ltd., Wuhan, China).The relative quantitative of gene expression was evaluated by the 2-ΔΔCt method (15).

Immunohistochemistry
The liver tissues were removed from the mice and xed with 10% formaldehyde and embedded with para n, and sectioned. Sections were incubated with primary antibody (Table IV) and the secondary antibody (Table V), and DAB solution (Zhongshang Jinqiao Biotechnology Co., Ltd., Beijing, China) was added to color.
Statistical analysis SPSS 25.0 was used for all statistical analyses. The data were expressed as means ± SD for each group. One-way analysis of variance (ANOVA) was among the groups. A value of P < 0.05 was considered to indicate statistically signi cant differences.

Effects of probiotics on liver function and lipid levels in serum
Serum ALT, AST, TC, and TG levels in the model group were considerably higher than those in the control group (Fig. 1). However, four-week probiotics administration lowered the serum levels of ALT, AST, TC, and TG.

Effect of probiotics on hepatic pathology
In the control group, H&E staining results showed orderly arrangement of hepatocytes, with no fat droplet deposition and in ammatory cells; the hepatic lobules had a normal structure (Fig. 2a and A). Nevertheless, the structure of the hepatic lobule was disordered and the arrangement of hepatocytes was unclear boundaries. Besides, there were more fat droplets and in ammatory cells in the cytoplasm in the model group ( Fig. 2c and C). Four-week probiotics treatment alleviated all these signs ( Fig. 2b and B).

Effect of probiotics on the intestinal barrier function and permeability
Serum D-Lac levels of and the serum and ileum levels of DAO were known to be important indicators for measurements of intestinal barrier function and permeability. In our study, the levels of D-Lac and DAO in the model group were signi cantly higher than those in the control group. However, the serum levels of D-Lac and DAO in the probiotics-treated group were lower than those in the model group. Moreover, the ileum level of DAO in the model group was signi cantly higher than that in the control group, but the treatment with probiotics considerably decreased this level, suggesting that probiotics could repair the intestinal barrier and reduce intestinal permeability (Fig. 3).
The pathological results in our investigation showed that the ileum mucosa in the control group was intact, and the intestinal villi were complete and neatly arranged, without any in ltration of in ammatory cells. However, in the model group the ileum mucosal structure was seriously damaged, with loose and disorderly villi arrangement. Importantly, the ileum mucosa in the probiotic group had fewer injuries and the villi were arranged more neatly (Fig. 4). Besides, the ileum tissue of the control group had the lowest Chiu's score range (0-1), whereas that of the model group was 3-4, and the one of the probiotic group was 1-2 (Fig. 5).
The tight junction protein ZO-1 was critically involved in intestinal barrier function maintenance and had been considered the most reliable marker protein for tight junction detection. Western blot results revealed obviously lower expression of ZO-1 in the model than in the control group. However, the expression of the intestinal tight junction protein ZO-1 was upregulated in the probiotic group in contrast to the model group (Fig. 6A,E).

Effect of probiotics on ROS/JNK signaling pathway in NAFLD
To verify the regulation effect of probiotics on the ROS/JNK signaling pathway in NAFLD mice, we detected the expression of ROS/JNK signaling pathway-associated proteins in the liver. The consequences demonstrated that ROS was observably added in the model group; moreover, it was decreased after four weeks of probiotics treatment (Fig. 7). Subsequently, we used Western blot analysis to explore the JNK signaling pathway mechanisms of the probiotics action in the treatment of NAFLD. We established a dramatically higher increase in the p-JNK, Bax, and Caspase-3 levels in the model group than in the control group, whereas probiotics obviously suppressed the expression of p-JNK, Bax, and Caspase-3 in the probiotics group (Fig. 6B-E). These results were also con rmed by immunohistochemical staining (Fig. 9). qPCR assays showed that probiotics suppressed apoptosis by inhibiting the expression of the related genes in the JNK signaling pathway (Fig. 8). These results indicated that the oxidative stress induced by ROS in MCD-induced NAFLD can cause apoptosis, but probiotics can effectively attenuate this liver injury.

Discussion
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 rst method is based on the establishment of animal NAFLD models through diet induction; the second is realized by speci c gene knockout through genetic modi cation (16)(17)(18)(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)(21)(22)(23)(24). The most commonly applied diet models are choline-de cient, L-amino acidde ned (CDAA) diet, high-cholesterol diet (HCD), MCD, high-sugar diet, etc. Unlike other diet models, MCD can lead to insulin resistance and a signi cant 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)(21)(22)(23)(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 in ammation by activating Toll-like receptors (29)(30)(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 signi cantly 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 bene cial 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 in ammation through the NF-κB signaling pathways and aggravates the in ammatory 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-speci c 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)(38)(39). In a previous study, the inhibition of JNK phosphorylation reduced liver fat deposition and improved liver function and in ammatory response in animals with MCDinduced NAFLD (40). To further con rm the anti-in ammatory 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 signi cantly 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, signi cantly 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 signi cance 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 antiapoptotic 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 veri ed 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 clari cation of the potential mechanisms of probiotics action in the treatment of NAFLD.