Establishment of animal models for NASH and utilization for mining associated gut microbiota

doi:10.21203/rs.3.rs-1826044/v1

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Establishment of animal models for NASH and utilization for mining associated gut microbiota

https://doi.org/10.21203/rs.3.rs-1826044/v1

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A high-fat diet (HFD) animal model has an indispensable role in the research of gut-liver axis by generating an imbalance of gut microbiota and intestinal leakage, resulting in translocation of harmful bacterial metabolites and lipopolysaccharides (LPSs) to the liver, triggering immune responses, and stimulating non-alcoholic steatohepatitis (NASH). Replacing trans-fat with palm oil in HFDs is an emerging animal model with human translatability in terms of liver biopsy phenotype and transcriptome changes. We developed the NASH and fibrosis mouse model using a palm oil-containing high-fat diet (P-HFD) combined with LPS to simulate gut dysbiosis and endotoxemia and examined the role of carbon tetrachloride (CCl₄) on the gut-liver axis. The mice were fed P-HFD with or without intraperitoneal LPS/CCl₄ injections. NASH/fibrosis progression and gut microbiota shift were examined at 4, 8, 12, 16, 20, and 24 weeks (n = 192). The P-HFD animal model showed obesogenic and metabolic changes, hepatomegaly, NASH phenotype, increased gut dysbiosis, changes in gut microbiota and its function, reduced beneficial bacteria, increased pathogenic related microbiome, intestinal leakage, and endotoxemia. P-HFD with LPS supplementation resulted in comparable NASH phenotypes compared with the P-HFD group, but worsened the degree of intestinal microbiota dysbiosis by increasing the occurrence of pathogenic bacteria and reducing beneficial microbiota. P-HFD with CCl₄ resulted in a liver fibrosis phenotype and produced a more severe gut microbiota shift. These mouse models could be informative for a researcher focused on the gut-liver axis and could be used as valuable tool for preclinical drug testing.

Non-alcoholic steatohepatitis

animal models

gut microbiome

dysbiosis

endotoxemia

palm oil

carbon tetrachloride

Chronic liver diseases, including non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), are common medical problems worldwide.¹ Hepatic steatosis is the initial phase that can develop into NASH, cirrhosis, and eventually hepatocellular carcinoma.² The spectrum of fatty liver disease is caused by multiple factors, such as frequent consumption of high-calorie food, high alcohol intake, and other chronic diseases. Dietary nutrients are essential for human health, and they are also crucial for the gut microbiota.³ An unhealthy diet induces an imbalance of gut microbiota, resulting in the production of harmful metabolites.⁴ Gut-derived harmful metabolites include lipopolysaccharides (LPS), flagellin, and peptidoglycan, which adversely affect the body’s immune response and induce hepatic inflammation. These metabolites subsequently translocate to the liver via the portal vein, activating the release of hepatic pro-inflammatory cytokines.^{5, 6} A previous study suggested that LPS administration via subcutaneous pumps with diethylnitrosamine/carbon tetrachloride (CCl₄) injection promoted hepatocarcinogenesis in mice.⁷ Gut microbiome-derived compounds affect hepatocytes, leading to activation of inflammatory signaling, gene expression changes, and alteration in metabolism and toxicity. Various Toll-like receptors (TLRs) in different liver cell types (Kupffer cells, hepatic stellate cells, and hepatocytes) sense bacterial products and trigger downstream inflammatory responses as well as cytokine production.⁵ Both the gut microbiota and an unhealthy diet could increase intestinal permeability.⁸ Maintaining the gut barrier could prevent the efflux of toxic metabolites to the liver.^{9, 10} Additionally, gut sterilization by using antibiotics reduces to late stages of hepatocarcinogenesis.^{11, 12}

There are several animal models for inducing NASH and liver fibrosis, such as using a high-fat-containing and choline-deficient diet.^{13, 14} The trans-fat-containing amylin liver NASH (AMLN) diet has been confirmed for inducing metabolic disease and NASH. The enhancement of dietary trans-fats intensifies the steatogenic and pro-fibrotic properties of obesogenic diets in mice.¹⁵ Since trans-fats have adverse effects on health, the US Food and Drug Administration (FDA) passed a rule in 2018 against the inclusion of artificial trans-fats in the food additives.^16–18 In a recent study utilizing a mouse model, the substitution of trans-fats with the palm oil Gubra Amylin NASH (GAN) diet promoted a similar phenotype of NASH compared to trans-fat induced NAFLD.¹⁹ Interestingly, liver biopsy histological analysis and transcriptome signatures of the mice treated with palm oil-containing high-fat diet (P-HFD) were similar to patients diagnosed with NASH.²⁰ Diet intervention for inducing NASH and fibrosis is a lengthy process. CCl₄ is a chemical that is well-known to accelerate NASH and liver fibrosis progression.²¹ A low weekly dose of intraperitoneal CCl₄ acts as a NASH accelerator in a western diet-fed animal model.²² CCl₄ can be converted to trichloromethyl (•CCl₃) and trichloromethyl peroxyl (•CCl₃O₂) radicals by the cytochrome P450 enzyme. These radicals damage the tissue by increasing oxidative stress and lipid peroxidation.²³

In the current literature, there are no preclinical animal models that could faithfully recapitulate gut dysbiosis and the consequent acceleration of NASH and liver fibrosis. Although the combined effects of the HFD and the LPS/CCl₄ mouse model are widely utilized, P-HFD application in the LPS/CCl₄ model to induce NAFLD and fibrosis has not been explored. This study developed a NASH and fibrosis mouse model using palm oil as a non-trans-fat in a diet with LPS intraperitoneal injection to simulate gut dysbiosis and endotoxemia. As such, changes in gut microbiota composition, intestinal permeability, and gut-derived LPS, including its function, could be investigated. We also examined the impact of CCl₄ on the gut-liver axis. This mouse model represents a potentially valuable experimental tool for preclinical drug evaluation.

P-HFD caused obesogenic and metabolic changes; supplementation of LPS or CCl ₄ injection in P-HFD-fed mice retarded the progress of obesity

The experimental design of the study is shown in Fig. 1a. The mice (n = 192) were fed with a control or P-HFD with or without LPS/CCl₄ injection. Mice were sacrificed at 4, 8, 12, 16, 20, and 24 weeks on a diet (n = 7–8/experimental group/time point) to investigate the progression of obesogenic, metabolic, and hepatic changes. The results demonstrated that P-HFD significantly induced a greater body weight increment compared to the control diet (p < 0.001) at every time point (Fig. 1b). The average body weight changes of the P-HFD group at 0, 4, 8, 12, 16, 20, and 24 weeks were 24.6 ± 1.4, 29.8 ± 2.2, 34.5 ± 3.2, 37.3 ± 4.7, 40.7 ± 4.6, 44.3 ± 5.1, and 47.0 ± 4.8 g, respectively, whereas the control group constantly maintained an average body weight of 27.2 ± 2.3 g. P-HFD induced a body weight gain of 3.7 ± 0.8 g per four-week period. Intraperitoneal injection of LPS or CCl₄ in P-HFD-fed mice resulted in a body weight decrement (p < 0.05 and p < 0.01, respectively) compared to the P-HFD group alone with an average difference of 4.1 ± 1.3 and 10.2 ± 4.2 g, respectively, partially due to the lower overall food and energy intake (Supplementary Fig. 1).

Relative total adipose tissue against bodyweight of the P-HFD group exhibited a result similar to bodyweight changes. P-HFD-fed mice displayed a more pronounced increase (p < 0.001) in whole-body adipose tissue compared to the control group (Fig. 1c). The longer the P-HFD feeding continued, the higher the total adipose tissue weight, from 9.8 ± 1.6% (week 4) to 19.0 ± 1.6% (week 24). WAT, including iWAT, eWAT, mWAT, pWAT, isWAT, and iBAT of the P-HFD group exhibited the highest increase compared to other groups (Fig. 1d-h and Supplementary Fig. 2–4). LPS and CCl₄ intraperitoneal injection resulted in declining adipose tissue weight. The impact of CCl₄ on reducing fat mass is comparatively more substantial than LPS injection. The adipose tissue of the CCl₄ group is relatively low and comparable to the control group.

Additionally, P-HFD promoted the degree of hypercholesterolemia and increased plasma LDL and HDL compared to the control group at all time points (p < 0.01). LPS injection reduced overall cholesterol, LDL, and HDL levels compared to only P-HFD-fed group (Fig. 1i-k). The cholesterol level of CCl₄ injected mice was significantly lower compared to the P-HFD group. CCl₄ group LDL concentration was elevated at the early stage of induction (fourth weeks), but gradually reduced from 16 weeks onward. Interestingly, the plasma HDL level of the CCl₄ group was the lowest compared to other groups. There were no significant changes in total triglyceride levels of the P-HFD-fed mice treated with LPS or CCl₄ compared to the mice solely fed with P-HFD (Fig. 1l). These results suggested that P-HFD enhanced lipidemia, and that LPS and CCl₄ in P-HFD feeding mice interfered with lipid metabolism, resulting in an alteration of circulating lipid levels.

P-HFD induced hepatomegaly and increase hepatic triacylglycerol levels, but LPS intervention slowed this progress

Plasma AST and ALT are helpful indicators for detecting hepatic injury. P-HFD did not elevate the AST and ALT levels at four weeks compared to control group but increased from eight weeks onward (Fig. 2a-b). LPS injection in P-HFD-fed mice exhibited lower AST and ALT levels compared to mice solely fed with P-HFD, but not to a large extent. CCl₄ intervention in P-HFD-fed mice exhibited a higher level of AST and ALT than the P-HFD group at the early stage of induction (fourth week), and the level of ALT in the P-HFD group significantly increased from 16 to 24 weeks. The relative liver weight and hepatic triacylglycerol level showed a comparable trend of liver damage. P-HFD gradually resulted in hepatomegaly during the feeding time, and at the conclusion of the study, the relative liver weight had significantly increased to 7.8 ± 1.2% (p < 0.0001), 1.8-fold larger than the control group compared at the same age (Fig. 2c and 2e). LPS injection showed a lower liver weight from week 12 to 24. The CCl₄ intervention significantly increased liver weight at the fourth and eighth week compared to the P-HFD group and gradually reduced from week 12 to 24, possibly due to the occurrence of liver fibrosis. The hepatic triacylglycerol levels were subsequently measured. The P-HFD group displayed a higher triacylglycerol level at every time point (p < 0.001), and its overall content was 4.6-fold higher than the control group. It reached a maximum level at the 12th week (213.1 ± 34.7 mg/g of the liver). Minor liver triglyceride content differences were found in the LPS group compared to the P-HFD group. The hepatic triglyceride content in the group receiving CCl₄ was significantly lower than the P-HFD group from eight to 24 weeks (p < 0.05). Collectively, the P-HFD caused hepatomegaly and accumulation of hepatic triacylglycerol, but LPS and CCl₄ injection resulted in decreased liver weight.

P-HFD and P-HFD + LPS induced NASH phenotype, whereas P-HFD + CCl₄ induced liver fibrosis

The left lateral lobe of the liver underwent histopathological analysis to evaluate the NASH and hepatic fibrosis characteristics (Fig. 3a). According to the NAS system, ≥ 5 scores were defined as NASH. Based on the NAS score, the results demonstrated that P-HFD and P-HFD + LPS groups induced NASH phenotype at eight weeks, whereas P-HFD + CCl₄ induced NASH and liver fibrosis earlier at four weeks (Fig. 3b and Supplementary Fig. 5a). Both P-HFD and P-HFD + LPS groups were capable of inducing NASH phenotype with NAS scores of up to 6, whereas the P-HFD + CCl₄ group induced NASH with a maximal NAS score of 8. According to histological results of the liver, the more extended NASH induction in the P-HFD group after 12 weeks did not increase the severity of the NASH phenotype. No liver fibrosis was observed in the P-HFD group at any given time point, whereas 14% liver fibrosis with zone 3 perisinusoidal and portal/periportal fibrosis were found in the P-HFD + LPS group at 24 weeks (Fig. 3c and Supplementary Fig. 5b). P-HFD + CCl₄ group exhibited liver fibrosis earlier at four weeks. The induction of liver fibrosis by CCl₄ was only up to bridging fibrosis (stage 3) and did not reach cirrhosis (stage 4). Supplemental injection of LPS in P-HFD-fed mice did not cause differences in steatosis, lobular inflammation, and hepatocyte ballooning score. CCl₄ injection accelerated steatosis and lobular inflammation during the first eight weeks compared to the P-HFD group (Fig. 3d-f and Supplementary Fig. 6). We also observed an increase in the overall pro-inflammatory cytokines, TNF-ɑ, IL-1β, and IL-6, in the P-HFD group compared to the control group. However, there was no significant difference in TNF-ɑ and IL-1β among the P-HFD with or without LPS/CCl₄ intervention groups suggesting these additional interventions did not increase the pro-inflammatory cytokines (Fig. 3g-i). TNF-ɑ, IL-1β, and IL-6 in the P-HFD-fed group reached a maximum level at 12 weeks, whereas IL-1β and IL-6 levels in the LPS/CCl₄ intervention groups reached a maximum level at eight weeks. This study observed an increase in relative spleen and kidney weight at the initial stage of LPS or CCl4 intervention. However, no abnormal findings were observed based on histological analysis (Supplementary Fig. 7–8). Interestingly, the relative cecum weight of the P-HFD group was lower than the control. According to histological analysis, no significant findings were observed in the intestine (Supplementary Fig. 9). Our results confirmed that P-HFD and P-HFD + LPS potentially induced NASH phenotype, whereas the P-HFD + CCl₄ induced liver fibrosis. P-HFD group produced the most elevated pro-inflammatory cytokine at 12 weeks.

P-HFD and P-HFD with LPS or CCl ₄ modified the gut microbiota composition, with the interventions having a greater influence on the gut microbiota than the feeding duration

To understand the impact of diet and intervention against NASH/liver fibrosis pathogenesis and gut microbiota alteration, we performed analysis of the feces microbiota based on V3V4 16S RNA amplicon sequencing, followed by bioinformatic analysis QIIME2 pipeline. The results demonstrated that P-HFD, LPS, and CCl₄ led to changes in ɑ-diversity indices of the gut microbiota, including observed ASV richness, Shannon diversity index, and Simpson diversity index (Fig. 4a-c). P-HFD exhibited a lower overall ASV richness compared to the control diet group, but a higher evenness based on the Shannon index (p < 0.0001 and p < 0.0029, respectively). The Simpson diversity index, a dominance index providing more weight to dominant species, was higher in the P-HFD-fed group (p < 0.0001). There was no significant difference in the three ɑ-diversity indices between the P-HFD and P-HFD + LPS groups, suggesting both groups were remarkably similar. The P-HFD + CCl₄ group had reduced ASV richness compared to the group solely fed with P-HFD. There was no significant difference in the Shannon and Simpson diversity indices among P-HFD + LPS/CCl₄ groups suggesting that diet was the primary factor that influenced the dominant gut microbiome, rather than the effect of LPS or CCl₄.

The PCoA based on the Bray–Curtis distance revealed that P-HFD + LPS/CCl₄ significantly altered the bacterial composition (ANOSIM: R = 0.574, P < 0.001) (Fig. 4d). The type of diet was the primary factor that differentiated the feces microbiota. LPS intraperitoneal injection had less influence on the overall microbiome composition. Interestingly, the CCl₄ injection caused the gut microbiome to shift, and its effect was more pronounced than that of the LPS injection. The duration of treatment also influenced the within-group gut microbiota shift based on PCoA (ANOSIM: R = 0.755, P < 0.001). Gut microbiota shifts based on the impact of the time for each group and the treatment effect of each time point are presented in Fig. 4g and Fig. 4h, respectively. Housing the animal receiving the intervention for four weeks was sufficient to reach the gut microbiota shift. Bray–Curtis dissimilarity index of intra-group analysis was significantly lower than the intergroup analysis (p < 0.0001), and the effect of time was less than the effect of treatment (p < 0.0001), implying the impact of feeding duration was less influential to change the gut microbiota composition compared to the actual treatment provided (P-HFD + LPS/CCl4) (Fig. 4e-f). Thus, we further compared the gut microbiota composition by treatment group without considering the time effect.

P-HFD and P-HFD with LPS or CCl ₄ increased gut dysbiosis by reducing the beneficial bacteria and increasing the pathogenic related microbiome

The statistical analysis of feces microbiota was performed and ranked from phylum to species level with bar plots and heat maps presented in Supplementary Fig. 10–15. At the phylum level, the P-HFD group saw an increase in Deferribacteres, Firmicutes, and Proteobacteria, but a decrease in Actinobacteria, Patescibacteria, and Tenericutes (q < 0.05) (Fig. 5a-g and Supplementary Fig. 10). P-HFD did not affect the overall Firmicutes to Bacteriodetes (F/B) ratio. CCl₄ caused an increase in Actinobacteria, Proteobacteria, and Verrucomicrobia. LPS injection had no effect on the gut microbiota at the phylum level compared to the P-HFD group. Interestingly, the Venn diagram showed 18 genera present in the LPS group (Fig. 5i). These genera lack information on how they influence animal and human health and disease. However, we found that Acetatifactor can be isolated from the intestine of an obese mouse.²⁴ Anaerospora and Rhodococcus are associated with human disease.^{25, 26} A heatmap (Fig. 5j) shows 73 significant differences in feces microbiota for each group at each time point at the genus level based on the Kruskal–Wallis test with FDR-adjusted P-value (q < 0.05). Hierarchical clustering of the genus was divided into two principal clusters, suggesting that the diet was the primary factor that had an impact on the gut microbiota. There were 30 genera significantly enriched in the P-HFD group at the genus level, and 26 genera were decreased compared to the control group. Interestingly, Lactobacillus and Bifidobacterium, which are considered to be part of a beneficial microbiome genus, were reduced in the P-HFD-fed group compared to the control group (Fig. 5k-l). Additionally, pathogenic genus and species, including, Clostridioides difficile, and Enterococcus faecalis were increased in the P-HFD group (Fig. 5m-o). P-HFD with LPS showed a more negligible effect on shaping the gut microbiota. A change in only two genera ([Eubacterium] ventriosum group↑ and Ruminococcaceae NK4A214 group↓) (Supplementary Fig. 14) was observed, suggesting that LPS injection did not have an enormous influence on the gut microbiome compared to the P-HFD group. However, according to the Venn diagram, LPS increased the degree of intestinal microbiota dysbiosis by increasing the occurrence of pathogenic bacteria and reducing beneficial microbiota. Additionally, Escherichia-Shigella tended to be increased in mice treated with LPS (Fig. 5m). In contrast, the CCl₄ intervention exhibited a more substantial effect on the gut microbiota. There were 24 genera increased and 28 genera decreased by CCl₄ injection. Interestingly, we observed that Escherichia-Shigella was increased in the P-HFD + CCl₄ group compared to the P-HFD group. Moreover, pathogenic C. difficile was substantially increased in the CCl₄ intervention group, indicating the CCl₄ increased gut dysbiosis (Fig. 5n and Supplementary Fig. 15e).

P-HFD and P-HFD with LPS or CCl ₄ adjusted the function of gut microbiota resulting in intestinal leakage and endotoxemia

The intestinal permeability test based on FITC-dextran was employed in this study. The result demonstrated that P-HFD was the main factor increasing the overall intestinal permeability (p < 0.0637) (Fig. 6a). Intraperitoneal injection of LPS and CCl₄ did not increase intestinal permeability compared to P-HFD alone. The intestinal leakage that resulted from increased intestinal permeability typically allowed leakage of microbiota molecules, such as LPS, which are constituted in the cell wall of Gram-negative bacteria. Gut microbiota metabolites could be conveyed via the portal vein and lymphatic system to the liver. Based on murine TLR4/NF-κB/SEAP reporter HEK293 cells assay, the overall plasma LPS in the P-HFD group was significantly higher than in the control group (p < 0.0080). LPS levels in the P-HFD group increased from 16 weeks onward, and the increase was approximately 1.5-fold higher than in the control group. The LPS injection induced a plasma LPS level similar to that of the P-HFD group, while CCl₄ decreased the LPS level.

To determine the functional capability of the feces microbiota, we employed PICRUSt2 analysis based on V3V4 16S rDNA amplicon to predict the relative abundance of the KEGG pathway. The predicted pathway based on the KO number was 10364 in total and was grouped into 322 pathways. The PCoA of the relative abundance of the KEGG pathway is shown in Fig. 6c. Similar to the gut microbiota composition, the type of diet affected functional capabilities. Among the four groups, we observed 296 different functions based on Kruskal–Wallis test with FDR-adjusted p-values (q < 0.05) (Fig. 6e and Supplementary Fig. 16). Several lipid metabolism-related pathways of the gut microbiota were increased in the P-HFD group, including biosynthesis of saturated and unsaturated fatty acids, lipid metabolism, and fat digestion and absorption. LPS intervention had a similar effect compared to the P-HFD group. CCl₄ injection also influenced the gut microbiota's lipid metabolism, including linoleic acid and steroid biosynthesis. LPS biosynthesis pathway proteins were elevated in the P-HFD with or without LPS/CCl₄ groups (Fig. 6f-g). The LPS biosynthesis pathway proteins show a positive correlation with plasma LPS (Fig. 6h-i). These results indicate that the functions of the gut microbiota and intestinal barrier strength were modified by diet and intervention. It consequently upregulated LPS biosynthesis resulting in higher levels of LPS, which subsequently translocated to the circulation, resulting in endotoxemia and enhancing the development of NASH and liver fibrosis.

In previous studies, the trans-fat-containing animal model has been widely used to induce obesity and NAFLD as a tool for preclinical drug screening.¹⁵ Due to the US FDA's prohibition of trans-fats in the food supply chain,^16–18 this animal model may not be appropriate for mimicking the cause of NAFLD in humans and animal-human phenotype translatability. In a recent study, the substitute of trans-fats with palm oil in animal feed developed an identical degree of steatosis and NASH comparable to the human NAFLD phenotype. P-HFD is more obesogenic compared to a trans-fat diet, with impaired glucose tolerance promoting a comparable phenotype with steatosis, lobular inflammation, hepatocyte ballooning, and fibrotic liver lesions based on liver biopsy in ob/ob and C57BL/6J mice; however, it promotes only a mild to moderate fibrotic NASH phenotype.¹⁹ Recently, research has focused on the gut microbiota-liver axis. Gut microbiota and its harmful metabolites have been proven to be one critical factor in NAFLD progression. Thus, gut dysbiosis simulation by injection of LPS may provide alternative means to understand how harmful gut metabolites enhance the degree of NASH. Additionally, the effects of the combination of P-HFD and NASH accelerators, such as CCl_4, on the development of NASH/liver fibrosis and gut microbiota shift, have not been explored.

Herein, we demonstrated the obesogenic effect, induction of NASH histological phenotype, and gut microbiota changes in C57BL/6J mice caused by a P-HFD combined with LPS or CCl₄ injection. P-HFD generated an obesogenic, metabolic, and NASH phenotype, including increases in bodyweight, relative fat mass, plasma lipids, AST, ALT, hepatomegaly, relative liver weight, hepatic triacylglycerol content, NAS score, and pro-inflammatory cytokines. These results are consistent with previous reports.^{19, 20} P-HFD induced in mice morphological characteristics comparable to the patient with NASH, including macrosteatosis, lobular inflammation, hepatocyte ballooning degeneration, and periportal/perisinusoidal fibrosis.²⁰ This indicates that the P-HFD, comprising a high amount of palm oil fat, fructose, and cholesterol, is a primary factor in the pathogenesis of NAFLD/NASH. Intake of dietary saturated fats and fructose has been connected to intrahepatic lipid accumulation, lipogenesis, insulin resistance, oxidative stress, and inflammation.^{27, 28} Palm oil has an exceptionally high concentration of saturated fat-palmitic acid (C16:0).²⁹ Palm and hybrid palm oils have been documented to induced liver fat accumulation, hepatocyte ballooning, and lobular inflammation.³⁰ Another study found that substitution of palm oil in a normocaloric and normolipidic diet could negatively affect metabolic parameters and glucose homeostasis, and upregulate cytokine gene expression in the liver and WAT, the effects of which are even worse in mice fed with interesterified palm oil.³¹

LPS injection in P-HFD fed mice retarded the severity of the obesogenic and metabolic phenotype; we observed uncomfortable behavior in the mice after injection of LPS and CCl₄. CCl₄ injection in P-HFD fed mice also reduced the increase in bodyweight and fat mass due to its toxicity. P-HFD and P-HFD + LPS induced a similar level of NASH phenotype based on the maximal NAS score of approximately 5 to 6 at all time points. In contrast, P-HFD + CCl₄ induced mild to moderate liver fibrosis with a maximal NAS score of 7–8. LPS plays a crucial role in fibrosis development and NASH progression by activating toll-like receptor 4 (TLR4) signaling.⁵ In a previous study, C57BL/6J mice were fed a choline-deficient l-amino-acid-defined diet combined with intraperitoneal low-dose LPS injection (0.5 mg/kg) three times a week for 16 weeks. Low-dose LPS enhanced pericellular fibrosis via TLR4/NF-κB signaling activation.³² Our study used the exact dosage of LPS injection but at a frequency of only once per week. Our results revealed that only 14% liver fibrosis occurred with zone 3 perisinusoidal and portal/periportal fibrosis in the P-HFD group with LPS injection at 24 weeks. Although liver fibrosis was observed in the P-HFD with LPS group during the last week of the study, the dosage and frequency of LPS injection may not have been sufficient for accelerating the progression of liver fibrosis. On the other hand, weekly intraperitoneal injection of CCl₄ for four weeks steadily developed liver fibrosis. This study did not examine the hepatic transcriptome analysis. We mainly focused on the changes in gut microbiota composition, as well as gut-derived LPS and intestinal permeability. Liver transcriptome changes in P-HFD fed ob/ob mice for a duration of 16 weeks have previously been reported. P-HFD induces changes to several genes resulting in modifications of fatty acid synthesis (Fasn, Scd1), reduction of fatty acid β-oxidation (Cpt-1), triglyceride synthesis (Gpat4), cholesterol synthesis (Hmgcr, Hmgcs1) and transport (ApoCIII, Ldlr, Lrp1, Scarb1), increase in pro-inflammatory signaling (Nfkb, P38, Tgfbr, Tnfa), inflammasome (Ipaf, Nlrp1b, Nlrp3, Tlr4), and pro-apoptotic activity (Casp-8, Rip-1, Rip-3).¹⁹ Several gene expression pathways are involved in NASH pathogenesis in P-HFD mice, which manifest with comparable dynamics in NASH patients.²⁰

Dietary nutrients are vital for both humans and residing gut microbiota.³ Symbiotic interactions of the gut microbiota affect human health.^{33, 34} Diet is a primary factor that modifies the gut microbiota’s composition. Our data demonstrated that P-HFD was the prime factor resulting in a shift in gut microbiota. Both ɑ- and β-diversity indices indicated the gut microbiota's shift since the first four weeks of P-HFD feeding. The feeding duration was another factor that affected the gut microbiota’s shift; however, it had less influence on the gut microbiota structure compared to the actual treatment. Several studies observed that the gut microbiota respond rapidly to dietary changes.³⁵ Some studies infer that the gut microbiota of older people differ from that of younger adults, indicating that a shift in gut microbiota occurs over time.³⁶ Injection of LPS or CCl₄ resulted in gut microbiota remodeling; however, their effect was lower compared to the diet itself. Interestingly, the CCl₄ resulted in a more severe gut microbiota shift compared to LPS injection, suggesting that exposure to chemical toxins via the non-gastrointestinal route affects the gut microbiota shift. However, the mechanism by which CCl₄ induced the modification of gut microbiota composition remains unknown.

P-HFD changed the core structure of the gut microbiota at the phylum level by increasing the overall relative abundance of Deferribacteres, Firmicutes, and Proteobacteria, while decreasing that of Actinobacteria, Patescibacteria, and Tenericutes. P-HFD did not changed the overall F/B ratio that is frequently used as the hallmark of obesity. A study suggests that it is currently difficult to define the F/B ratio as a hallmark of obesity since it cannot specifically reflect health status.³⁷ The gut microbiome at the phylum level may not exert specific functions on health. Thus, genera and species, even strain level could be more informative to describe its function in health and diseases. The feces microbiome composition in P-HFD mice had an increased relative abundance of Akkermansia, Bacteroides, and Parasutterella, but decreased abundance of Clostridiales, Porphyromonadaceae, and Lactobacillus.¹⁹ Our study exhibited comparable results, verifying the role of P-HFD in modifying gut microbiota composition. In our study, the P-HFD and P-HFD with LPS or CCl₄ amplified the gut dysbiosis by lessening the beneficial bacteria, including Lactobacillus and Bifidobacterium. Diverse Lactobacillus strains have a reported anti-NAFLD function: Lactobacillus casei Shirota prevents fructose-induced liver steatosis through the hepatic TLR4 signaling cascade.³⁸ Supplementation of L. rhamnosus GG in fructose-induced NAFLD mice reduced portal LPS, restored gut barrier function, and attenuated liver inflammation and steatosis.³⁹ L. plantarum protects the liver against NAFLD, improves liver function in HFD feeding rodents, restoring intestinal barrier integrity, and increases intestinal probiotics Akkermansia and Lactococcus.^{40, 41} Bifidobacterium adolescentis and L. rhamnosus relieve NAFLD induced by a high-fat, high-cholesterol diet through modulation of gut microbiota-dependent pathways.⁴² B. lactis V9 attenuates liver steatosis and inflammation in rodents with NAFLD.⁴³ Compared to Bifidobacteria and Lactobacilli in children with NAFLD, Bifidobacteria has been shown to possess a protective function against NAFLD development and obesity.⁴⁴

In our study, the P-HFD and P-HFD with LPS or CCl₄ also enriched the pathogenic related microbiome, including Escherichia-Shigella, E. faecalis, and C. difficile. Facultative pathogens such as Enterobacteriaceae or Escherichia-Shigella are associated with metabolic syndromes such as fatty liver disease and diabetes mellitus.^{45, 46} E. faecalis promotes NAFLD progression in mice.⁴⁷ Additionally, E. faecalis can promote alcoholic liver disease.⁴⁸ NAFLD is a novel risk factor for C. difficile infection. C. difficile infection is the leading cause of healthcare-associated diarrhea, with rising prevalence and death rates.^{49, 50} C. difficile can also cause liver injury through inflammation and hepatocyte damage in mice.⁵¹ In our study, the pathogenic C. difficile was substantially enriched in the CCl₄ intervention group, resulting in gut dysbiosis and infection.

The gut microbiota core structure of the P-HFD and P-HFD + LPS groups were relatively similar according to PCoA and heatmap. However, at the genus level in the P-HFD + LPS group, [Eubacterium] ventriosum group was significantly increased, and Ruminococcaceae NK4A214 was decreased. Eubacterium ventriosum group was enriched in obese individuals⁵² and positively correlated with BMI differences in adult monozygotic twins.⁵³ Ruminococcaceae NK4A214 group is a short-chain fatty acid producer that is enriched in normotensive subjects compared to hypertensive subjects.⁵⁴ Ruminococcaceae NK4A214 group was also discovered to be less abundant in patients with chronic obstructive pulmonary disease.⁵⁵ Ruminococcaceae NK4A214 group was lower in hens with hepatic steatosis.⁵⁶ Additionally, the Venn diagram exposed 18 genera present in the LPS group, including Acetatifactor, Anaerospora, and Rhodococcus, among others. Acetatifactor can be obtained in the intestines of obese mice.²⁴ Anaerospora hongkongensis has been discovered in intravenous drug abusers with pseudobacteremia.²⁶ Rhodococcus equi is one of the most significant pathogens exhibiting pneumonia potential in the equine breeding industry.²⁵ Therefore, the injection of LPS in P-HFD mice did not impact the microbiota core but increased the pathogenic bacteria and lessened the beneficial bacteria.

High-fat or western-style diet is associated with the production of harmful metabolites by gut microbiota, including LPS. It subsequently translocates to the liver via the portal vein and triggers body immune responses.⁵⁷ Gut leakage also increase translocation of gut microbiota metabolites to the body.⁵⁸ NASH patients have higher levels of circulatory LPS, hepatocyte LPS localization, and TLR4 + macrophages compared to patients with simple steatosis or controls.⁵⁹ Gut leakage was observed in the P-HFD fed group. Intraperitoneal injection of LPS or CCl₄ did not increase intestinal permeability, suggesting that exposure to these toxins did not result in gut leakage. The intestinal leakage typically allowed gut microbe biomolecules to translocate through the portal vein. Gut-derived LPS trigger hepatic NF-κB and recruit inflammatory cells by raising TNF-ɑ and IL-1β, consequently stimulating fibrosis.⁵⁷ TLR4/NF-κB/SEAP reporter HEK293 cells results suggested that the plasma LPS in the P-HFD group was increased. The P-HFD group's LPS level initially became elevated from 16 weeks onward, coinciding with the shift in gut microbiota composition that displayed separation at the same time in the P-HFD group (Fig. 4g and Fig. 6b). Previously, a rodent model supplied with LPS through subcutaneous pumps with diethylnitrosamine/CCl₄ injection to induce hepatocarcinoma, was reported.⁶⁰ Our study demonstrated that the LPS group did not exhibit a substantial increase in plasma LPS level compared to the P-HFD fed group, suggesting that additional LPS administration did not induce the gut microbiota changes, and that enhanced LPS production as a result of the diet was the major factor affecting gut microbiota. Moreover, the dosage and frequency of LPS were possibly insufficient to produce endotoxemia and induce the extraordinary severity of NASH. Interestingly, we observed that the CCl₄ group had slightly decreased LPS levels, possibly due to the gut microbiota shift because of P-HFD.

Gut microbiota dysbiosis has been frequently investigated in obesity and type 2 diabetes mellitus, both diseases strongly intertwined with NAFLD.⁶¹ To understand the gut microbiota function, we predicted the functional composition of the gut microbiome based on a computational approach (PICRUSt2).^{62, 63} We demonstrated that P-HFD modified the gut microbiota function, including lipid metabolism and LPS-related pathways. P-HFD has changed several fat-related metabolic processes, including biosynthesis of saturated and unsaturated fatty acids, lipid metabolism, and fat digestion and absorption. A previous study also found that the gut microbiome functionals of NASH patients are enriched in carbohydrate, lipid, and amino acid metabolism, and secondary metabolism. The lipid metabolism function, including fatty acid and lipid biosynthesis, is also enriched in patients with more severe fibrosis compared to patients with/without mild fibrosis.⁶⁴ The upregulation of LPS-related pathways of the gut microbiome could reflect gut microbiome community shifting and how it utilizes food nutrients to produce LPS. The LPS biosynthesis pathway of the gut microbiota increases in NASH patients.⁶⁵ Our study demonstrated that the LPS biosynthesis pathway positively correlated with plasma LPS, implying LPS was highly produced in the gut, after which it translocated to the circulation, causing endotoxemia.

In summary, this study demonstrated the effect of P-HFD, LPS, and CCl₄ on NASH and liver fibrosis development, proving that P-HFD administration for eight weeks sufficiently induced the NASH phenotype in mice. However, we suggested continuing administration of the diet for 12 weeks since it could be more effective for inducing NASH, as evident by the pro-inflammatory cytokines and hepatic triglycerides reaching their maximum level at this time point. According to gut microbiota data, four weeks of P-HFD with or without LPS/CCl₄ resulted in an alteration of the gut microbiome and dysbiosis by reducing beneficial bacteria and increasing pathogenic bacteria. The elevation of plasma LPS was observed at 16 weeks. Nevertheless, this study provided scientific evidence for induction of NASH and liver fibrosis. There is no perfect experimental animal model in the current literature that imitates gut dysbiosis and accelerates NASH and liver fibrosis. This study developed the NASH and fibrosis mouse model by using a non-trans-fat P-HFD with LPS intraperitoneal injection simulating gut dysbiosis and endotoxemia, as well as reporting the changes in gut microbiota composition, intestinal permeability, and gut-derived LPS, including its function. Additionally, we demonstrated the effect of CCl₄ on the gut-liver axis (Fig. 7). The novel P-HFD with or without LPS/CCl₄ injection mouse model could be a valuable preclinical model for drug screening purposes and drug therapies for NASH and liver fibrosis.

Animal model

The use of animals was in accordance with the guidelines established by the Institutional Animal Care and Use Committee of National Taiwan University (IACUC) (Approval No: NTU-110-EL-00060). Six-week-old male C57BL/6J mice were purchased from Taiwan National Laboratory Animal Center and housed in an alternating light/dark cycle (12 h) room with a temperature of 23 ± 2°C and relative humidity of 50–70%.^{66, 67} After two weeks of adaptive feeding, the mice (n = 192) were randomly assigned to four groups: (1) control diet (Research Diets, Inc., NJ, USA; D12450K) + intraperitoneal injection of phosphate buffer saline (PBS); (2) Palm oil-containing high-fat diet (P-HFD; Gubra Amylin NASH (GAN) diet; Research Diets, Inc., NJ, USA; D09100310) + intraperitoneal injection of PBS; (3) P-HFD + intraperitoneal injection of LPS (500 µg/kg bw/week; LPS from Escherichia coli O55:B5; Sigma-Aldrich; L2880); and (4) P-HFD + intraperitoneal injection of CCl₄ (320 µg/kg bw/week; Merck Millipore; code 1.02209.1000). Control diet comprises 10 kcal% fat and carbohydrates, mainly in the form of corn starch. P-HFD comprises 40 kcal% fat (mainly palm oil), 20% fructose, and 2% cholesterol.^{19, 20} Bodyweight and food intake were measured weekly during the experiment. Mice (n = 7–8) were sacrificed by carbon dioxide asphyxiation at 4, 8, 12, 16, 20, and 24 weeks. Blood was collected after carbon dioxide asphyxiation by a heparin-coated syringe and then transferred to a collection tube. The mice were dissected to extract specimens. The liver, spleen, kidney, cecum, white adipose tissue (WAT) including inguinal WAT (iWAT), epididymal WAT (eWAT), mesenteric WAT (mWAT), perirenal WAT (pWAT), inguinal subcutaneous WAT (isWAT), and interscapular brown adipose tissue (iBAT) were harvested, imaged, and weighed after sacrificing the mice. The colon feces content was collected for DNA sequencing.

Liver histopathological analysis

Histopathological analyses were performed using the formalin-fixed paraffin-embedded method. The largest liver lobe was fixed with a 10% neutral-buffered formalin solution and then embedded in paraffin, serially sectioned at 5 µm intervals, and stained with hematoxylin and eosin for observing and interpreting fibrosis, steatosis, lobular inflammation, and hepatocyte ballooning. Hepatic histological scoring was performed according to the NAFLD activity score (NAS) and fibrosis staging⁶⁸ by an experienced pathologist from the College of Veterinary Medicine Animal Disease Diagnostic Center (National Chung Hsing University, Taichung, Taiwan). Scores of 5–8 were considered diagnostic for NASH.

Plasma biochemical analyses

Plasma was extracted by centrifuging the blood at 1000 g for 15 min at 4°C. The biochemical parameters of the plasma, including the levels of total cholesterol, total triglycerides, (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), were analyzed using Beckman Coulter AU 5800 (Beckman Coulter Inc., USA).

Liver triacylglycerol, pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 analyses

A liver homogenate was prepared by homogenizing 0.1 g of liver tissue with 1 mL of cold buffer (8 mM KH₂PO₄, 12 mM K₂HPO₄, and 1.5% KCl, pH 7.4) at 4°C, followed by centrifugation at 10,000 g for 30 min at 4°C. The supernatant was obtained and stored at -80°C.⁶⁹ The liver triacylglycerol content was analyzed using kits obtained from Cayman Chemical Co. (Ann Arbor, MI). The hepatic pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were analyzed using a commercial enzyme-linked immunosorbent assay kit (Invitrogen, USA).

Fecal DNA extraction, 16S rRNA amplicon sequencing, sequencing analysis, metagenome prediction, and functional profiling

Mouse fecal samples were obtained from the colon when mice were killed. The samples were snap-frozen using liquid nitrogen and stored at -80°C before use. Fecal genomic DNA was extracted using the QIAmp Power Fecal Pro-DNA Kit (QIAGEN, Netherlands) and quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific).

The V3-V4 hypervariable region of the 16S rRNA gene was amplified using the primer pair ((Forward = 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3′) and Reverse = 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′)). Polymerase chain reaction (PCR) amplification was conducted in a 25 µL reaction mixture containing 5 ng of DNA template, 0.2 µM forward and reverse primers, and 12.5 µL of 2×Taq Master Mix (KAPA HiFi HotStart ReadyMix, Roche, Switzerland). The PCR conditions included an initial step at 95°C for 3 min, followed by 25 cycles of 95°C, 55°C, and 72°C for 30 s each, and a final extension at 72°C for 5 min. Amplified products were subsequently visualized by using 2% agarose gel electrophoresis. Dual index and Illumina sequencing adapters were joined using a Nextera XT Index Kit via PCR. PCR product cleanup was conducted using AMPure XP beads to purify amplicon. The sizes of PCR products were validated using the Bioanalyzer DNA 1000 chip. Library quantification was carried out for quality control before sequencing using the Agilent Technologies 2100 Bioanalyzer. The pooled libraries were subjected to paired-end sequencing (2×300 bps) using the Illumina MiSeq platform.

The raw sequences were processed according to QIIME2 pipeline. Demultiplexed sequences from each sample were quality filtered and trimmed, de-noised, merged, and then the chimeric sequences were identified and removed using the QIIME2 dada2 plugin. The QIIME2 feature-classifier plugin was subsequently used to align amplicon sequence variant (ASV) sequences against the SILVA database (version 132).⁷⁰ For metagenome prediction and functional profiling, the 16S rRNA sequencing dataset of the SILVA reference sequence collection was used to determine the functional profiles of the microbial communities using the PICRUSt2 plugin to obtain predictions of the KEGG orthology (KO) pathway.⁶²

The ASV and predicted function table subsequently performed the statistical analysis and visualization using R software. The vegan package in R software was used to calculate the ɑ-diversity, including the observed ASVs, Shannon index, and Simpson index. Principal coordinate analysis (PCoA) was performed using the Bray–Curtis distance. Analysis of similarities (ANOSIM) was calculated to determine the heterogeneity of the feces microbiota among the groups. Heatmap was plotted using the heatmap3 packages in R software.

Plasma lipopolysaccharide quantification

Plasma LPS was quantified by using a cell-based colorimetric assay for the detection of biologically active endotoxin. Plasma samples were added (10 µL) to murine TLR4/NF-κB/SEAP reporter HEK293 cells for 24 h. After incubation, the medium was collected and the HEK-Blue™ LPS Detection Kit (InvivoGen, USA) was used to detect TLR4, and NF-kB-inducible secreted embryonic alkaline phosphatase (SEAP) was measured by a spectrophotometer at 620 nm.⁷¹

Intestinal permeability analysis

Fluorescein isothiocyanate–dextran (FITC-dextran) with an average molecular weight of 4,000 (Sigma-Aldrich, 46944) was used as a permeability probe. A solution of 15 mg of FITC-dextran in 0.2 mL of PBS was gavaged to mice 3 h before killing them. Plasma FITC-dextran measurements were performed using a fluorometer at 485 nm of excitation and 538 nm of emission (Fluoroskan Ascent FL, Thermo Fisher Scientific, USA) in black 96-well plates.⁶⁷

Statistical Analysis

Data are represented as mean ± standard deviation (SD). An unpaired two-tailed Student’s t-test or one-way ANOVA with Tukey’s range test was used to compare group means. The Wilcoxon signed-rank test, Kruskal–Wallis test, Dunn's multiple comparison test with false discovery rate (FDR), unpaired two-tailed Student’s t-test, and one-way ANOVA with Tukey’s range test were used for the analysis of the fecal microbiome data set. All statistics were analyzed using Graphpad Prism (version 9.1.2), R (version 3.6.1), or R Studio (version 1.2.5001).

Data availability

The raw 16s rRNA sequencing data used to produce all figures are accessible at the NCBI Short Read Archive under the following accession numbers: BioProject: PRJNA841777 and BioSample: SAMN28616626, and SRA: SRR19372785-967.

Code availability

Bioinformatic tools, software version, parameters, and open-source code used in this present study are described in the "Methods" section. More details regarding the code to reproduce the analyses are available upon request.

Acknowledgements

This study was financed by the Ministry of Science and Technology (Taiwan) (109-2327-B-002-005 and 110-2327-B-002-007).

Competing interests

The authors declare that there are no competing interests.

Authors contributions

S.P. designed, instructed, and assisted with the experiment, performed the bioinformatics and statistical analysis, and drafted the manuscript; W.K.W. instructed the experiments and reviewed the manuscript; Y.C.H., Y.J.T., and S.Y.P. performed the animal experiments; R.A.C. and H.S.H. assisted with performing the animal experiments; Y.H.C. assisted with animal and LPS quantification experiments; T.C.D.S., C.T.H., C.J.L., H.L.C., and C.C.H. critically reviewed the manuscript; M.S.W. and L.Y.S. designed the experiments, provided the funding for the study, and critically revised the manuscript.

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Establishment of animal models for NASH and utilization for mining associated gut microbiota

Status:

Version 1

Abstract

Figures

Introduction

Results

P-HFD induced hepatomegaly and increase hepatic triacylglycerol levels, but LPS intervention slowed this progress

P-HFD and P-HFD + LPS induced NASH phenotype, whereas P-HFD + CCl₄ induced liver fibrosis

Discussion

Methods

Animal model

Liver histopathological analysis

Plasma biochemical analyses

Liver triacylglycerol, pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 analyses

Fecal DNA extraction, 16S rRNA amplicon sequencing, sequencing analysis, metagenome prediction, and functional profiling

Plasma lipopolysaccharide quantification

Intestinal permeability analysis

Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Establishment of animal models for NASH and utilization for mining associated gut microbiota

Status:

Version 1

Abstract

Figures

Introduction

Results

P-HFD induced hepatomegaly and increase hepatic triacylglycerol levels, but LPS intervention slowed this progress

P-HFD and P-HFD + LPS induced NASH phenotype, whereas P-HFD + CCl4 induced liver fibrosis

Discussion

Methods

Animal model

Liver histopathological analysis

Plasma biochemical analyses

Liver triacylglycerol, pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 analyses

Fecal DNA extraction, 16S rRNA amplicon sequencing, sequencing analysis, metagenome prediction, and functional profiling

Plasma lipopolysaccharide quantification

Intestinal permeability analysis

Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

P-HFD and P-HFD + LPS induced NASH phenotype, whereas P-HFD + CCl₄ induced liver fibrosis