Onset Of Non-alcoholic Steatohepatitis Research Articles

Potent Efficacy of 3-Amino-4-hydroxy Benzoic Acid, a Small Molecule Having Anti-fibrotic Activity, in a Mouse Model of Non-alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH), which is on the rise due to the increasing obese population and changing lifestyles, causes fibrosis over time and carries the risk of progression to cirrhosis and hepatocellular carcinoma. However, there are no approved effective treatments for NASH. Recent studies suggest that increased lipid metabolism and reduced nitric oxide content are responsible for NASH; 3-amino-4-hydroxy benzoic acid (AHBA) was identified as an inhibitor for the phosphatase activity of soluble epoxy hydrolase, which in turn inhibits lipid metabolism and endothelial nitric oxide synthase activity. The aim of this study was to assess the efficacy of AHBA in a mouse model of NASH. NASH was induced in mice by streptozotocin administration and a high-fat diet loading. The efficacy of AHBA was determined by measuring liver function using serum and liver samples and conducting a morphological assessment. AHBA considerably attenuated the increase in the liver weight and alkaline phosphatase content, which occurred due to the progression of NASH. Hepatocellular steatosis, inflammatory cell infiltration, and hepatocellular ballooning of hepatocytes remained unaltered. In contrast, AHBA treatment significantly ameliorated the fibrotic alterations within liver tissue that were induced by the onset of NASH. These results demonstrate the potential of AHBA as a therapeutic pharmaceutical compound that can treat NASH.

Bone marrow monocytes sustain NK cell-poiesis during non-alcoholic steatohepatitis

Natural killer (NK) cells are the predominant lymphocyte population in the liver. At the onset of non-alcoholic steatohepatitis (NASH), an accumulation of activated NK cells is observed in the liver in parallel with inflammatory monocyte recruitment and an increased systemic inflammation. Using invivo and invitro experiments, we unveil a specific stimulation of NK cell-poiesis during NASH by medullary monocytes that trans-present interleukin-15 (IL-15) and secrete osteopontin, a biomarker for patients with NASH. This cellular dialogue leads to increased survival and maturation of NK precursors that are recruited to the liver, where they dampen the inflammatory monocyte infiltration. The increase in the production of both osteopontin and the IL-15/IL-15Rα complex by bone marrow monocytes is induced by endotoxemia. We propose a tripartite gut-liver-bone marrow axis regulating the immune population dynamics and effector functions during liver inflammation.

UDP-glucuronate metabolism controls RIPK1-driven liver damage in nonalcoholic steatohepatitis

Hepatocyte apoptosis plays an essential role in the progression of nonalcoholic steatohepatitis (NASH). However, the molecular mechanisms underlying hepatocyte apoptosis remain unclear. Here, we identify UDP-glucose 6-dehydrogenase (UGDH) as a suppressor of NASH-associated liver damage by inhibiting RIPK1 kinase-dependent hepatocyte apoptosis. UGDH is progressively reduced in proportion to NASH severity. UGDH absence from hepatocytes hastens the development of liver damage in male mice with NASH, which is suppressed by RIPK1 kinase-dead knockin mutation. Mechanistically, UGDH suppresses RIPK1 by converting UDP-glucose to UDP-glucuronate, the latter directly binds to the kinase domain of RIPK1 and inhibits its activation. Recovering UDP-glucuronate levels, even after the onset of NASH, improved liver damage. Our findings reveal a role for UGDH and UDP-glucuronate in NASH pathogenesis and uncover a mechanism by which UDP-glucuronate controls hepatocyte apoptosis by targeting RIPK1 kinase, and suggest UDP-glucuronate metabolism as a feasible target for more specific treatment of NASH-associated liver damage.

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.

Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.

Effect of daikenchuto (TU-100) on carcinogenesis in non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is associated with a higher risk of hepatocellular carcinoma (HCC), and the importance of the gut?liver axis has been recognized in NASH-associated HCC. We investigated the effect of TU-100 on the intestinal microbiome and hepatocarcinogenesis in a NASH model.Seven-week-old Tsumura Suzuki obese diabetes mice, a model that shows the spontaneous onset of NASH and HCC, were used. They were divided into a TU-100 treated group and a control group. Mice were sacrificed at 24 and 48 weeks to evaluate hepatic steatosis, fibrosis, carcinogenesis, cytokine expression, and microbiome abundance.At 24 weeks, the TU-100 group showed significantly lower expression of IL6, IL1B, and ACTA2 mRNA in the liver (P?<?0.05). At 48 weeks, the TU-100 group showed significantly lower levels of serum alanine aminotransferase. The TU-100 group also showed a lower rate of NASH than the control group (28% vs 72%?;?P?=?0.1). Tumor diameter was significantly smaller in the TU-100 group compared with that in the control group (P?<?0.05). Regarding the intestinal microbiome, the genera Blautia and Ruminococcus were increased in the TU-100 group (P?<?0.05), whereas Dorea and Erysipelotrichaceae were decreased in the TU-100 group (P?<?0.05).TU-100 regulates the intestinal microbiome and may suppress subsequent hepatocarcinogenesis in the NASH model. J. Med. Invest. 70 : 66-73, February, 2023.

Corydalis saxicola Bunting Total Alkaloids ameliorate diet-induced non-alcoholic steatohepatitis by regulating hepatic PI3K/Akt and TLR4/NF-κB pathways in mice

Corydalis saxicola Bunting (Yanhuanglian), distributed in Southwest China, is mainly used for treatment of hepatitis, oral mucosal erosion, conjunctivitis, dysentery, acute abdominal pain and hemorrhoids in the folk. Corydalis saxicola Bunting Total Alkaloids (CSBTA) are the active ingredients extracted from the root of C. saxicola bunting. Non-alcoholic steatohepatitis (NASH) is the hinge between steatosis and cirrhosis in the spectrum of Non-alcoholic fatty liver disease (NAFLD), which has become one of the most common chronic liver diseases in the world. CSBTA can reduce tumors and brain diseases through anti-inflammatory and antioxidant pathways. Our study was designed to clarify the effects of CSBTA on the HFHC (High fat and high carbohydrate drinking) diet induced mice. In our research, A HFHC diet induced NASH mice model was applied to investigate the effects of CSBTA in vivo and obeticholic acid (OA) was set as positive control. Moreover, the underlying mechanisms were explored by palmitic acid (PA) and lipopolysaccharide (LPS) stimulated HepG2 cells in vitro. The in vivo study illustrated that CSBTA could alleviate mice away from the onset of NASH, and reduce intrahepatocellular lipid accumulation and hepatocyte inflammation under high fat condition. Further in vitro analysis confirmed that CSBTA attenuated inflammation and hepatic lipid accumulation by improving hepatic PI3K/Akt and suppressing hepatic TLR4/NF-κB pathways. In summary, this study demonstrated that CSBTA might be a promising compound for the treatment of NAFLD.

Identification cholesterol metabolites altered before the onset of nonalcoholic steatohepatitis by targeted metabolomics

Nonalcoholic steatohepatitis (NASH) is a disease with symptoms similar to those of alcoholic liver inflammation without alcohol intake. As an effective treatment strategy has not been established for this disease, a detailed understanding of the pathological progression mechanism is required. We focused on cholesterol metabolites, which are suspected to regulate NASH pathology, and investigated their relationship with the pathological progression in the early stages of NASH.First, the LC/MS/MS methods for bile acids and sterols were optimized and validated. Next, NASH model mice were established by feeding a choline-deficient, methionine-reduced high-fat diet, and the levels of hepatic cholesterol metabolites were measured. As a result, before the onset of NASH, desmosterol, 4β-hydroxycholesterol, campesterol, sitosterol, secondary bile acids such as taurodeoxycholic acid significantly decreased by up to 1/38 of NASH model group. Autoxidation-generated sterols significantly increased 2- to 5-fold, and various primary bile acids such as conjugated β-muricholic acids and cholic acids significantly increased 2- to 7-fold.In this study, the levels of cholesterol metabolites changed in the before the onset of NASH. These metabolic alterations involved in inflammation induction and detoxification for NASH may help the discovery of early diagnostic biomarkers in the future.

Caspase-Cleaved Keratin 18 Measurements Identified Ongoing Liver Injury after Bariatric Surgery.

Bariatric surgery has emerged as an effective treatment option in morbidly obese patients with non-alcoholic fatty liver disease (NAFLD). However, worsening or new onset of non-alcoholic steatohepatitis (NASH) and fibrosis have been observed. Caspase-cleaved keratin 18 (ccK18) has been established as a marker of hepatocyte apoptosis, a key event in NASH development. Thus, ccK18 measurements might be feasible to monitor bariatric surgery patients. Clinical data and laboratory parameters were collected from 39 patients undergoing laparoscopic Roux-en-Y gastric bypass at six timepoints, prior to surgery until one year after the procedure. ccK18 levels were measured and a high-throughput analysis of serum adipokines and cytokines was carried out. Half of the cohort’s patients (20/39) presented with ccK18 levels indicative of progressed liver disease. 21% had a NAFLD-fibrosis score greater than 0.676, suggesting significant fibrosis. One year after surgery, a mean weight loss of 36.87% was achieved. Six and twelve months after surgery, ccK18 fragments were significantly reduced compared to preoperative levels (p < 0.001). Yet nine patients did not show a decline in ccK18 levels ≥ 10% within one year postoperatively, which was considered a response to treatment. While no significant differences in laboratory parameters or ccK18 could be observed, they presented with a greater expression of leptin and fibrinogen before surgery. Consecutive ccK18 measurements monitored the resolution of NAFLD and identified non-responders to bariatric surgery with ongoing liver injury. Further studies are needed to elicit the pathological mechanisms in non-responders and study the potential of adipokines as prognostic markers.

Extracellular Vesicles as Inflammatory Drivers in NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent chronic liver disease in most parts of the world affecting one-third of the western population and a growing cause for end-stage liver diseases such as hepatocellular carcinoma (HCC). Majorly driven by obesity and diabetes mellitus, NAFLD is more of a multifactorial disease affected by extra-hepatic organ crosstalk. Non-alcoholic fatty liver (NAFL) progressed to non-alcoholic steatohepatitis (NASH) predisposes multiple complications such as fibrosis, cirrhosis, and HCC. Although the complete pathogenic mechanisms of this disease are not understood, inflammation is considered as a key driver to the onset of NASH. Lipotoxicity, inflammatory cytokines, chemokines, and intestinal dysbiosis trigger both hepatic and systemic inflammatory cascades simultaneously activating immune responses. Over a few years, extracellular vesicles studied extensively concerning the pathobiology of NAFLD indicated it as a key modulator in the setting of immune-mediated inflammation. Exosomes and microvesicles, the two main types of extracellular vesicles are secreted by an array of most mammalian cells, which are involved mainly in cell-cell communication that are unique to cell type. Various bioactive cargoes containing extracellular vesicles derived from both hepatic and extrahepatic milieu showed critical implications in driving steatosis to NASH reaffirming inflammation as the primary contributor to the whole process. In this mini-review, we provide brief insights into the inflammatory mediators of NASH with special emphasis on extracellular vesicles that acts as drivers of inflammation in NAFLD.

Human hepatic in vitro models reveal distinct anti-NASH potencies of PPAR agonists.

Non-alcoholic steatohepatitis (NASH) is a highly prevalent, chronic liver disease characterized by hepatic lipid accumulation, inflammation, and concomitant fibrosis. Up to date, no anti-NASH drugs have been approved. In this study, we reproduced key NASH characteristics in vitro by exposing primary human hepatocytes (PHH), human skin stem cell-derived hepatic cells (hSKP-HPC), HepaRG and HepG2 cell lines, as well as LX-2 cells to multiple factors that play a role in the onset of NASH. The obtained in vitro disease models showed intracellular lipid accumulation, secretion of inflammatory chemokines, induced ATP content, apoptosis, and increased pro-fibrotic gene expression. These cell systems were then used to evaluate the anti-NASH properties of eight peroxisome proliferator-activated receptor (PPAR) agonists (bezafibrate, elafibranor, fenofibrate, lanifibranor, pemafibrate, pioglitazone, rosiglitazone, and saroglitazar). PPAR agonists differently attenuated lipid accumulation, inflammatory chemokine secretion, and pro-fibrotic gene expression.Based on the obtained readouts, a scoring system was developed to grade the anti-NASH potencies. The in vitro scoring system, based on a battery of the most performant models, namely PHH, hSKP-HPC, and LX-2 cultures, showed that elafibranor, followed by saroglitazar and pioglitazone, induced the strongest anti-NASH effects. These data corroborate available clinical data and show the relevance of these in vitro models for the preclinical investigation of anti-NASH compounds.

Hepatocyte Injury and Hepatic Stem Cell Niche in the Progression of Non-Alcoholic Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by lipid accumulation in hepatocytes in the absence of excessive alcohol consumption. The global prevalence of NAFLD is constantly increasing. NAFLD is a disease spectrum comprising distinct stages with different prognoses. Non-alcoholic steatohepatitis (NASH) is a progressive condition, characterized by liver inflammation and hepatocyte ballooning, with or without fibrosis. The natural history of NAFLD is negatively influenced by NASH onset and by the progression towards advanced fibrosis. Pathogenetic mechanisms and cellular interactions leading to NASH and fibrosis involve hepatocytes, liver macrophages, myofibroblast cell subpopulations, and the resident progenitor cell niche. These cells are implied in the regenerative trajectories following liver injury, and impairment or perturbation of these mechanisms could lead to NASH and fibrosis. Recent evidence underlines the contribution of extra-hepatic organs/tissues (e.g., gut, adipose tissue) in influencing NASH development by interacting with hepatic cells through various molecular pathways. The present review aims to summarize the role of hepatic parenchymal and non-parenchymal cells, their mutual influence, and the possible interactions with extra-hepatic tissues and organs in the pathogenesis of NAFLD.

Novel Chemical Scaffolds to Inhibit the Neutral Amino Acid Transporter B0AT1 (SLC6A19), a Potential Target to Treat Metabolic Diseases

Lack of B0AT1 (SLC6A19) partially protects mice against the onset of non-alcoholic steatohepatitis (NASH). To achieve a similar outcome through pharmacological treatment, we improved previously identified inhibitors of B0AT1 by medicinal chemistry and identified second generation inhibitors by high through-put screening. Modified diarylmethine compounds inhibited B0AT1 with IC50 values ranging from 8–90 μM. A second generation of inhibitors was derived from high-throughput screening and showed higher affinity (IC50 of 1–15 μM) and strong selectivity against amino acid transporters with similar substrate specificity, such as ASCT2 (SLC1A5) and LAT1 (SLC7A5). All compounds were unrelated to B0AT1 substrates, but were likely to bind in the vicinity of the substrate binding site.

Plasma metabolic alterations in patients with severe obesity and non-alcoholic steatohepatitis.

Obesity can influence hepatic mitochondrial function, and cause non-alcoholic steatohepatitis (NASH). Diagnosis and follow-up rely on invasive liver biopsy so blood-based markers are urgently required. To investigate whether values of circulating metabolites from energy and one-carbon (1-C) metabolism may: (a) reflect hepatic mitochondrial flexibility failure and (b) act as NASH biomarkers. Patients with severe obesity undergoing bariatric surgery (n=270) were investigated using quantitative targeted plasma metabolomics. Comparisons were with non-obese controls without liver disease (n=50). Obese patients with NASH (n=53) and without NASH (n=130) representing extreme groups of liver disease were assessed to test the diagnostic ability of the measured circulating metabolites. Paired liver biopsy and plasma samples from NASH patients were available 1year post-surgery and were evaluated to monitor metabolomic changes with liver damage resolution. We identified correlations between human liver metabolism and obesity. High-plasma α-ketoglutarate (α-KG) and lactate concentrations in NASH patients indicating citric acid cycle replenishment via glutaminolysis might also be a crucial point in NASH onset. Plasma measurements of α-KG, β-hydroxybutyrate, pyruvate and oxaloacetate reduced the uncertainty in clinical diagnosis of NASH [area under receiver operating characteristic curve (AUC) of 0.826] and predicted NASH resolution without ambiguity (AUC of 0.999). Changes in plasma mitochondrial metabolites appear to be associated with NASH. These metabolic responses may be dynamically remodelled following resolution of liver damage through massive weight loss.

Agaricus brasiliensis KA21 May Prevent Diet-Induced Nash Through Its Antioxidant, Anti-Inflammatory, and Anti-Fibrotic Activities in the Liver.

Non-alcoholic steatohepatitis (NASH) is a progressive disease that occurs in the liver. As the number of people with NASH has increased, effective prevention and treatment strategies are needed. Agaricus brasiliensis KA21 (AGA) is a mushroom native to Brazil and is considered a healthy food because of its purported health benefits, including its antioxidant properties. In this study, we focused on the oxidative stress that accompanies the onset of NASH and examined whether AGA can prevent NASH development through its antioxidant activity. We used a mouse model of NASH in which pathogenesis was promoted by dietary induction. Supplementation with AGA attenuated the development of hepatic fibrosis, which is a characteristic feature of late-stage NASH. This effect appeared to be mechanistically linked to an AGA-promoted reduction in hepatic oxidative stress. These results demonstrate a novel role for AGA in NASH prevention.

Melon GliSODin® Prevents Diet-Induced NASH Onset by Reducing Fat Synthesis and Improving Liver Function.

A high-calorie diet causes fat accumulation and oxidative stress in the liver, leading to fatty liver and eventually non-alcoholic steatohepatitis (NASH). Melon GliSODin® is used as a nutritional supplement because of its antioxidant activity. This study aimed to assess the antioxidant activity of Melon GliSODin® and its effectiveness in preventing NASH, which primarily results from oxidative stress. Furthermore, we verified the protective effect of Melon GliSODin® by administering it to a mouse model of diet-induced NASH. Melon GliSODin® suppressed liver fibrosis and fat accumulation, which is characteristic of the NASH phenotype. Gene expression analysis confirmed the suppression of fat synthesis and activation of antioxidative mechanisms. These results show that Melon GliSODin® mitigates NASH onset at the molecular level, suggesting its potential application as a NASH preventive agent.

Dietary Glycotoxins Impair Hepatic Lipidemic Profile in Diet-Induced Obese Rats Causing Hepatic Oxidative Stress and Insulin Resistance.

Nonalcoholic fatty liver disease (NAFLD) is caused by excessive liver lipid accumulation, but insulin resistance is specifically associated with impaired lipid saturation, oxidation, and storage (esterification), besides increased de novo lipogenesis. We hypothesized that dietary glycotoxins could impair hepatic lipid metabolism in obesity contributing to lipotoxicity-driven insulin resistance and thus to the onset of nonalcoholic steatohepatitis (NASH). In diet-induced obese rats with methylglyoxal-induced glycation, magnetic resonance spectroscopy, mass spectrometry, and gas chromatography were used to assess liver composition in fatty acyl chains and phospholipids. High-fat diet-induced obesity increased liver lipid fraction and suppressed de novo lipogenesis but did not change fatty acid esterification and saturation or insulin sensitivity. Despite a similar increase in total lipid fraction when supplementing the high-fat diet with dietary glycotoxins, impairment in the suppression of de novo lipogenesis and decreased fatty acid unsaturation and esterification were observed. Moreover, glycotoxins also decreased polyunsaturated cardiolipins and caused oxidative stress, portal inflammation, and insulin resistance in high-fat diet-induced obese rats. Dietary glycated products do not change total lipid levels in the liver of obese rats but dramatically modify the lipidemic profile, leading to oxidative stress, hepatic lipotoxicity, and insulin resistance in obesity and thus contribute to the onset of NASH.

BET Inhibition Improves NASH and Liver Fibrosis

Non-alcoholic fatty liver disease (NAFLD) is a leading form of chronic liver disease with large unmet need. Non-alcoholic steatohepatitis (NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. To identify potential new therapeutics for NASH, we used a computational approach based on Connectivity Map (CMAP) analysis, which pointed us to bromodomain and extra-terminal motif (BET) inhibitors for treating NASH. To experimentally validate this hypothesis, we tested a small-molecule inhibitor of the BET family of proteins, GSK1210151A (I-BET151), in the STAM mouse NASH model at two different dosing timepoints (onset of NASH and progression to fibrosis). I-BET151 decreased the non-alcoholic fatty liver disease activity score (NAS), a clinical endpoint for assessing the severity of NASH, as well as progression of liver fibrosis and interferon-γ expression. Transcriptional characterization of these mice through RNA-sequencing was consistent with predictions from the CMAP analysis of a human NASH signature and pointed to alterations in molecular mechanisms related to interferon signaling and cholesterol biosynthesis, as well as reversal of gene expression patterns linked to fibrotic markers. Altogether, these results suggest that inhibition of BET proteins may present a novel therapeutic opportunity in the treatment of NASH and liver fibrosis.

Histological effects and pharmacokinetics of lipopolysaccharide derived from Porphyromonas gingivalis on rat maxilla and liver concerning with progression into non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is one of the chronic liver diseases that can develop into hepatocirrhosis. The purpose of the present study was to investigate the impact of lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis) on NASH onset, and to determine the biodistribution of double-radiolabeled LPS (R-LPS) biosynthesized in P. gingivalis. Rats fed a basal diet (BD) or a high-fat diet (HD) were injected with P. gingivalis-LPS or R-LPS into the palatine gingiva around the right maxillary first molar, and were classified into the following 4 groups: BD/LPS (-), BD/LPS (+), HD/LPS (-), and HD/LPS (+) or 2 groups: BD/R-LPS and HD/R-LPS. Inflammation in the gingiva of the LPS (+) groups progressed significantly more than the LPS (-) groups. Furthermore, in the HD/LPS (+) liver, histologic analysis confirmed the presence of NASH, characterized by large fat droplets, ballooning degeneration, and infiltration of inflammatory cells. When 3 H, 14 C-R-LPS was injected into the palatine gingiva, radioactivity in the right palatal mucosa of HD/R-LPS rats was the highest in comparison with other regions and was significantly elevated after 24hours compared to BD/R-LPS rats. Autoradiographic analysis of the maxilla showed distributions from the palatal mucosa to the hard palate and the interdental region. Radioactivity in organs of both BD/R-LPS and HD/R-LPS rats were mostly localized to the liver even after 24hours. The present study suggests that the transfer of P. gingivalis-LPS from the oral cavity to the liver plays an important role in disease exacerbation of NASH.

Pathophysiological characteristics of non-alcoholic steatohepatitis-like changes in cholesterol-loaded type 2 diabetic rats.

Spontaneously Diabetic Torii (SDT) fatty rats, a new obese diabetic model, reportedly presented with features of non-alcoholic steatohepatitis (NASH) after 32 weeks of age. We tried to accelerate the onset of NASH in SDT fatty rats using dietary cholesterol loading and noticed changes in the blood choline level which is expected to be a NASH biomarker. Body weight and biochemical parameters were measured from 8 to 24 weeks of age. At 16, 20, 24 weeks, pathophysiological analysis of the livers were performed. Hepatic lipids, lipid peroxides, and the expression of mRNA related to triglyceride (TG) synthesis, inflammation, and fibrosis were evaluated at 24 weeks. Hepatic fibrosis was observed in SDT fatty rats fed cholesterol-enriched diets (SDT fatty-Cho) from 16 weeks. Furthermore, hepatic lipids and lipid peroxide were significantly higher in SDT fatty-Cho than SDT fatty rats fed normal diets at 24 weeks. Hepatic mRNA expression related to TG secretion decreased in SDT fatty-Cho, and the mRNA expression related to inflammation and fibrosis increased in SDT fatty-Cho at 24 weeks. Furthermore, SDT fatty-Cho presented with increased plasma choline, similar to human NASH. There were no significant changes in the effects of feeding a cholesterol-enriched diet in Sprague-Dawley rats. SDT fatty-Cho has the potential to become a valuable animal model for NASH associated with type 2 diabetes and obesity.