Non-alcoholic Steatohepatitis Activity Score Research Articles

Machine learning-based algorithm identifies key mitochondria-related genes in non-alcoholic steatohepatitis

BackgroundEvidence suggests that hepatocyte mitochondrial dysfunction leads to abnormal lipid metabolism, redox imbalance, and programmed cell death, driving the onset and progression of non-alcoholic steatohepatitis (NASH). Identifying hub mitochondrial genes linked to NASH may unveil potential therapeutic targets.MethodsMitochondrial hub genes implicated in NASH were identified via analysis using 134 algorithms.ResultsThe Random Forest algorithm (RF), the most effective among the 134 algorithms, identified three genes: Aldo–keto reductase family 1 member B10 (AKR1B10), thymidylate synthase (TYMS), and triggering receptor expressed in myeloid cell 2 (TREM2). They were upregulated and positively associated with genes promoting inflammation, genes involved in lipid synthesis, fibrosis, and nonalcoholic steatohepatitis activity scores in patients with NASH. Moreover, using these three genes, patients with NASH were accurately categorized into cluster 1, exhibiting heightened disease severity, and cluster 2, distinguished by milder disease activity. ConclusionThese three genes are pivotal mitochondrial genes implicated in NASH progression.

The promising role of CCL2 as a noninvasive marker for nonalcoholic steatohepatitis diagnosis in Egyptian populations.

Nonalcoholic fatty liver disease (NAFLD) is a common liver problem, including both nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). In this study, we investigated the role of CCL2 and IL6 as a noninvasive tool for the diagnosis of NASH in clinical practice and to establish criteria for discrimination NASH from NAFL in Egyptian populations with NAFLD. In addition to 30 healthy controls, serum samples from 66 NAFLD patients histologically diagnosed by biopsy (32 NAFL and 34 NASH) were analyzed for serum IL6, CCL2, liver biomarkers, complete blood count and lipid profile. Serum IL6 or CCL2 levels were tested for correlation with the NASH activity score (NAS score). Both IL6 and CCL2 were significantly upregulated in NASH patients compared with NAFL patients or control. Serum CCL2 was significantly correlated with the degree of hepatocytes ballooning (the diagnostic endpoint for NASH) without any significant correlation with steatosis or lobular inflammation. Serum IL6 was not correlated with the NAS score. The ROC curve analysis of CCL2 for NASH diagnosis revealed an area under curve (AUROC) of 0.959 at cutoff ≥227 pg/ml. While IL6 revealed an (AUROC) of 0.790. Serum CCL2 but not IL6 is a promising noninvasive tool for NASH diagnosis and CCL2 can provide a reliable, validated scoring system to discriminate NAFL from NASH in the Egyptian population confirming the role of CCL2 in NASH pathogenesis. These findings will aid in the development of innovative NASH treatment strategies in Egypt and improve the quality of clinical care.

Epigallocatechin-3-Gallate Dampens Non-Alcoholic Fatty Liver by Modulating Liver Function, Lipid Profile and Macrophage Polarization.

Epigallocatechin-3-gallate (EGCG) has been shown to attenuate obesity, fatty liver disease, hepatic inflammation and lipid profiles. Here, we validate the efficacy of EGCG in a murine model of non-alcoholic fatty liver disease (NAFLD) and extend the mechanistic insights. NAFLD was induced in mice by a high-fat diet (HFD) with 30% fructose. EGCG was administered at a low dose (25 mg/kg/day, EGCG-25) or high dose (50 mg/kg/day, EGCG-50) for 8 weeks. In HFD-fed mice, EGCG attenuated body and liver weight by ~22% and 47%, respectively, accompanied by ~47% reduction in hepatic triglyceride (TG) accumulation and ~38% reduction in serum cholesterol, resonating well with previous reports in the literature. In EGCG-treated mice, the hepatic steatosis score and the non-alcoholic steatohepatitis activity score were both reduced by ~50% and ~57%, respectively, accompanied by improvements in hepatic inflammation grade. Liver enzymes were improved ~2–3-fold following EGCG treatment, recapitulating previous reports. Hepatic flow cytometry demonstrated that EGCG-fed mice had lower Ly6C+, MHCII+ and higher CD206+, CD23+ hepatic macrophage infiltration, indicating that EGCG impactedM1/M2 macrophage polarization. Our study further validates the salubrious effects of EGCG on NAFLD and sheds light on a novel mechanistic contribution of EGCG, namely hepatic M1-to-M2 macrophage polarization. These findings offer further support for the use of EGCG in human NAFLD.

The Impact of Modern Chemotherapy and Chemotherapy-Associated Liver Injuries (CALI) on Liver Function: Value of 99mTc-Labelled-Mebrofenin SPECT-Hepatobiliary Scintigraphy.

Chemotherapy is increasingly used before hepatic resection, with controversial impact regarding liver function. This study aimed to assess the capacity of 99mTc-labelled-mebrofenin SPECT-hepatobiliary scintigraphy (HBS) to predict liver dysfunction due to chemotherapy and/or chemotherapeutic-associated liver injuries (CALI), such as sinusoidal obstruction syndrome (SOS) and nonalcoholic steatohepatitis (NASH) activity score (NAS). From 2011 to 2015, all consecutive noncirrhotic patients scheduled for a major hepatectomy (≥ 3 segments) gave informed consent for preoperative SPECT-HBS allowing measurements of segmental liver function. As primary endpoint, HBS results were compared between patients with versus without (1) preoperative chemotherapy (≤ 3months); and (2) CALI, mainly steatosis, NAS (Kleiner), or SOS (Rubbia-Brandt). Secondary endpoints were (1) other factors impairing function; and (2) impact of chemotherapy, and/or CALI on hepatocyte isolation outcome via liver tissues. Among 115 patients, 55 (47.8%) received chemotherapy. Sixteen developed SOS and 35 NAS, with worse postoperative outcome. Overall, chemotherapy had no impact on liver function, except above 12 cycles. In patients with CALI, a steatosis ≥ 30% significantly compromised function, as well as NAS, especially grades 2-5. Conversely, SOS had no impact, although subjected to very low patients number with severe SOS. Other factors impairing function were diabetes, overweight/obesity, or fibrosis. Similarly, chemotherapy in 73 of 164 patients had no effect on hepatocytes isolation outcome; regarding CALI, steatosis ≥ 30% and NAS impaired the yield and/or viability of hepatocytes, but not SOS. In this first large, prospective study, HBS appeared to be a valuable tool to select heavily treated patients at risk of liver dysfunction through steatosis or NAS.

SUN-694 Case Study of Nonalcoholic Steatohepatitis Reversibility in Type Two Diabetic Patient with Weight Loss Using Liraglutide

Introduction Nonalcoholic fatty liver disease (NAFLD) is a common form of chronic liver disease.1 NAFLD prevalence is likely to be between 75- 100% in the morbidity obese. Where Nonalcoholic steatohepatitis (NASH) in turns develops in 30% of patients with NAFLD. Obesity prevalence in Kuwait is estimated to be 39% for adults male and 52% for adults females.2 No pharmacotherapy is approved for NAFLD treatment, and the basis treatment is lifestyle modifications focusing on body fat loss.3 A study showed that may take 10% or more weight loss to have an impact on NASH Activity Scores as assessed by liver biopsy. Glucagon-like peptide-1 receptor agonists (GLP-1 RA) are approved for the treatment of T2D and obesity and have also been shown to reduce liver inflammation and fibrosis.3 Methods A 54 years old male presented to our clinic with a history of hypothyroidism, diabetes with Hemoglobin A1C (HbA1c) of 7%, hypercholesterolemia and overweight with Body Mass Index (BMI) of 27.6 kg/m2Patient’s investigations showed high Gamma-glutamyl transferase (GGT) and ferritin level, and due to his abnormal liver function test patient was referred for abdominal ultra sound that showed fatty liver disease. With a high NAFLD score, he was referred for Fibroscan that showed fibrosis score of F3; which indicates a sever liver scarring.GLP-1 RA was started for weight management and a better glycemic control in a setting of multidisciplinary team, including endocrinologist, diabetes educator, dietitian and a physical trainer. Results In a six months’ period of time, patient was able to lose 15.3% of his total body weight, with better glycemic control of HbA1c of 5.6% from 7% and his repeated Fibroscan showed improvement in his score from F3 to F0 with other clinically important outcomes. Conclusion GLP-1 RA seems to be safe to use for patients with NASH, and it might have benefits of reversing fibrosis in addition to other benefits such as weight reduction and HbA1c improvement. References 1- Babusik P, Bilal M, Duris I. Nonalcoholic fatty liver disease of two ethnic groups in Kuwait: comparison of prevalence and risk factors. Medical Principles and Practice. 2012;21(1):56-62.2- Jamal MH. The Status of Organ Transplantation in Kuwait.3- Seghieri M, Christensen AS, Andersen A, Solini A, Knop FK, Vilsbøll T. Future Perspectives on GLP-1 Receptor Agonists and GLP-1/glucagon Receptor Co-agonists in the Treatment of NAFLD. Frontiers in endocrinology. 2018;9.

334 OBETICHOLIC ACID IMPROVES EXPERIMENTAL NON-INVASIVE MARKERS OF NON-ALCOHOLIC STEATOHEPATITIS AND ADVANCED FIBROSIS; RESULTS OF A SECONDARY ANALYSIS FROM THE MONTH-18 INTERIM ANALYSIS OF THE REGENERATE STUDY

In the REGENERATE 18-month interim analysis, obeticholic acid (OCA) improved surrogate endpoints of liver fibrosis in patients with non-alcoholic steatohepatitis (NASH). New biomarker indices are being developed, including FibroMeter (FM), which is designed to predict fibrosis stage ≥2 using age, gender, alpha-2-macroglobulin, international normalized ratio, platelets, urea, and gamma-glutamyltransferase. FM Vibration-Controlled Transient Elastography (VCTE) uses the same biomarkers (excluding urea) with liver stiffness (LS). The FibroScan AST (FAST™) score uses LS by VCTE, Controlled Attenuation Parameter score, and aspartate aminotransferase to identify patients with NASH and NAFLD Activity Score (NAS) ≥4 and fibrosis stage ≥2. NASH patients with fibrosis stages 2 and 3 were randomized (1:1:1) to placebo (N=311), OCA 10 mg (N=312), or OCA 25 mg (N=308) once daily. Changes in FM (N=604), FM VCTE (N=604), and FAST (N=391) were analyzed using a mixed-effect repeated measures model (MRMM), with factors of treatment, baseline, visit, visit-by-treatment interaction, and stratification. Least square means and p-values were based on MMRM. At baseline, no significant differences were observed in scores across treatment groups (figure 1). Patients with stage 3 fibrosis at baseline had higher scores than those with stage 2 fibrosis. OCA-treated patients experienced improvements in FM, FM VCTE, and FAST at Month 6 through Month 18. No improvements were observed with placebo (figure 1). OCA treatment resulted in early and sustained improvements in non-invasive assessments of fibrosis in NASH. Improvements in FM and FM VCTE are consistent with OCA’s anti-fibrotic effect, while improvements in FAST are consistent with amelioration of NASH inflammation and fibrosis.

PNPLA3 polymorphism influences the association between high-normal TSH level and NASH in euthyroid adults with biopsy-proven NAFLD

AimWe aimed to evaluate the association between serum thyroid stimulating hormone (TSH) levels, within the reference range, and the histological severity of nonalcoholic fatty liver disease (NAFLD), and whether this association was modulated by the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism. Materials and methodsWe enrolled 327 euthyroid individuals with biopsy-proven NAFLD, who were subdivided into two groups, i.e., a ‘strict-normal’ TSH group (TSH level 0.4 to 2.5mIU/L; n=283) and a ‘high-normal’ TSH group (TSH level 2.5 to 5.3mIU/L with normal thyroid hormones; n=44). Logistic regression analyses were performed to assess the association between TSH status and presence of nonalcoholic steatohepatitis (NASH) after stratifying subjects by PNPLA3 genotypes. ResultsCompared to strict-normal TSH group, patients with high-normal TSH levels were younger and had a greater prevalence of NASH and higher histologic NAFLD activity score. After stratifying by PNPLA3 genotypes, the significant association between high-normal TSH levels and presence of NASH was restricted only to carriers of the PNPLA3 G risk allele and remained significant even after adjustment for potential confounding factors (adjusted-odds ratio: 3.279; 95% CI: 1.298–8.284; P=0.012). ConclusionIn euthyroid individuals with biopsy-proven NAFLD, we found a significant association between high-normal TSH levels and NASH. After stratifying by PNPLA3 rs738409 genotypes, this association was observed only among carriers of the PNPLA3 G risk allele.

Increased Expression of RUNX1 in Liver Correlates with NASH Activity Score in Patients with Non-Alcoholic Steatohepatitis (NASH).

Given the important role of angiogenesis in liver pathology, the current study investigated the role of Runt-related transcription factor 1 (RUNX1), a regulator of developmental angiogenesis, in the pathogenesis of non-alcoholic steatohepatitis (NASH). Quantitative RT-PCRs and a transcription factor analysis of angiogenesis-associated differentially expressed genes in liver tissues of healthy controls, patients with steatosis and NASH, indicated a potential role of RUNX1 in NASH. The gene expression of RUNX1 was correlated with histopathological attributes of patients. The protein expression of RUNX1 in liver was studied by immunohistochemistry. To explore the underlying mechanisms, in vitro studies using RUNX1 siRNA and overexpression plasmids were performed in endothelial cells (ECs). RUNX1 expression was significantly correlated with inflammation, fibrosis and NASH activity score in NASH patients. Its expression was conspicuous in liver non-parenchymal cells. In vitro, factors from steatotic hepatocytes and/or VEGF or TGF-β significantly induced the expression of RUNX1 in ECs. RUNX1 regulated the expression of angiogenic and adhesion molecules in ECs, including CCL2, PECAM1 and VCAM1, which was shown by silencing or over-expression of RUNX1. Furthermore, RUNX1 increased the angiogenic activity of ECs. This study reports that steatosis-induced RUNX1 augmented the expression of adhesion and angiogenic molecules and properties in ECs and may be involved in enhancing inflammation and disease severity in NASH.

1139 High Prevalence of Non-Alcoholic Steatohepatitis in Veterans With Non Alcoholic Fatty Liver Disease Undergoing Sleeve Gastrectomy

INTRODUCTION: Sleeve gastrectomy leads to significant weight loss and improves features of metabolic syndrome. The prevalence of non-alcoholic fatty liver disease (NAFLD) in the general population undergoing bariatric surgery is reported at 84% -95%. Prevalence of Non Alcoholic Steatohepatitis (NASH) in this population is 25%- 50%. Around 10% of these patients have advanced fibrosis at the time of surgery. The prevalence of NAFLD/NASH or advanced fibrosis in the veteran population undergoing sleeve gastrectomy has not been previously reported. NASH may be underrecognized prior to time of surgery, particularly if there are no associated laboratory abnormalities. This study aims to assess the prevalence of NASH and advanced fibrosis in veterans undergoing sleeve gastrectomy. METHODS: This is a retrospective study of 39 patients who underwent sleeve gastrectomy between January 1, 2018- April 30, 2019 at a Veterans Affairs Hospital. Patients with known alcohol use, cirrhosis or without liver biopsy at the time of surgery were excluded. A single pathologist reviewed the liver biopsies and provided a NASH Activity Score (NAS) for each sample. A Fib 4 score and NAFLD fibrosis score were calculated. Summary statistics was used to determine the prevalence of NAFLD at time of surgery. RESULTS: Patients baseline characteristics are outlined in Table 1. Only 13% of patients had an abnormal ALT at the time of surgery. There was no evidence of liver synthetic dysfunction with normal INR, bilirubin and albumin. Median NAFLD fibrosis score was -0.8 and median FIB-4 was 0.9 which rules out advance fibrosis. 59% of patients had NAFLD and 61% of these patients had evidence of NASH on pathologic exam. Advanced fibrosis was seen in 8% of patients. 70% of patients had NAS score of 4 or below. Percentage of steatosis, inflammation, ballooning and fibrosis score have been summarized in Table 2. CONCLUSION: More than half of our NAFLD patients had characteristics features of NASH on biopsy. This is higher than reported in the general population and close to what has been reported in diabetic patients. However, prevalence of diabetes in the NAFLD patients was only 26%, which would not explain the high number of NASH patients. Advanced fibrosis was only seen in 8% of patients at the time of surgery, which is similar to previous studies. This is the first study to our knowledge to report high prevalence of NASH in a VA population undergoing surgery in the absence of high rate of concomitant diabetes.

High Throughput Screening of Serum γ-Glutamyl Dipeptides for Risk Assessment of Nonalcoholic Steatohepatitis with Impaired Glutathione Salvage Pathway.

Nonalcoholic fatty liver disease (NAFLD) is the most common preventable chronic liver disorder in developed countries, the prevalence of which is increasing worldwide due to its association with obesity and type 2 diabetes. However, the exact mechanisms of NAFLD pathophysiology remain poorly understood including its progression to the more severe nonalcoholic steatohepatitis (NASH). New advances for early detection and monitoring of NASH progression are limited due to the lack of specific blood biomarkers, thus requiring invasive liver biopsies for histopathology. Herein, multisegment injection-capillary electrophoresis-tandem mass spectrometry (MSI-CE-MS/MS) is validated as a high throughput, robust, and quantitative platform for targeted analysis of a panel of 16 serum γ-glutamyl dipeptides from a cohort of NASH adult patients from Japan (median age = 53 years, median BMI = 27 kg/m2, n = 116). Multiplexed separations based on MSI-CE-MS/MS enable the design of unique data workflows that rely on customizable serial sample injection formats for accurate determination of γ-glutamyl dipeptides with quality control. Also, the introduction of a liquid coolant device to the capillary outlet improves long-term migration time stability in CE. Unsupervised pattern recognition methods revealed two distinctive NASH subgroups based on their contrasting γ-glutamyl dipeptide status despite patients having similar clinical phenotypes and NASH activity scores (median NAS ≈ 6.0). There was an inverse correlation between serum γ-glutamyl dipeptide concentrations and γ-glutamyltransferease (GGT) enzyme activity (r = -0.46; p = 2.5 × 10-7), which was indicative of a low-risk (n = 64) as compared to a high-risk (n = 52) patient subgroup with impaired glutathione salvage pathway and likely poor clinical prognosis. Our findings highlight the key role of defects in the γ-glutamyl cycle for differentiation of NASH patients, which may enable better risk assessment of long-term survivorship as a complement to standard liver enzyme screens and histopathology.

Human amnion epithelial cells and their soluble factors reduce liver fibrosis in murine non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma. Currently, lifestyle modification is the only effective treatment. We have shown that human amnion epithelial cells (hAECs) reduce inflammation and fibrosis in toxin-induced liver injury models. We examined the effect of these cells and the soluble factors released by the cells into culture medium (hAEC conditioned medium [hAEC-CM]) in a diet-induced murine NASH model. C57BL/6J male mice received a Western "fast food diet" for 42weeks. Group 1 received an intraperitoneal injection of 2×106 hAECs at week 34, group 2 received an additional hAEC dose at week 38, and group 3 received thrice weekly hAEC-CM injections intraperitoneal for 8weeks from week 34. Liver fibrosis area, inflammation, and fibrosis regulators were measured by immunohistochemistry, qPCR, and gelatin zymography. Metabolic parameters were also assessed. Fast food diet-fed mice demonstrated peri-cellular hepatic fibrosis, inflammation, and steatosis typical of NASH. Liver fibrosis area was reduced by 40% in hAEC-treated and hAEC-CM-treated mice. hAEC treatment significantly reduced pSMAD 2/3 signaling and the number of activated hepatic stellate cells and liver macrophages. Matrix metalloproteinase 2 and 9 gene and protein expression were variably affected. hAEC treatment did not alter the NASH activity score or metabolic parameters such as bodyweight, total cholesterol, or glucose tolerance. Human amnion epithelial cell and hAEC-CM significantly reduced hepatic inflammation and fibrosis in a diet-induced non-alcoholic fatty liver disease model. Although hAEC and hAEC-CM did not affect the metabolic components of NASH, their therapeutic potential is promising and warrants further investigation.

Augmented liver inflammation in a microsomal prostaglandin E synthase 1 (mPGES-1)-deficient diet-induced mouse NASH model

In a subset of patients, non-alcoholic fatty liver disease (NAFLD) is complicated by cell death and inflammation resulting in non-alcoholic steatohepatitis (NASH), which may progress to fibrosis and subsequent organ failure. Apart from cytokines, prostaglandins, in particular prostaglandin E2 (PGE2), play a pivotal role during inflammatory processes. Expression of the key enzymes of PGE2 synthesis, cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1), was increased in human NASH livers in comparison to controls and correlated with the NASH activity score. Both enzymes were also induced in NASH-diet-fed wild-type mice, resulting in an increase in hepatic PGE2 concentration that was completely abrogated in mPGES-1-deficient mice. PGE2 is known to inhibit TNF-α synthesis in macrophages. A strong infiltration of monocyte-derived macrophages was observed in NASH-diet-fed mice, which was accompanied with an increase in hepatic TNF-α expression. Due to the impaired PGE2 production, TNF-α expression increased much more in livers of mPGES-1-deficient mice or in the peritoneal macrophages of these mice. The increased levels of TNF-α resulted in an enhanced IL-1β production, primarily in hepatocytes, and augmented hepatocyte apoptosis. In conclusion, attenuation of PGE2 production by mPGES-1 ablation enhanced the TNF-α-triggered inflammatory response and hepatocyte apoptosis in diet-induced NASH.

Soybean Oil-Derived Poly-Unsaturated Fatty Acids Enhance Liver Damage in NAFLD Induced by Dietary Cholesterol.

While the impact of dietary cholesterol on the progression of atherosclerosis has probably been overestimated, increasing evidence suggests that dietary cholesterol might favor the transition from blunt steatosis to non-alcoholic steatohepatitis (NASH), especially in combination with high fat diets. It is poorly understood how cholesterol alone or in combination with other dietary lipid components contributes to the development of lipotoxicity. The current study demonstrated that liver damage caused by dietary cholesterol in mice was strongly enhanced by a high fat diet containing soybean oil-derived ω6-poly-unsaturated fatty acids (ω6-PUFA), but not by a lard-based high fat diet containing mainly saturated fatty acids. In contrast to the lard-based diet the soybean oil-based diet augmented cholesterol accumulation in hepatocytes, presumably by impairing cholesterol-eliminating pathways. The soybean oil-based diet enhanced cholesterol-induced mitochondrial damage and amplified the ensuing oxidative stress, probably by peroxidation of poly-unsaturated fatty acids. This resulted in hepatocyte death, recruitment of inflammatory cells, and fibrosis, and caused a transition from steatosis to NASH, doubling the NASH activity score. Thus, the recommendation to reduce cholesterol intake, in particular in diets rich in ω6-PUFA, although not necessary to reduce the risk of atherosclerosis, might be sensible for patients suffering from non-alcoholic fatty liver disease.

Circulating PCSK9 levels are not associated with the severity of hepatic steatosis and NASH in a high-risk population

Background and aimsSome studies suggested that proprotein convertase subtilisin kexin type 9 (PCSK9) is linked to liver steatosis severity and non-alcoholic steatohepatitis (NASH). We aimed to assess whether circulating PCSK9 levels are associated with either liver fat content (LFC) or histological markers of NASH in high-risk patients. MethodsWe present results from three cross-sectional studies from two French Hospitals: Dijon and Numevox (departments of Endocrinology) and Angers (department of Hepatology). Only patients without lipid-lowering therapy were included. All 132 patients had type 2 diabetes in Dijon, compared to 55/224 in Numevox (25%) and 39/122 in Angers (32%). LFC was assessed on MRI (Dijon and Numevox), and NASH lesion on liver biopsy (Angers). Additionally, we included mRNA results from 138 overweight patients of a Belgian Hospital (Antwerp). ResultsWhile circulating levels of PCSK9 were positively correlated with total cholesterol, LDL-C, triglycerides and non-HDL-C in all 3 cohorts, no significant association was found between PCSK9 and transaminases. Furthermore, no association was found between plasma PCSK9 levels and LFC in both Numevox (βadjusted = 0.71 ± 1.33, p = 0.60) and Dijon (βadjusted = −1.03 ± 0.90, p = 0.25). There was no correlation between circulating PCSK9 and histological liver lesions: steatosis severity (βadjusted = −3.95 ± 2.75, p = 0.15), NASH activity score (βadjusted = −0.31 ± 0.17, p = 0.082), lobular (β = −0.067 ± 0.055, p = 0.22) or portal inflammation (β = −0.088 ± 0.079, p = 0.27), ballooning (β = −0.025 ± 0.065, p = 0.70) and fibrosis (β = −0.17 ± 0.11, p = 0.12). Finally, hepatic PCSK9 mRNA levels were not correlated with NASH histological severity. ConclusionsCirculating PCSK9 concentrations are not associated with the severity of liver steatosis or histological markers of NASH. These data are reassuring regarding the clinical use of PCSK9 inhibitors in cardiovascular diseases.

Overexpression of the Vitronectin V10 Subunit in Patients with Nonalcoholic Steatohepatitis: Implications for Noninvasive Diagnosis of NASH.

Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL). Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment of the ECM protein vitronectin (VTN) was then selected as a promising circulating biomarker in discriminating NASH. The analysis of sera of patients provided these major findings: the circulating VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits. In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive discrimination of NASH from simple steatosis.

Tualang honey improves non alcoholic steatohepatitis animal model

Introduction: Non-alcoholic Steatohepatitis (NASH) is an emerging chronic liver disease with limited therapy available. Studies utilizing animal models induced with cholesterol diet ranging from 1-2% are hampered by inconsistent yield of NASH features. Therefore, we aimed to establish a NASH animal model utilizing 12% cholesterol diet (CD) and to investigate the effects of Tualang honey (TH) known for its anti-inflammatory and antioxidative properties in this model. Methods: Twenty-four Sprague-Dawley rats were divided into 2 groups (12% CD and standard diet) and were fed for 6 weeks. Following the establishment of NASH, the rats in the 12% CD group were subsequently divided into 3 groups. The first group was continued with only 12% CD. In the other 2 groups in addition to the 12% CD they were given TH treatment at different concentrations (1.2 and 2.4 g/kg/day) for 4 weeks. Blood biochemical analysis and histological assessment of liver were subsequently performed. Results: The liver histological sections of the rats fed with 12% CD showed macrovesicular steatosis, ballooning degeneration with lobular and portal inflammation. They also had increased serum alanine aminotransferase (ALT), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), fasting insulin, HOMA-IR and reduced high density lipoprotein cholesterol (HDL-C). Meanwhile, the TH treatment groups exhibited significant improvement in both the NASH grading and activity scores. The ALT, LDL-C, TC, triglyceride (TG), fasting insulin and HOMA-IR levels were reduced significantly. Conclusions: The 12% CD was able to induce NASH in the animal model. Tualang honey improved insulin sensitivity, dyslipidaemia, steatohepatitis.

Hepatic gene expression profile characterizes high levels of liver regeneration related genes in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a chronic hepatic disease caused by abnormal or excessive accumulation of fat in hepatocytes. NAFLD covers a spectrum of liver disorders ranging from simple steatosis (S) to nonalcoholic steatohepatitis (NASH) and cirrhosis, and is associated with changes in gene expression profiles. Tissue gene expression profiling has been precious for developing diagnostic and predictive biomarkers as well as for improving therapeutical strategies in many diseases. Therefore, the aim of this study was to use microarray analysis to characterize a liver gene expression profile that differentiates individuals diagnosed with steatosis or steatohepatitis. The discovery cohort (n = 22) including well-characterized human liver samples, categorized as normal (n = 7), steatosis (n = 7), and nonalcoholic steatohepatitis (n = 8), was analyzed by Affymetrix GeneChip and qRT-PCR. A gene set enrichment analysis was performed to identify processes that distinguish between steatosis/NASH and normal groups and hence, might be novel biomarkers or treatment targets. Among 1624 genes, Gene Ontology (GO) analysis identified six genes, involved in tissue repair/regeneration, which were differentially expressed and significantly upregulated in patients with NASH. This subset of the top-regulated liver regeneration-related genes includes amphiregulin (AREG), plasminogen activator receptor urokinase-type (PLAUR), thrombospondin 1 (THBS1), chemokine CC ligand 20 (CCL20), aldo-keto reductase family 1 member B10 (AKR1B10) and interleukin 32 (IL-32). These findings were validated in a second, independent and large cohort (n = 112) and confirmed by quantitative real-time PCR (qRT-PCR). Patients with NASH (n = 43) had significantly higher levels of AREG, PLAUR, THBS1, CCL20, AKR1B10 and IL-32 expression compared to steatotic (n = 37) and/or normal (n = 32) liver tissue. Subsequently, in this study we aim to correlate the mRNA expression of these genes with steatosis grade, NASH activity score, fibrosis, adipositas, BMI, Diabetes Mellitus and hypercholesterolemia.

Omega-3 polyunsaturated fatty acids in treating non-alcoholic steatohepatitis: A randomized, double-blind, placebo-controlled trial

& aims: Few clinical trials have addressed the potential benefits of omega-3 polyunsaturated fatty acids (PUFAs) on non-alcoholic steatohepatitis (NASH). We evaluated the effects of supplementation with omega-3 PUFAs from flaxseed and fish oils in patients with biopsy-proven NASH. Patients received three capsules daily, each containing 0.315g of omega-3 PUFAs (64% alpha-linolenic [ALA], 16% eicosapentaenoic [EPA], and 21% docosahexaenoic [DHA] acids; n-3 group, n=27) or mineral oil (placebo group, n=23). Liver biopsies were evaluated histopathologically by the NASH activity score (NAS). Plasma levels of omega-3 PUFAs were assessed as a marker of intake at baseline and after 6 months of treatment. Secondary endpoints included changes in plasma biochemical markers of lipid metabolism, inflammation, and liver function at baseline and after 3 and 6 months of treatment. At baseline, NAS was comparable between the groups (p=0.98). After intervention with omega-3 PUFAs, plasma ALA and EPA levels increased (p≤0.05). However in the placebo group, we also observed increased EPA and DHA (p≤0.05), suggesting an off-protocol intake of PUFAs. NAS improvement/stabilization was correlated with increased ALA in the n-3 group (p=0.02) and with increased EPA (p=0.04) and DHA (p=0.05) in the placebo group. Triglycerides were reduced after 3 months in the n-3 group compared to baseline (p=0.01). In NASH patients, the supplementation of omega-3 PUFA from flaxseed and fish oils significantly impacts on plasma lipid profile of patients with NASH. Plasma increase of these PUFAs was associated with better liver histology. (ID 01992809).

PPARα gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis

Peroxisome proliferator-activated receptors (PPARs) have been implicated in non-alcoholic steatohepatitis (NASH) pathogenesis, mainly based on animal data. Gene expression data in NASH patients are scarce. We studied liver PPARα, β/δ, and γ expression in a large cohort of obese patients assessed for presence of NAFLD at baseline and 1 year follow-up. Patients presented to the obesity clinic underwent a hepatic work-up. If NAFLD was suspected, liver biopsy was performed. Gene expression was studied by mRNA quantification. Patients were reassessed after 1 year. 125 patients were consecutively included in the study, of which 85 patients had paired liver biopsy taken at 1 year of follow-up. Liver PPARα expression negatively correlated with the presence of NASH (p=0.001) and with severity of steatosis (p=0.003), ballooning (p=0.001), NASH activity score (p=0.008) and fibrosis (p=0.003). PPARα expression was positively correlated to adiponectin (R(2)=0.345, p=0.010) and inversely correlated to visceral fat (R(2)=-0.343, p<0.001), HOMA IR (R(2)=-0.411, p<0.001) and CK18 (R(2)=-0.233, p=0.012). Liver PPARβ/δ and PPARγ expression did not correlate with any histological feature nor with glucose metabolism or serum lipids. At 1 year, correlation of PPARα expression with liver histology was confirmed. In longitudinal analysis, an increase in expression of PPARα and its target genes was significantly associated with histological improvement (p=0.008). Human liver PPARα gene expression negatively correlates with NASH severity, visceral adiposity and insulin resistance and positively with adiponectin. Histological improvement is associated with an increase in expression of PPARα and its target genes. These data might suggest that PPARα is a potential therapeutic target in NASH.

Neonatal monosodium glutamate treatment causes obesity, diabetes, and macrovesicular steatohepatitis with liver nodules in DIAR mice.

Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome (MS). Monosodium glutamate (MSG)-treated ICR mice is a useful model of MS and NASH, but it shows the different patterns of steatosis from human NASH. Because inbred aged DIAR (ddY, Institute for Animal Reproduction) mice spontaneously show the similar pattern of steatosis as NASH, we analyzed their liver pathology after administering MSG. MSG-treated DIAR mice (DIAR-MSG) and untreated DIAR mice (DIAR-controls) were sacrificed and assessed histopathologically at 29, 32, 40, 48, and 54 weeks of age. The NASH activity score, body mass index, blood glucose level, and oral glucose tolerance test were also assessed. The body mass index and blood glucose levels of DIAR-MSG were significantly higher than controls. The oral glucose tolerance test revealed a type 2 diabetes pattern in DIAR-MSG. The livers of DIAR-MSG mice showed macrovesicular steatosis, lobular inflammation with neutrophils, and ballooning degeneration after 29 weeks. At 54 weeks, mild fibrosis was observed in 5/6 DIAR-MSG and 2/5 DIAR-control mice. In imaging mass spectrometry analysis, cholesterol as well as triglyceride accumulated in the liver of DIAR-MSG mice. Atypical liver nodules were also observed after 32 weeks in DIAR-MSG, some with cellular and structural atypia mimicking human hepatocellular carcinoma. The NASH activity score of DIAR-MSG after 29 weeks was higher than that of control mice, suggesting the development of NASH. DIAR-MSG had NASH-like liver pathology and liver nodules typically associated with MS symptoms. DIAR-MSG provides a valuable animal model to analyze NASH pathogenesis and carcinogenesis.