Оценка эффективности гепатопротекторов при неалкогольной жировой болезни печени

Изучены структуры и содержание липидов в гепатоцитах мышей линии C57Bl/6 при моделировании неалкогольной жировой болезни печени (НАЖБП) гиперкалорийным кормом с последующим применением ремаксола (25 мл/кг 1 раз в сутки через день на протяжении 20 дней), фосфогливом (лиофилизат для приготовления раствора для внутривенного введения 0,1 мг/кг 1 раз в сутки через день на протяжении 20 дней), а также их комбинацией. Оценку эффективности проводили в ходе исследования гистологической структуры срезов печени мышей линии C57Bl6 при помощи шкалы оценки активности НАЖБП (NAFLD activity score — NAS). Установлены признаки положительного влияния фосфоглива и ремаксола (величина NAS 3,7 ± 1,1 и 3,3 ± 0,9 балла, по сравнению с исходными 6,7 ± 0,7 баллами, p < 0,05). Выраженность стеатоза в группе фосфоглива составляла 29 ± 7 %, а ремаксола — 26 ± 8 % в сравнении с исходными 82 ± 15 % (p < 0,05). Хотя величина индекса NAS и процент стеатоза в этих группах не имели статистически значимого отличия между собой, показатели распространённости балонной дистрофии различались и составляли 35 ± 9 % (по сравнению с исходными 75 ± 10) в группе фосфоглива и 12,5 ± 6 % в группе ремаксола, что свидетельствует о большей эффективности последнего (p < 0,05). Наилучший эффект был достигнут при использовании комбинации обоих препаратов: индекс NAS — 2,5 ± 0,7, балонная дистрофия затрагивала 11,1 ± 4 % гепатоцитов, что отличалось от значений до применения изученных препаратов и других схем терапии (p < 0,05).Оценка эффективности исследуемых препаратов у 30 человек с высоким индексом стеатоза (FLI) показала, что индекс стеатоза достоверно снижался после использования всех трёх исследуемых схем терапии, достигая 77,3 ± 7,7 в сравнении с 96,9 ± 2,35 (p < 0,05) при применении фосфоглива (внутривенно 20 мг/мл + 50 мг/мл 2 раза в сутки в течение 10 дней), 67,4 ± 7,4 в сравнении с 96,5 ± 2,6 (p < 0,05) при использовании ремаксола (400 мл внутривенно 1 раз в сутки в течение 10 дней) и 62,7 ± 7,7 в сравнении с 96,4 ± 2,2 (p < 0,05) при комбинированном назначении обоих препаратов. Было выявлено статистически значимое преимущество более, чем на 10 % по этому показателю при использовании схем терапии, содержащих ремаксол по сравнению с монотерапией фосфогливом (p < 0,05). Различий в снижении индекса стеатоза между группами больных, получивших ремаксол и комбинированную терапию, выявлено не было.

Association between Equol Production Status and Nonalcoholic Steatohepatitis

Equol is a metabolite of daidzein, a major soybean isoflavone with estrogenic and antioxidant activities. As the production of equol depends on the presence of certain members of the intestinal microflora, not all individuals can produce equol. We examined the relationship between NASH histological features and equol production. In an animal model, obese OLETF rats were intraperitoneally injected with a porcine serum to augment liver fibrogenesis. Equol-rich soy product, SE5-OH was orally administered during the experimental period. Treatment with SE5-OH markedly attenuated the development of liver fibrosis and expression of alpha-smooth muscle actin. In clinical research, 38 NAFLD patients (13 men and 25 women) were included. The degree of fibrosis and ballooning in equol-nonproducers was significantly higher than in equol-producers in women. The percentage of nonproducers with NAFLD activity score (NAS) ≥5 was significantly higher than that of producers. None of the histological features were significantly different between nonproducers and producers in men. Decision tree analysis identified predictors for NAS ≥5 in women. The status of equol production was the strongest predictor, followed by fasting glucose. Since equol can be noninvasively detected in urine, it can be applied as a screening tool for the progression of NASH in women.

Use of GP73 in the diagnosis of non-alcoholic steatohepatitis and the staging of hepatic fibrosis

Objective To evaluate the utility of Golgi protein 73 (GP73) in the diagnosis of non-alcoholic steatohepatitis (NASH) and hepatic fibrosis (HF) staging. Methods Ninety-one patients with non-alcoholic fatty liver disease (NAFLD) were allocated to NAFL (n = 46) and NASH (n = 45) groups according to their NAFLD activity score (NAS), and there were 30 healthy controls. Serum GP73 was measured by ELISA, GP73 protein expression was evaluated using immunohistochemistry, and FibroScan was used to determine liver hardness. Results The serum GP73 concentrations of the NAFL and NASH groups were significantly higher than those of controls. GP73 expression in the liver of the patients gradually progressed from absent or low to moderate or high. Serum GP73 positively correlated with liver expression, and the serum and liver GP73 of the patients positively correlated with FibroScan value and HF stage. There was a strong positive correlation of the combination of alanine aminotransferase, gamma glutamyl transferase and GP73 with NASH. The combination of serum GP73 and FibroScan value was found to predict NASH (NAS > 4) and advanced HF (stage ≥2) in patients with NAFLD using receiver operating characteristic analysis. Conclusion Serum GP73 may be useful in the diagnosis of NASH and the staging of HF.

Correlation between HDL2, HDL3 and serum ferritin levels with fatty liver and NAFLD activity score (NAS) in liver histology of organ donors

Background Nonalcoholic fatty liver disease (NAFLD) is one of the most important liver diseases. High-density lipoprotein (HDL) has anti-atherogenic properties and its reduction can be associated with fatty liver. Serum ferritin levels are usually elevated in patients with NAFLD. This study aimed to evaluate the correlation between HDL subtypes and serum ferritin levels with evidence of NAFLD in liver histology of organ donors. Methods One hundred organ donor patients who were eligible for the study were included in the study and ferritin; HDL2 and HDL3 were measured in blood samples. Donated liver tissue biopsy specimens were evaluated for fatty liver and NAFLD activity score (NAS). In addition, AST and ALT were measured in recipients 24 h after transplant. All data abstracted and analyzed statistically. Results Serum HDL2 levels and HDL2/HDL3 ratio in patients with NAS > 1 were significantly lower (P < 0.05). Serum levels of HDL3 and ferritin were not significantly associated with NAS >1 (P > 0.05). In addition, serum ferritin > 1000 ng/ml in organ donors associated with increased AST and ALT levels 24 h after transplantation in the liver organ recipient. Conclusions Lower HDL2 values and HDL2/HDL3 ratio were associated with increased NAFLD activity score, but HDL3 and ferritin did not show such a relationship. In addition, higher levels of ferritin in organ donors may be associated with increased AST and ALT 24 h after liver transplantation in the organ recipient.

Adiponutrin and Adiponectin Gene Variants in Indonesian Patients with Non-Alcoholic Fatty Liver Disease: a Preliminary Study

BackgroundVariants of adiponutrin (PNPLA3) and adiponectin (ADIPOQ) genes were considered to be associated with non-alcoholic fatty liver disease (NAFLD). Although the prevalence of NAFLD is increasing, there are limited numbers of studies about the association in Indonesian population.ObjectiveTo confirm whether specific variants of adiponutrin (PNPLA3) and adiponectin (ADIPOQ) genes are associated with NAFLD in Indonesian patients.MethodsData and DNA of 152 participants were obtained from a previous study in Dr. Kariadi Hospital, Semarang, Indonesia. PCR-RFLP analysis was performed for detection of PNPLA3 rs738409 and ADIPOQ rs2241767 variants. The diagnosis and severity of NAFLD were assessed according to NAFLD activity score (NAS) based on histopathology assessment of liverbiopsy.ResultsAllele G of PNPLA3rs738409 was associated with NAFLD in both bivariate (p=0.009, OR 2.52, CI 95% 1.25–5.07) and multivariate (p=0.008, OR 2.62, CI 95% 1.29%–5.32%) analysis, while ADIPOQ rs2241767 had no significant association. In NAFLD participants, both genotypes showed allele G was higher in the group of possible non-alcoholic steatohepatitis (NASH) – NASH (NAS >2) than in the simple steatosis group (NAS <2) i.e. 40.0% vs. 3.75% for the rs2241767 variant and 23.75% vs. 1.25% for the rs738409 variant, without significant association.ConclusionVariant PNPLA3 rs738409 was associated with NAFLD incidence in studied population. Among NAFLD participants, the frequency of both variants were found higher in the possible NASH – NASH group, yet needs to be confirmed with more participants and a multicenter study.Data and DNA of 152 participants were obtained from a previous study in Dr. Kariadi Hospital, Semarang, Indonesia. PCR-RFLP analysis was performed for detection of PNPLA3 rs738409 and ADIPOQ rs2241767 variants. The diagnosis and severity of NAFLD were assessed according to NAFLD activity score (NAS) based on histopathology assessment of liverbiopsy.

Hepatocyte TLR4 triggers inter-hepatocyte Jagged1/Notch signaling to determine NASH-induced fibrosis

Aberrant hepatocyte Notch activity is critical to the development of nonalcoholic steatohepatitis (NASH)–induced liver fibrosis, but mechanisms underlying Notch reactivation in developed liver are unclear. Here, we identified that increased expression of the Notch ligand Jagged1 (JAG1) tracked with Notch activation and nonalcoholic fatty liver disease (NAFLD) activity score (NAS) in human liver biopsy specimens and mouse NASH models. The increase in Jag1 was mediated by hepatocyte Toll-like receptor 4 (TLR4)–nuclear factor κB (NF-κB) signaling in pericentral hepatocytes. Hepatocyte-specific Jag1 overexpression exacerbated fibrosis in mice fed a high-fat diet or a NASH-provoking diet rich in palmitate, cholesterol, and sucrose and reversed the protection afforded by hepatocyte-specific TLR4 deletion, whereas hepatocyte-specific Jag1 knockout mice were protected from NASH-induced liver fibrosis. To test therapeutic potential of this biology, we designed a Jag1-directed antisense oligonucleotide (ASO) and a hepatocyte-specific N-acetylgalactosamine (GalNAc)–modified siRNA, both of which reduced NASH diet–induced liver fibrosis in mice. Overall, these data demonstrate that increased hepatocyte Jagged1 is the proximal hit for Notch-induced liver fibrosis in mice and suggest translational potential of Jagged1 inhibitors in patients with NASH.

Equol Production Status is An Effective Predictor of Nonalcoholic Steatohepatitis in Women

Equol is a metabolite of daidzein, a major soybean isoflavone with estrogenic and antioxidant activities. As the production of equol depends on the presence of certain members of the intestinal microflora, not all individuals can produce equol. The incidence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) increases in menopausal women, and estrogen is associated with the progression of NAFLD/NASH. Therefore, the status of equol production might also be related to the pathogenesis of NAFLD/NASH in women. We, thus, examined the relationship between NASH histological features and equol production. Thirty-eight NAFLD patients who underwent liver biopsy were included in this study. In women, the degree of fibrosis and ballooning in nonproducers was significantly higher than that in producers. The percentage of nonproducers with NAFLD activity score (NAS) ≥5 was significantly higher than that of producers. None of the histological features were significantly different between nonproducers and producers in men. Decision tree analysis identified the predictors of NAS ≥5 in women. The status of equol production was the strongest predictor, followed by fasting glucose. Equol can be noninvasively detected in urine, suggesting its application as a screening test for predicting the diagnosis and progression of NASH in women.

Hepatic Macrophages Express Melanoma Differentiation-Associated Gene 5 in Nonalcoholic Steatohepatitis

The activation of innate immune system is essential for the pathogenesis of nonalcoholic steatohepatitis (NASH). Among pattern recognition receptors, it is well-characterized that toll-like receptors (TLRs) are deeply involved in the development of NASH to reflect exposure of the liver to gut-driven endotoxins. In contrast, it has not been elucidated whether retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are similarly implicated in the disease progression. In the present study, we examined the expression of melanoma differentiation-associated antigen 5 (MDA5), known to be a member of RLRs, in a diet-induced murine model of NASH using western blotting and immunohistochemistry (IHC). The results of western blotting showed that hepatic expression of MDA5 was significantly increased 6 weeks after choline-deficient L-amino acid-defined high-fat diet (CDAHFD). In IHC, MDA5-positive cells co-express F4/80 and CD11b, indicating they were activated macrophages, and these cells began to appear at 1 week after CDAHFD. Additionally, we performed IHC using liver biopsy specimens collected from eight patients with nonalcoholic fatty liver diseases (NAFLD), and found that MDA5 was expressed in CD11b-positive macrophages in six out of eight patients. The lobular inflammation score of NAFLD activity score tended to be higher in MDA5-positive cases than in MDA5-negative cases. Our findings suggest that MDA5 may be involved in the inflammation of NASH.

Hepatocyte Nuclear Factor 4 Alpha Attenuated Lipotoxicity, But Increased Bile Acid Toxicity in Non-Alcoholic Fatty Liver Disease Model.

Background: It is known that hepatocyte nuclear factor 4 alpha (HNF4α) is key master nuclear receptor for hepatic fat and bile acid metabolic pathways. But the role of HNF4α in non-alcoholic fatty liver disease (NAFLD) is complex. The current study aimed to investigate role of HNF4α in NAFLD. Methods: Hepatic HNF4α expression evaluated in human NAFLD subjects. Free fatty acid induced lipotoxicity evaluated under HNF4α over- and down regulation. Chenodeoxy cholic acid (CDCA) induced bile acid toxicity evaluated under HNF4α in In Vitro NAFLD model. NAFLD activity score and fibrosis assessed after HNF4α silencing in methionine choline deficiency diet fed mice. Results: Hepatic HNF4α expression was higher in NAFLD than in control group. Overexpression of HNF4α reduced intracellular lipid contents via increasing mitochondria beta-oxidation and hepatic fat excretion. HNF4α overexpression attenuated palmitic acid (PA) induced lipotoxicity. Protective effects of HNF4α on cell death were reversed when CDCA co-treated with PA. CDCA mono-treatment did not affect cell viability, but co-treatment with PA and CDCA decreased cell viability. Bile acid toxicity of HNF4α was exaggerated under PA co-treatment with CDCA. HNF4α knock down using small interfering RNA recovered cell apoptosis and increased cell proliferation from PA and CDCA co-treatment condition. Inhibition of HNF4α using sh-HNF4α adenovirus vector did not reduce hepatic fat accumulation, but decreased intrahepatic inflammation and NAFLD activity score compared to control.Conclusions: HNF4α increased free fatty acid oxidation and attenuated lipotixicity, but increased bile acid toxicity in NAFLD animal model. Inhibition of HNF4α attenuated In vivo NAFLD model.