Palmitate-induced Activation of Mitochondrial Metabolism Promotes Oxidative Stress and Apoptosis in H4IIEC3 Rat Hepatocytes

Objective

Hepatic lipotoxicity is characterized by reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and excessive apoptosis, but the precise sequence of biochemical events leading to oxidative damage and cell death remains unclear. The goal of this study was to delineate the role of mitochondrial metabolism in mediating hepatocyte lipotoxicity.

Materials/Methods

We treated H4IIEC3 rat hepatoma cells with free fatty acids in combination with antioxidants and mitochondrial inhibitors designed to block key events in the progression toward apoptosis. We then applied ¹³C metabolic flux analysis (MFA) to quantify mitochondrial pathway alterations associated with these treatments.

Results

Treatment with palmitate alone led to a doubling in oxygen uptake rate and in most mitochondrial fluxes. Supplementing culture media with the antioxidant N-acetyl-cysteine (NAC) reduced ROS accumulation and caspase activation and partially restored cell viability. However, ¹³C MFA revealed that treatment with NAC did not normalize palmitate-induced metabolic alterations, indicating that neither elevated ROS nor downstream apoptotic events contributed to mitochondrial activation. To directly limit mitochondrial metabolism, the complex I inhibitor phenformin was added to cells treated with palmitate. Phenformin addition eliminated abnormal ROS accumulation, prevented the appearance of apoptotic markers, and normalized mitochondrial carbon flow. Further studies revealed that glutamine provided the primary fuel for elevated mitochondrial metabolism in the presence of palmitate, rather than fatty acid beta-oxidation, and that glutamine consumption could be reduced through co-treatment with phenformin but not NAC.

Conclusion

Our results indicate that ROS accumulation in palmitate-treated H4IIEC3 cells occurs downstream of altered mitochondrial oxidative metabolism, which is independent of beta-oxidation and precedes apoptosis initiation.     

Using intestinal flora to distinguish non-alcoholic steatohepatitis from non-alcoholic fatty liver

ObjectiveTo explore specific flora in mouse models of non-alcoholic steatohepatitis (NASH) to improve NASH diagnostic protocols.MethodsSixty mice were divided into normal diet (ND, 20 mice) and high-fat/high-sugar diet (HFSD) groups (40 mice). After 8 weeks of feeding, 10 mice in the ND group and 20 mice in the HFSD group were sacrificed to create the short-term ND and non-alcoholic fatty liver (NAFL) groups, respectively. After 16 weeks of feeding, the remaining mice were sacrificed to create the long-term ND and NASH groups, respectively. We then examined fecal flora, serum biochemical indices, and lipopolysaccharide and tumor necrosis factor-α levels and analyzed liver tissue.ResultsThe relative abundance of Lactobacillus, Desulfovibrio, Ruminiclostridium 9, and Turicibacter differed between NASH and NAFL mice, and the areas under the receiver operating characteristic curve of the four genera for diagnosing NASH were 0.705, 0.734, 0.737, and 0.937. The non-alcoholic fatty liver disease activity score was positively correlated with the relative abundance of Desulfovibrio (r = 0.353), Ruminiclostridium 9 (r = 0.431), and Turicibacter (r = 0.688).ConclusionsThe relative abundance of Lactobacillus, Desulfovibrio, Ruminiclostridium, and Turicibacter may help distinguish NASH from NAFL.     

Schisandra Chinensis Baill,A Chinese Medicinal Herb,Alleviates High-Fat-Diet-Inducing Non-Alcoholic Steatohepatitis in Rats

Background

he present study aims to explore whether Schisandra chinensis Baill, a Chinese, medicinal herb can alleviates high-fat-diet-inducing non-alcoholic steatohepatitis in rats.

Materials and Methods

In the study, 24, male Wister rats with body weight between 180-220g, were included. The rats were randomly divided into four groups: model group, normal control group, rosiglitazone group, and Schisandra chinensis Baill group. The treatment lasted for 56, days. The high-fat diet used in the present study includes 25% lard, 2%, cholesterol 0.5%, sodium cholate, and 25%, Tween-80. The hepatic levels of superoxide dismutase (SOD), and malondialdehyde (MDA); the serum levels of total cholesterol (TC), and low-density lipoprotein cholesterol (LDLC), were detected.

Results

We found that the hepatic levels of SOD were significantly lower, and the serum levels of TC, LDLC as well as, the hepatic levels of MDA in model group were significantly higher than those of normal control group; rosiglitazone group and Schisandra chinensis Baill group (P<0.05), indicates that non-alcoholic steatohepatitis rats were successfully induced by high-fat diet. Schisandra chinensis Baill group presented a significant lower serum levels of LDLC, than rosiglitazone group (P<0.05); and the hepatic levels of SOD in Schisandra chinensis Baill group were significantly lower than rosiglitazone group (P<0.05). However, no significant difference existed between Schisandra chinensis Baill group, and rosiglitazone group on the hepatic levels of MDA and the serum levels of TC (P>0.05).

Conclusion

It is then concluded that Schisandra chinensis Baill can significantly alleviate the non-alcoholic steatohepatitis of the rats induced by high-fat diet, and it may be used as a complementary therapy for rosiglitazone.     

Myeloperoxidase gene polymorphism predicts fibrosis severity in women with hepatitis C

Oxidative stress plays an important role on liver fibrosis progression in the course of hepatitis C virus (HCV) infection. Myeloperoxidase (MPO) is an enzyme released by neutrophils and macrophages, responsible for generating hypochlorous acid and reactive oxygen species (ROS) that may lead to liver injury in HCV infection. On the other hand, antioxidant enzymes such as manganese superoxide dismutase (SOD) controls ROS-mediated damage. The aim of the present study was to investigate the influence of MPO G-463A and SOD2 Ala16Val polymorphisms in the severity of liver fibrosis in individuals with chronic HCV infection. The present study included 270 patients with chronic HCV recruited from the Gastrohepatology Service of the Oswaldo Cruz University Hospital/Liver Institute of Pernambuco (Recife, Northeastern Brazil). All patients underwent liver biopsy, which was classified according METAVIR score. The SNPs were determined by real-time PCR. After multivariate analysis adjustment, the GG genotype of MPO and the presence of metabolic syndrome were independently associated with fibrosis severity in women (P = 0.025 OR 2.25 CI 1.10–4.59 and P = 0.032 OR 2.32 CI 1.07–5.01, respectively). The presence of the GG genotype seems to be a risk factor for fibrosis severity in women with HCV.     

Fatty liver disease

It is now widely accepted that fatty liver disease is one of the commonest causes of cirrhosis and liver cell cancer (even in the absence of cirrhosis), in its own right as well as being an important cofactor for the progression of other diseases e.g. viral hepatitis. While much work has been done on developing non-invasive techniques for assessing liver disease, the liver biopsy remains the benchmark against which these tests have to be validated as well as providing information that cannot be obtained in any other way. This review describes the histological features that alcoholic and non-alcoholic liver disease have in common (e.g. fatty change, ballooning and Mallory–Denk bodies) as well as identifying those that are more characteristic of each of them (e.g. nuclear vacuolation in non-alcoholic fatty liver disease and a florid fatty liver hepatitis in alcoholic fatty liver disease). Recent developments in the assessment of the degree of fatty change are described.     

Investigating pharmacological mechanisms of andrographolide on non-alcoholic steatohepatitis (NASH): A bioinformatics approach of network pharmacology

Objective: To investigate the mechanisms of andrographolide against non-alcoholic steatohepatitis (NASH) based on network pharmacology, so as to provide a reference for further study of andrographolide in the treatment of NASH and other metabolic diseases. Methods: The methionine- and choline-deficient (MCD) diet-induced NASH mice were treated by administration of andrographolide, and serum transaminase and pathological changes were analyzed. The network pharmacology-based bioinformatic strategy was then used to search the potential targets, construct protein-protein interaction (PPI) network, analyze gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment, and conduct molecular docking to explore the molecular mechanisms. Results: The predicted core targets TNF, MAPK8, IL6, IL1B and AKT1 were enriched in non-alcoholic fatty liver disease (NAFLD) signaling pathway and against NASH by regulation of de novo fatty acids synthesis, anti-inflammation and anti-oxidation. Conclusion: This work provides a scientific basis for further demonstration of the anti-NASH mechanisms of andrographolide.     

Risk Factors and Ultrasound Can Predict Chronic Hepatitis Caused by Nonalcoholic Fatty Liver Disease

The diagnosis of nonalcoholic fatty liver disease (NAFLD) is underrecognized. The aim of this study was to develop a scoring system that separates NAFLD diagnosis as a cause of chronic hepatitis from controls by using clinical features and liver ultrasound. A retrospective review of consecutive NAFLD cases and other liver disease controls was undertaken selecting patients from an abnormal liver function test code. To qualify for analysis all patients had to have elevated liver injury tests for more then 6 months, a biopsy-confirmed diagnosis, and an ultrasound as part of the evaluation. There were 84 cases of NAFLD and 75 liver disease controls. The NAFLD group had a larger body mass index (BMI) (34.9 versus 26.1; P ≤ 0.0001), a larger liver span (9.8 versus 8.1 cm; P ≤ 0.0001), and higher triglycerides (252 versus 142.6; P ≤ 0.0001). The ultrasound reports recorded features consistent with fatty infiltration in 65.5% of NAFLD cases, compared to 5.3% of other liver diseases (P ≤ 0.0001). Diabetes mellitus was found in 35% of NAFLD and 6.7% of other cases (P ≤ 0.0001). The BMI was >30 in 79.8% of NAFLD cases and 22.7% of other liver disease cases (P ≤ 0.0001). The liver span was >8 cm in 78.6% of NAFLD cases and in only 16% of controls (P = 0.0001). On multivariate analysis using logistic regression, the odds ratio of having ultrasound report findings suggestive of fatty infiltration was 15.9 (CI, 4.1–60). The odds ratio was 9.4 (CI, 2.3–37.9) for diabetes, 5.0 (CI, 1.7–14.6) for BMI >30, and 2.3 for liver span >8 cm (CI, 1.36–3.90). A scoring system using clinical features and ultrasound was shown to reliably separate NAFLD from other cases of chronic hepatitis.     

非酒精性脂肪性肝炎发病机制探讨

目的建立大鼠非酒精性脂肪性肝炎（NASH）动物模型,探讨脂质过氧化、一氧化氮（NO）和一氧化氮合酶（NOS）以及肿瘤坏死因子α（TNF-α）、转化生长因子-β（TGF-β）等细胞因子在NASH致病中的作用.方法通过持续16周的高脂肪、高胆固醇饮食,建立大鼠NASH模型,造模结束时检测模型组和正常组血清转氨酶,血清及肝组织丙二醛（MDA）、超氧化物歧化酶（SOD）、NO、NOS、谷胱甘肽（GSH）含量.免疫组化标记α平滑肌肌动蛋白（α-SMA）、KP-1、TNF-α、TGF-β.结果实验结束时,模型组动物体重、肝脏指数比正常组显著增高（P值分别＜0.05和0.01）.血清转氨酶较正常组显著升高（P＜0.01）.除血清NO显著升高外（P＜0.05）,MDA、SOD、NOS、GSH均与正常组差异无统计学意义（P＞0.05）;而肝匀浆中,MDA、NO较正常组显著升高（P值分别＜0.05和＜0.01）,SOD、NOS、GSH则较正常组显著下降（P值分别＜0.05和0.01）.模型组α-SMA、KP-1、TNF-α、TGF-β免疫组化显示阳性细胞较正常组显著增加（P值均＜0.01）.结论脂质过氧化损伤增强,抗氧化能力降低,以及TNF-α、TGF-β、NO等炎性介质活化,共同参与了NASH的发病,并预示NASH可发展为肝纤维化、肝硬化.