Folic acid ameliorates palmitate-induced inflammation through decreasing homocysteine and inhibiting NF-κB pathway in HepG2 cells.

OBJECTIVE: Prevention of inflammation is one of the possible remedy procedure for steatohepatitis during NAFLD. In this study, we researched the folic acid (FA) potency to attenuate the inflammation of palmitate-treated HepG2 cells and the related signalling pathways. METHODS: The molecular mechanisms related to FA anti-inflammatory effect in palmitate and Hcy-treated HepG2 cell line were assessed. RESULTS: The results indicated that while palmitate enhances the expression and secretion of TNF-α, IL-6, and IL-1ß, and also intracellular ROS level, FA at concentrations of 25, 50, and 75 µg/mL significantly reversed these effects in HepG2 cells. In addition, FA could ameliorate inflammation and decrease ROS production induced by Hcy. Furthermore, FA pre-treatment suppress palmitate -induced (NF-κB) p65 level in palmitate or Hcy stimulated cells. CONCLUSIONS: Overall, these results suggest that FA reduces inflammation in HepG2 cells through decreasing ROS and Hcy concentration level resulting in inhibiting the NF-κB pathway.

Vitamin D3 and estradiol alter PAD2 expression and activity levels in C6 glioma cells.

Multiple Sclerosis (MS) is known as a chronic demyelinating disease with multifactorial etiology. It is suggested that the deimination of myelin basic proteins (MBPs) by peptidyl arginine deiminase 2 (PAD2) may increase citrulline residues resulting in the reduction of myelin sheath density and the progression of multiple sclerosis. The aim of this study was to investigate the effects of vitamin D (25-hydroxy cholecalciferol (D3)) and estradiol on PAD2 gene expression level and its catalytic activity in rat C6 glioma cells. C6 glioma cells were cultured in DMEM medium and were treated with vitamin D (10 and 100 ng/ml) and estradiol (10 and 100 µM) based on the cellular viability. Then, the PAD2 gene expression and catalytic activity were evaluated using real-time qRT-PCR and spectrophotometry techniques, respectively. The PAD2 gene expression level and its catalytic activity increased significantly in estradiol-treated cells (P = 0.0435 and P = 0.0015, respectively). Conversely, vitamin D downregulated significantly the PAD2 gene expression level (P < 0.015) and its activity (P < 0.017). The study results suggested that estradiol conversely with vitamin D increases the activity of the PAD2 enzyme so that it might develop multiple sclerosis, especially in women.

Anti-Inflammatory Effects of Plasma Circulating Exosomes Obtained from Normal-Weight and Obese Subjects on Hepatocytes.

INTRODUCTION: Obesity is a disorder with low-grade chronic inflammation that plays a key role in hepatic inflammation and steatosis. Moreover, there are studies to support the role of exosomes in cellular communications, the regulation of metabolic homeostasis and immunomodulatory activity. Accordingly, we aimed to evaluate the influence of plasma circulating exosomes derived from females with normal-weight and obesity on the secretion of inflammatory cytokines in human liver cells. METHODS: Plasma circulating exosomes were isolated from four normal (N-Exo) and four obese (OExo) women. The exosomes were characterized and approved for CD63 expression (common exosomal protein marker) and morphology/size using the western blot and TEM methods, respectively. The exosomes were used for the stimulation of HepG2 cells in vitro. After 24 h of incubation, the protein levels of TNF-α, IL-6, and IL-1ß were measured in the culture supernatant of HepG2 cells using the ELISA kit. RESULTS: The protein levels of IL-6 and TNF-α in the cells treated with O-Exo and N-Exo reduced significantly in comparison with the control group (P=0.039 and P<0.001 respectively), while significant differences were not found between normal and obese groups (P=0.808, and P=0.978 respectively). However, no significant differences were found among the three groups in terms of IL-1ß levels (P=0.069). Based on the correlation analysis, the protein levels of IL-6 were positively correlated with TNF-α (r 0.978, P<0.001). CONCLUSION: These findings suggest that plasma circulating exosomes have probably antiinflammatory properties independent of body mass index and may decrease the secretion of inflammatory cytokines in the liver. However, further in vitro and in vivo investigations are needed to address the anti-inflammatory function of N-Exo and O-Exo in human liver cells and/or other cells.

Combination of metformin and genistein alleviates non-alcoholic fatty liver disease in high-fat diet-fed mice.

Metformin (MET) and genistein (GEN) have a beneficial role in alleviating non-alcoholic fatty liver disease (NAFLD), but their combined effect on this disease has not yet been studied. The present study aimed to investigate the potential protective effects of combined MET and GEN on NAFLD in high-fat diet (HFD) fed mice. C57BL/6 male mice were fed on an HFD for 10 weeks. Animals were then divided into different groups and treated with MET (0.23%), GEN (0.2%) and MET+GEN (0.23% + 0.2%) for 3 months. Treatment with MET and GEN, alone or in combination significantly lowered body and liver weights and fasting blood glucose (FBG) in HFD mice. Combination therapy reduced liver triglyceride (TG) level and this effect was correlated with increased expression of carnitine palmitoyl transferase 1 (CPT1) gene, and reduced expression of fatty-acid synthase (FAS)and sterol regulatory element-binding protein-1c (SREBP-1c) genes. Combination therapy also affects gluconeogenesis pathway through decreasing expression of Glucose 6-phosphatase (G6Pase) and increasing phosphorylation of Glycogen synthase kinase 3ß (GSK-3ß). Furthermore, combination of MET and GEN ameliorates liver inflammation by switching macrophage into M2 phenotype, decreasing macrophage infiltration, reducing expression of pro-inflammatory cytokines and decreasing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity. In addition, combination therapy enhances phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK). Taken together, these findings suggest that the combination of MET and GEN have beneficial effects against NAFLD in HFD-fed model.

Evaluation of eight formulas for LDL-C estimation in Iranian subjects with different metabolic health statuses.

BACKGROUND: Considering the crucial role of low-density lipoprotein-cholesterol (LDL-C) concentration in determining cardiovascular risk, the accuracy of LDL-C estimation is essential. To date, various types of formulae have been introduced, albeit their accuracy has not been assessed in varied populations. In this study, the accuracy of eight formulae for LDL-C estimation was evaluated in an Iranian population. METHODS: A data set of 2752 individuals was included in the study and all samples were analyzed in term of lipid profiles using direct homogeneous assay. The population was divided into various subgroups based on the triglyceride (TG), high-density lipoprotein- cholesterol (HDL-C), total cholesterol (TC), fasting blood sugar (FBS) and age values and estimated LDL-C values by Friedewald, Chen, de Cordova, Vujovic, Anandaraja, Hattori, Ahmadi, and Puavillai equations were compared to the directly measured LDL-C in each subgroup. RESULTS: Estimated LDL-C values by Puavillai formulae showed an insignificant difference compared to the directly measured LDL-C in subjects with high level of TG. However, for TG range < 3.38 mmol/L and high levels of HDL-C, the difference between the means of estimated LDL-C by Hattori and de Cordova formulas, and directly measured LDL-C was relatively lower than other equations. In addition, estimated LDL-C by Hattori and de Cordova formulae had insignificant differences as compared to the direct LDL-C at some levels of cholesterol, the normal level of FBS and some age ranges. CONCLUSIONS: Therefore, it seems that Hattori and de Cordova formulas can be considered as the best alternatives for LDL-C direct measurement in the Iranian population, especially for healthy subjects.

Calcium and TNFα additively affect the chondroitinase ABC I activity.

Experimental models have repeatedly shown the therapeutic potential of cABC I in spinal cord injury (SCI) by degrading chondroitin sulfate proteoglycans that hinder neurite outgrowth. Following SCI, some molecules are released from injured cells. This study is designed to determine the effects of some of these molecules at the SCI loci on activity, stability and structure of wild type and Q140A variant of cABC I. The effect of Ca2+, ATP, adenosine, Asp, Glu, Gln, TNFα, and a combination of them in physiologic and pathologic concentrations was assessed. The results showed that Ca2+ and TNFα have increasing and additive effects on the enzymes activity. Meanwhile, the other molecules had neither considerable effect on the activity nor on thermal stability of the enzymes, significantly. Structural analyses of wild type and mutant cABC I in the presence and absence of Ca2+ were also carried out using fluorescence and far-UV circular dichroism techniques. Although, the secondary and tertiary structure of enzymes showed no significant alterations in the presence of Ca2+, but fluorescence quenching data indicated that calcium increases flexibility of the wild type enzyme, slightly. Therefore, it can be concluded that this ion affect enzyme activity without remarkable conformational changes.

Erythrocytes Membrane Alterations Reflecting Liver Damage in CCl4-Induced Cirrhotic Rats: The Ameliorative Effect of Naltrexone.

Cirrhosis is the consequence of chronic liver disease. Deleterious effects of oxidative stress on hepatocytes may be reflected in the erythrocyte membrane. Naltrexone (NTX) has been shown to attenuate hepatocellular injury in fibrotic animal models. The aim of this study was to investigate the progressive effect of CCl4 on the liver and whether the improvement of liver cirrhosis can be monitored through alterations in the erythrocyte membrane. In this study, 84 male Wistar rats were divided into 4 groups and received reagents (i.p.) as follows: 1- CCl4, 2- NTX + CCl4, 3- Mineral Oil (M), and 4- NTX + M. After 2, 6 and 8 weeks, the blood and liver tissue samples were collected. Plasma enzyme activities, the content of erythrocyte GSH and some membrane compositions, including protein carbonyl, protein sulfhydryl, and malondialdehyde were assessed. After 6 and 8 weeks, plasma enzyme activities and the content of protein carbonyl were higher in CCl4 group significantly, as compared to other groups (P<0.001). NTX significantly diminished protein carbonyl and plasma enzyme activities (P<0.001). GSH did not change until the 6th week. However, CCl4+NTX increased it significantly as compared to CCl4 group (P<0.05). Protein sulfhydryl showed changes in NTX+CCl4 group which indicated a significant increase in protein sulfhydryl content in a 6th week compared to CCl4 group (P<0.05). MDA did not show any significant alteration. CCl4-induced cirrhosis is accompanied by increased content of oxidative stress markers, especially protein carbonyl of RBC membrane and plasma enzyme activities. This study shows that the progression of liver cirrhosis and the ameliorative effect of NTX can be followed through alterations of these markers.