HFD-induced metabolic and histological alterations in the liver and the reversal effects of curcumin.

(A) Top: Hematoxylin and eosin staining of liver sections from normal chow (NC)-fed and high-fat diet (HFD)-fed mice with or without curcumin supplementation (10X and 40X magnification). Curcumin prevented HFD-induced steatosis, ballooning and liver inflammation. Bottom: Masson's trichrome staining of liver sections from HFD-fed mice reveals signs of mild perisinusoidal fibrosis (10X and 40X magnification). (B) Body weight gain in NC- and HFD-fed mice with or without curcumin supplementation, starting at week 4 (arrow) (*p(C) The blood glucose concentrations were measured upon conclusion of the dietary treatments, and glycemia was determined at basal conditions (Basal) and after glucose administration. Left: a similar blood glucose concentration was observed among the four groups. Right: hyperglycemia was observed in HFD-fed mice at 120 min after an intraperitoneal glucose injection (2 g/kg). Curcumin ameliorated the hyperglycemic conditions. (*p(D) Total serum cholesterol levels were measured upon conclusion of the dietary treatments. The HFD induced higher levels of cholesterol than the normal chow regardless of curcumin administration (*p<0.05). The box and whiskers show the non-parametric statistics: the median, lower and upper quartiles and confidence interval around the median. The Kruskal-Wallis test with Dunn’s post-test was performed.</p

Effect of linoleic acid on reactive oxygen species production in human monocytes and liver macrophages.

<p>(A) The stimulation index for reactive oxygen species production in monocytes was higher in patients with NAFLD (n = 12) than in control subjects (n = 10). The box and whiskers indicate the non-parametric statistics: the median, lower and upper quartiles and confidence interval around the median. A two-tailed Mann-Whitney U test was used; *p = 0.036. (B) DCF-MFI, 2', 7’-dichlorofluorescein median fluorescence intensity. Linoleic acid increased reactive oxygen species production in liver macrophages from patients with NAFLD (n = 12). Lines connect the “Basal” and “Linoleic acid” values for each patient. A Wilcoxon matched-pairs signed rank test was performed; *p = 0.001. (C) The stimulation index in monocytes and liver macrophages from patients with NAFLD were positively correlated. Spearman´s rank correlation coefficients test was used.</p

Demographic, anthropometric and biochemical baseline data of patients with NAFLD and control individuals.

<p>Demographic, anthropometric and biochemical baseline data of patients with NAFLD and control individuals.</p

The effects of leptin on IFN-γ production and T cell-associated alterations in liver samples from patients with NAFLD.

<p>(A) The fold of increase index for IFN-γ production in leptin-stimulated circulating CD4⁺ cells was higher in patients with NAFLD (n = 10) than in control subjects (n = 10; *p = 0.011). <b>(B)</b> The percentage of CD4⁺ cells among the total non-parenchymal cell population was higher in patients with NAFLD (n = 10) than in control subjects (n = 10), *p = 0.030. <b>(C)</b> Compared with control subjects (n = 9), patients with NAFLD (n = 9) showed increased hepatic mRNA expression levels of IFN-γ (*p = 0.012), T-bet (*p = 0.020) and CCL20 (*p = 0.007) as measured by quantitative PCR. The 2^-ΔΔCt method was used to calculate the mRNA fold change. The box and whiskers indicate the non-parametric statistics: the median, lower and upper quartiles and confidence interval around the median. A two-tailed Mann-Whitney U test was used for the statistical analysis.</p

Curcumin effects on linoleic acid- and leptin-induced the production of reactive oxygen species and cytokines as well as the infiltration of CD4⁺ cells in HFD-fed mice.

<p><b>(A)</b> After they were treated with linoleic acid ex vivo, liver macrophages from HFD-fed mice showed a higher stimulation index for reactive oxygen species production (*p<0.05 vs. normal chow-fed mice). In vivo curcumin administration of HFD-fed mice (HFD+curcumin) prevented the increase in the stimulation index (*p<0.05 vs. HFD-fed mice). <b>(B)</b> Ex vivo linoleic acid stimulation of hepatocytes from all the experimental groups resulted in similar stimulation indexes for reactive oxygen species production. <b>(C)</b> TNF-α production induced by ex vivo leptin treatment was higher in liver macrophages from HFD-fed mice (*p<0.05 vs. normal chow-fed mice). In vivo curcumin treatment of HFD-fed mice also prevented the increase in TNF- α production (*p<0.05, HFD+curcumin vs. HFD). <b>(D)</b> The percentage of CD4⁺ cells among the non-parenchymal cell populations was higher in HFD-fed mice (*p<0.01 vs. normal chow-fed mice). In vivo curcumin treatment also prevented the increase in CD4⁺ cell recruitment (*p<0.01, HFD+curcumin vs. HFD). The box and whiskers show the non-parametric statistics: the median, lower and upper quartiles and confidence interval around the median. The Kruskal-Wallis test with Dunn’s post-test was performed.</p

Diagram of the experimental design.

<p>Experimental design using human peripheral blood mononuclear cells (PBMCs), human or mouse liver cells. PMA: phorbol myristate acetate, H₂DCFDA: 2’7’-dichlorofluorescein diacetate.</p

The reversal effects of curcumin on peripheral immunological cells.

<p>Ex vivo curcumin treatment of PBMCs from patients with NAFLD resulted in decreases in <b>(A)</b> linoleic acid-induced reactive oxygen species generation (n = 9, *p = 0.011) and <b>(B)</b> leptin-induced TNF-α production (n = 9, *p = 0.016) by monocytes. <b>(C)</b> Ex vivo curcumin treatment of PBMCs from patients with NAFLD resulted in decreased IFN-γ production in CD4⁺ cells (n = 9, *p = 0.048). Lines connect the “Linoleic acid” and “Linoleic acid+Curcumin” stimulation indexes or the “Leptin” and "Leptin+Curcumin" fold of increase indexes for each patient. A Wilcoxon matched-pairs signed rank test was performed.</p

Effect of leptin on TNF-α and reactive oxygen species production in human monocytes.

<p>(A) The fold of increase index for TNF-α production was higher in monocytes from patients with NAFLD (n = 10) than those from control subjects (n = 10); however, when monocytes were stimulated with leptin, the stimulation index for reactive oxygen species production (B) was similar in patients with NAFLD (n = 10) and control subjects (n = 10). The box and whiskers indicate the non-parametric statistics: median, lower and upper quartiles and confidence interval around the median. A two-tailed Mann-Whitney U test was used, *p = 0.004.</p

Data_Sheet_1_Bayesian network analysis of panomic biological big data identifies the importance of triglyceride-rich LDL in atherosclerosis development.docx

IntroductionWe sought to explore biomarkers of coronary atherosclerosis in an unbiased fashion.MethodsWe analyzed 665 patients (mean ± SD age, 56 ± 11 years; 47% male) from the GLOBAL clinical study (NCT01738828). Cases were defined by the presence of any discernable atherosclerotic plaque based on comprehensive cardiac computed tomography (CT). De novo Bayesian networks built out of 37,000 molecular measurements and 99 conventional biomarkers per patient examined the potential causality of specific biomarkers.ResultsMost highly ranked biomarkers by gradient boosting were interleukin-6, symmetric dimethylarginine, LDL-triglycerides [LDL-TG], apolipoprotein B48, palmitoleic acid, small dense LDL, alkaline phosphatase, and asymmetric dimethylarginine. In Bayesian analysis, LDL-TG was directly linked to atherosclerosis in over 95% of the ensembles. Genetic variants in the genomic region encoding hepatic lipase (LIPC) were associated with LIPC gene expression, LDL-TG levels and with atherosclerosis.DiscussionTriglyceride-rich LDL particles, which can now be routinely measured with a direct homogenous assay, may play an important role in atherosclerosis development.Clinical trial registrationGLOBAL clinical study (Genetic Loci and the Burden of Atherosclerotic Lesions); [https://clinicaltrials.gov/ct2/show/NCT01738828?term=NCT01738828&rank=1], identifier [NCT01738828].</p