New evidence for the therapeutic potential of curcumin to treat nonalcoholic fatty liver disease in humans - PubMed (original) (raw)
New evidence for the therapeutic potential of curcumin to treat nonalcoholic fatty liver disease in humans
María Eugenia Inzaugarat et al. PLoS One. 2017.
Abstract
Introduction: The immune system acts on different metabolic tissues that are implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Leptin and linoleic acid have the ability to potentially affect immune cells, whereas curcumin is a known natural polyphenol with antioxidant and anti-inflammatory properties.
Aims: This study was designed to evaluate the pro-inflammatory and pro-oxidant effects of leptin and linoleic acid on immune cells from patients with NAFLD and to corroborate the modulatory effects of curcumin and its preventive properties against the progression of NAFLD using a high-fat diet (HFD)-induced NAFLD/nonalcoholic steatohepatitis mouse model.
Results: The ex vivo experiments showed that linoleic acid increased the production of reactive oxygen species in monocytes and liver macrophages, whereas leptin enhanced tumor necrosis factor-α (TNF-α) production in monocytes and interferon-γ production in circulating CD4+ cells. Conversely, oral administration of curcumin prevented HFD-induced liver injury, metabolic alterations, intrahepatic CD4+ cell accumulation and the linoleic acid- and leptin- induced pro-inflammatory and pro-oxidant effects on mouse liver macrophages.
Conclusion: Our findings provide new evidence for the therapeutic potential of curcumin to treat human NAFLD. However, the development of a preventive treatment targeting human circulating monocytes and liver macrophages as well as peripheral and hepatic CD4+ cells requires additional research.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Fig 1. Diagram of the experimental design.
Experimental design using human peripheral blood mononuclear cells (PBMCs), human or mouse liver cells. PMA: phorbol myristate acetate, H2DCFDA: 2’7’-dichlorofluorescein diacetate.
Fig 2. Effect of linoleic acid on reactive oxygen species production in human monocytes and liver macrophages.
(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.
Fig 3. Effect of leptin on TNF-α and reactive oxygen species production in human monocytes.
(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.
Fig 4. The effects of leptin on IFN-γ production and T cell-associated alterations in liver samples from patients with NAFLD.
(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) 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. (C) 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.
Fig 5. The reversal effects of curcumin on peripheral immunological cells.
Ex vivo curcumin treatment of PBMCs from patients with NAFLD resulted in decreases in (A) linoleic acid-induced reactive oxygen species generation (n = 9, *p = 0.011) and (B) leptin-induced TNF-α production (n = 9, *p = 0.016) by monocytes. (C) 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.
Fig 6. 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<0.05, **p<0.01 and ***p<0.001, HFD vs. NC). Curcumin prevented HFD-induced weight gain (#p<0.05, ##p<0.01 and ###p<0.001, HFD vs. HFD+curcumin). (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<0.01). (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.
Fig 7. 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.
(A) 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) Ex vivo linoleic acid stimulation of hepatocytes from all the experimental groups resulted in similar stimulation indexes for reactive oxygen species production. (C) 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). (D) 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.
References
- Tilg H, Moschen AR. Evolution of inflammation in nonalcoholic fatty liver disease: The multiple parallel hits hypothesis. Hepatology (Baltimore, Md). 2010;52(5):1836–46. - PubMed
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