Anti-Inflammatory and Anti-Aging Evaluation of Pigment-Protein Complex Extracted from Chlorella Pyrenoidosa

Abstract

Oxidative stress contributes to chronic inflammatory processes implicated in aging, referred to as "inflamm-aging." In this study, the potential anti-inflammatory and anti-aging effects of a pigment-protein complex (PPC) from Chlorella pyrenoidosa were investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and D-galactose (D-gal)-induced aging in a murine model. Results indicated that PPC inhibits the production of the inflammatory cytokines TNF-α and IL-6, and the inflammatory mediator nitric oxide (NO) in LPS-stimulated RAW 264.7 cells. It also protected mice from D-gal induced informatory aging by increasing the activity of the antioxidant enzyme, such as superoxide dismutase (SOD), inhibiting D-gal-induced NF-κB upregulation, and increasing PPARs expression in the brain and gut. The findings indicated that PPC has favorable anti-inflammatory and anti-aging properties, and could be useful in the treatment of acute inflammation and senescence diseases.

Keywords: Chlorella pyrenoidosa; NF-κB; PPARs; anti-aging; anti-inflammation; pigment–protein complex.

Figure 1

Purification and characterization of the pigment–protein mixture from Chlorella pyrenoidosa. (a) Sephadex G-25 gel filtration chromatography; (b) Absorption spectrum of the pigment–protein complex (PPC); (c) HPLC analysis.

Figure 2

Effect of PPC on RAW 264.7 cells. (a) The viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The control group consisted of untreated cells and was considered as 100% of viable cells. Results were expressed as a percentage of viable cells when compared with the control group. (b) Nitric oxide (NO) production levels; (c) Expression levels of tumor necrosis factor (TNF)-α; (d) The levels of interleukin (IL)-6 production; (e) The levels of IL-10 production; (f) Phagocytosis rate. All the experiments were performed in triplicate. Duncan’s new multiple range test was performed to determine the significance differences. The values are expressed as the as mean ± SD. ^# p < 0.05 vs. the blank group, ^## p < 0.01 vs. the blank group, * p < 0.05 vs. the LPS group, and ** p < 0.05 vs. the lipopolysaccharide (LPS) group.

Figure 3

D-galactose (D-gal) induced KMB17 cells premature senescence in vitro. (A) Cells morphology changes: normal group (a), Model group D-gal-stimulated (b), Ascorbic acid group (c), 200 μg/mL PPC (d), 400 μg/mL PPC (e); (B) The effects of PPC on KMB17 cell viability. The experiments were performed in triplicate, and the values are expressed as the as mean ± SD. Results were expressed as a percentage of viable cells when compared with the control group.

Figure 4

Effect of PPC on superoxide dismutase (SOD), malondialdehyde (MDA), NO, IL-6, TNF-α levels, and the morphological features of gut in D-gal-induced mice. (a) Changes in MDA, (b) SOD, and (c) NO production levels; (d) The levels of IL-6 production; (e) Expression levels of TNF-α; (f) The morphological features of hematoxylin and eosin (H&E) stained gut sections, Scale bar = 50 μm. A one-way ANOVA was performed to compare the three experimental groups. All the data are mean ± SD of three independent experiments. ^# p < 0.05 vs. the control group, ^## p < 0.01 vs. the control group, * p < 0.05 vs. the model group, and ** p < 0.01 vs. the model group.

Figure 4

Effect of PPC on superoxide dismutase (SOD), malondialdehyde (MDA), NO, IL-6, TNF-α levels, and the morphological features of gut in D-gal-induced mice. (a) Changes in MDA, (b) SOD, and (c) NO production levels; (d) The levels of IL-6 production; (e) Expression levels of TNF-α; (f) The morphological features of hematoxylin and eosin (H&E) stained gut sections, Scale bar = 50 μm. A one-way ANOVA was performed to compare the three experimental groups. All the data are mean ± SD of three independent experiments. ^# p < 0.05 vs. the control group, ^## p < 0.01 vs. the control group, * p < 0.05 vs. the model group, and ** p < 0.01 vs. the model group.

Figure 5

(a) Effects of PPC on the expression levels of nuclear factor κB (NF-κB), PPARα, PPARγ, and p53 in mice, the proteins of brain and gut in each group were processed by Western blotting, and all data performed in triplicate; (b) Potential regulation pathways by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software (http://string.embl.de/); (c) Proposed mechanism of action on brain and intestine.