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. 2019 Aug 2;8(8):270.
doi: 10.3390/antiox8080270.

Inhibition of LPS-Induced Oxidative Damages and Potential Anti-Inflammatory Effects of Phyllanthus emblica Extract via Down-Regulating NF-κB, COX-2, and iNOS in RAW 264.7 Cells

Hui Min-David Wang  1   2   3   4 , Ling Fu  2 , Chia Chi Cheng  5 , Rong Gao  6 , Meng Yi Lin  7 , Hong Lin Su  5 , Nathania Earlene Belinda  8 , Thi Hiep Nguyen  9 , Wen-Hung Lin  10 , Po Chun Lee  11 , Liang Po Hsieh  12
Affiliations

Inhibition of LPS-Induced Oxidative Damages and Potential Anti-Inflammatory Effects of Phyllanthus emblica Extract via Down-Regulating NF-κB, COX-2, and iNOS in RAW 264.7 Cells

Hui Min-David Wang et al. Antioxidants (Basel). .

Abstract

Phyllanthus emblica is an edible nutraceutical and functional food in the Asia area with medicinal and nutritive importance. The fruit extract of P. emblica is currently considered to be one of the effective functional foods for flesh maintenance and disease treatments because of its antioxidative and immunomodulatory properties. We examined the antioxidant abilities of the fruit extract powder by carrying out 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, iron reducing power, and metal chelating activity analysis and showed excellent antioxidative results. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the result showed that the samples had no cytotoxic effect on RAW 264.7 cells even at a high concentration of 2 mg/mL. To investigate its immunomodulatory function, our estimation was to treat it with lipopolysaccharide (LPS) in RAW 264.7 cells to present anti-inflammatory capacities. The extract decreased reactive oxygen species (ROS) production levels in a dose-dependent manner measured by flow cytometry. We also examined various inflammatory mRNAs and proteins, including nuclear factor-κB (NF-κB), inducible nitric oxide synthases (iNOS), and cyclooxygenase-2 (COX-2). In quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting assay, all three targets were decreased by the extract, also in a dose-dependent manner. In conclusion, P. emblica fruit extract powder not only lessened antioxidative stress damages, but also inhibited inflammatory reactions.

Keywords: COX-2; NF-κB; Phyllanthus emblica; antioxidant; iNOS.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The cell viability of RAW 264.7 cells was measured by MTT assay after 24 h treatment of P. emblica samples. The data represented mean ± S.D. of three independent experiments performed. The red dash line is the trend line for cell survival rate of 100%. * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
The reactive oxygen species (ROS) percentage measured by a flow cytometry. RAW 264.7 cells were pretreated with P. emblica samples (0.125–2 mg/mL) for 1 h, and then stimulated with LPS (5 μg/mL) for 6 h. The data represented mean ± S.D of three independent experiments performed. * p < 0.05, ** p < 0.01.
Figure 3
Figure 3
The inflammation-related mRNA expressions in RAW 264.7 cells. RNA expression levels of NF-κB, iNOS, COX-2 in RAW 264.7 cells treated with different concentrations of P. emblica samples (0.25–2 mg/mL) were evaluated by qRT-PCR and normalized to the GAPDH gene. The data represented mean ± S.D of three independent experiments performed. * p < 0.05, ** p < 0.01.
Figure 4
Figure 4
The inflammation-related protein expressions. (A) NF-κB, iNOS, and COX-2 expressions in RAW 264.7 cells were pretreated with P. emblica samples (0.25–2 mg/mL) for 1 h, and then were stimulated with LPS (5 μg/mL) for 6 h. (B1) Protein quantification of NF-κB (B2) iNOS (B3) COX-2 in western blotting. β-Actin was viewed as an internal control. * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
The cartoon illustration of antioxidation and anti-inflammation mechanisms of P. emblica fruit extract powder.
See this image and copyright information in PMC

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