Nutr Res Pract.  2023 Oct;17(5):827-843. 10.4162/nrp.2023.17.5.827.

Effects of Pogonatherum paniceum (Lamk) Hack extract on antimitochondrial DNA mediated inflammation by attenuating Tlr9 expression in LPS-induced macrophages

• Thongboontho R
• Petcharat K
• Munkong N
• Khonthun C
• Boondech A
• Phromnoi K
• Thim-uam A

• KMID: 2546942
• DOI: http://doi.org/10.4162/nrp.2023.17.5.827

Abstract

BACKGROUND/OBJECTIVES
Mitochondrial DNA leakage leads to inflammatory responses via endosome activation. This study aims to evaluate whether the perennial grass water extract (Pogonatherum paniceum) ameliorate mitochondrial DNA (mtDNA) leakage.
MATERIALS/METHODS
The major bioactive constituents of P. paniceum (PPW) were investigated by high-performance liquid chromatography, after which their antioxidant activities were assessed. In addition, RAW 264.7 macrophages were stimulated with lipopolysaccharide, resulting in mitochondrial damage. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay were used to examine the gene expression and cytokines.
RESULTS
Our results showed that PPW extract-treated activated cells significantly decrease reactive oxygen species and nitric oxide levels by reducing the p22 ^phox and iNOS expression and lowering cytokine-encoding genes, including IL-6, TNF-α, IL-1β, PG-E2 and IFN-γ relative to the lipopolysaccharide (LPS)-activated macrophages. Furthermore, we observed that LPS enhanced the mtDNA leaked into the cytoplasm, increasing the transcription of Tlr9 and signaling both MyD88/Irf7-dependent interferon and MyD88/NF-κb p65-dependent inflammatory cytokine mRNA expression but which was alleviated in the presence of PPW extract.
CONCLUSIONS
Our data show that PPW extract has antioxidant and anti-inflammatory activities by facilitating mtDNA leakage and lowering the Tlr9 expression and signaling activation.

Keyword

Medicinal plants; antioxidants; inflammation; mitochondrial DNA; endosome

Figure

• 

  Fig. 1 HPLC chromatograms of polyphenolic constituents in PPW. (A) Gallic acid, (B) Catechin, and (C) Quercetin.HPLC, high-performance liquid chromatography; PPW, Pogonatherum paniceum (Lamk) Hack extract.

• 

  Fig. 2 Cytotoxic and antioxidant effects of PPW on macrophages. Macrophages were cultured and treated with different concentrations of PPW extract (10–800 ug/mL) for 24 and 48 h. (A and D) Cell viability was determined with the MTS assay using untreated cells as the control. Cells and supernatants were collected and analyzed for (B) ROS and (C) NO (n = 3 per group). mRNA expression of (E) p22phox , (F) iNOS, (G) Catalase, (H) Nrf2, and (I) Sod2 were investigated using qPCR (n = 3–6 per group). Data are presented as mean ± SEM. No significant differences are indicated between LPS-stimulated cells with or without PPW treatment.PPW, Pogonatherum paniceum (Lamk) Hack extract; MTS, 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium; ROS, reactive oxygen species; NO, nitric oxide; qPCR, quantitative polymerase chain reaction; SEM, standard error of mean; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; ns, not significant.*P < 0.05, **P < 0.01, and ***P < 0.001.###P < 0.001, untreated cells compared with LPS-stimulated cells.

• 

  Fig. 3 Effects of PPW on anti-proinflammatory cytokine production. Cells were collected and examined the mRNA expression of (A) IL-6, (B) TNF-α, (C) IL-1β, and (G) Cox2 by qPCR (n = 3–4 per group). The concentration of pro-inflammatory cytokines (D) IL-6, (E) TNF-α, (F) IL-1β, and (H) PGE2 (n = 3–4 per group) in the supernatant were examined by ELISA. Data are presented as mean ± SEM.PPW, Pogonatherum paniceum (Lamk) Hack extract; IL, interleukin; TNF-α, tumor necrosis factor α; COX6, cytochrome c oxidase subunit 6; qPCR, quantitative polymerase chain reaction; PGE2, prostaglandin E2; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of mean.*P < 0.05; **P < 0.01, and ***P < 0.001.

• 

  Fig. 4 Effects of PPW on mitochondrial DNA leakage. Cells were collected after the treatment of PPW with or without LPS and determined (A) the mtDNA leakage in cytoplasm (n = 4–6 per group). The mtDNA transcription level of (B) Cox6 and (C) ND1 was examined by qPCR (n = 3). Data are presented as mean ± SEM.PPW, Pogonatherum paniceum (Lamk) Hack extract; LPS, lipopolysaccharide; mtDNA, mitochondrial DNA; COX6, cytochrome c oxidase subunit 6; ND1, NADH-ubiquinone oxidoreductase chain 1; qPCR, quantitative polymerase chain reaction; SEM, standard error of mean.*P < 0.05, **P < 0.01, and ***P < 0.001.

• 

  Fig. 5 The effect of PPW on endosome activation. Cells were collected and the mRNA transcription level of (A) Tlr9, (B) Tlr7, (C) Tlr8, (D) MyD88, (E) Irf7, (F) Nf-Kb p65, and (G) Ifnγ was examined by qPCR (n = 3–5 per group). (H) Supernatants were collected to measure the level of IFN-γ production (n = 3–5 per group). Data are presented as mean ± SEM. No significant differences are indicated between LPS-stimulated cells with or without PPW extract.PPW, Pogonatherum paniceum (Lamk) Hack extract; qPCR, quantitative polymerase chain reaction; IFN, interferon; ns, not significant; SEM, standard error of mean; LPS, lipopolysaccharide.*P < 0.05, **P < 0.01, and ***P < 0.001.

• 

  Fig. 6 Schematic summary of PPW on anti-inflammation in LPS-stimulated macrophages. PPW reduced the inflammatory response by decreasing the expression of Tlr9 expression and signaling. This figure was created using Biorender.com.PPW, Pogonatherum paniceum (Lamk) Hack extract; LPS, lipopolysaccharide; TLR, Toll-like receptor; ROS, reactive oxygen species; dsDNA, double stranded DNA; MYD88, myeloid differentiation primary response 88; NF-κB, nuclear factor kappa B; IRF, interferon regulatory factor; IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; iNOS, inducible nitric oxide synthase; COX2, cytochrome c oxidase subunit 2; NO, nitric oxide.

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