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. 2019 Mar 1;24(5):875.
doi: 10.3390/molecules24050875.

In Vitro and In Vivo Anti-Inflammatory Effects of Polyphyllin VII Through Downregulating MAPK and NF-κB Pathways

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Free PMC article

In Vitro and In Vivo Anti-Inflammatory Effects of Polyphyllin VII Through Downregulating MAPK and NF-κB Pathways

Chao Zhang et al. Molecules. .
Free PMC article

Abstract

Background: Polyphyllin VII (PP7), a steroidal saponin from Paris polyphylla, has been found to exert strong anticancer activity. Little is known about the anti-inflammatory property of PP7. In this study, the anti-inflammatory activity and its underlying mechanisms of PP7 were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in multiple animal models. Methods: The content of nitric oxide (NO) was determined by spectrophotometry. The levels of prostaglandin E2 (PGE₂) and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) assay. The mRNA expression of pro-inflammatory genes was determined by qPCR. The total and phosphorylated protein levels were examined by Western blotting. The in vivo anti-inflammatory activities were evaluated by using mouse and zebrafish models. Results: PP7 reduced the production of NO and PGE₂ and the protein and mRNA expressions of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and enzymes (inducible NO synthase [iNOS], cyclooxygenase-2 [COX-2], and Matrix metalloproteinase-9 [MMP-9]) in LPS-induced RAW264.7 cells by suppressing the NF-κB and MAPKs pathways. Notably, PP7 markedly inhibited xylene-induced ear edema and cotton pellet-induced granuloma formation in mice and suppressed LPS and CuSO₄-induced inflammation and toxicity in zebrafish embryos. Conclusion: This study demonstrates that PP7 exerts strong anti-inflammatory activities in multiple in vitro and in vivo models and suggests that PP7 is a potential novel therapeutic agent for inflammatory diseases.

Keywords: anti-inflammation; macrophage; mice; polyphyllin VII; zebrafish.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effects of Polyphyllin VII (PP7) on lipopolysaccharide (LPS)-induced nitric oxid (NO) production (A), prostaglandin E2 (PGE2) release (B), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA expression were evaluated in RAW264.7 cells. RAW264.7 cells were pre-treated with different concentrations (0.75, 1.00, 1.25, and 1.50 μM) of PP7 for 1 h and then incubated with or without 1 μg/mL LPS for a further 24 h. NO and PGE2 production in the medium was determined by using Griess reagent and enzyme immunoassay, respectively. iNOS and COX-2 protein levels were determined via Western blotting (C). The mRNA levels of iNOS and COX-2 were determined by qPCR (D,E). Values represent the means ± SD of at least three independent experiments. * p < 0.05 and ** p < 0.01 were compared with LPS-alone group.
Figure 2
Figure 2
Effect of PP7 on the LPS-induced pro-inflammatory cytokines protein and mRNA expression in RAW264.7 cells. RAW264.7 cells were pretreated with different concentrations (0.75, 1.00, 1.25, and 1.50 μM) of PP7 for 1 h and then incubated with or without 1 μg/mL LPS for a further 24 h. Supernatants were collected, and the tumor necrosis factor (TNF)-α (A), interleukin (IL)-1β (B), and IL-6 (C) production in the supernatants were determined by ELISA. The mRNA levels of TNF-α (D), IL-1β (E), and IL-6 (F) were determined by RT-PCR. Values represent the means ± SD of triplicate experiments. * p < 0.05 and ** p < 0.01 were compared with LPS-alone group.
Figure 3
Figure 3
PP7 suppresses LPS-induced NF-κB p65 nuclear translocation and IκB-α degradation and phosphorylation in RAW264.7 cells. Cells were pre-treated with or without different concentrations (0.75, 1.00, 1.25, and 1.50 μM) of PP7 for 1 h, LPS (1 μg/mL) was then added and cells were incubated for 30 min. Western blot analysis for NF-κB p65 in the nuclear (A) and cytoplasmic (B) extracts of RAW264.7 cells. (C,D) were densitometric analysis of (A,B). The levels of p-IκB-α and total IκB-α in the cytoplasm of RAW264.7 cells were determined by Western blotting (E,F). Values are expressed as means ± SD of triplicate experiments. * p < 0.05 and ** p < 0.01 were compared with LPS-alone group.
Figure 4
Figure 4
Inhibitory effect of PP7 on the LPS-induced MAPKs and MMP-9 proteins in RAW264.7 cells. Cells were pre-treated for 1 h with or without PP7 at indicated concentrations (0.75, 1.00, 1.25, and 1.50 μM) and then stimulated with LPS (1 μg/mL) for 15 min (BD) or 24 h (E). (A) The levels of p-ERK, ERK, p-JNK, JNK, p-p38, p38, and MMP-9 were determined by Western blotting. (BE) were densitometric analysis of (A). Values are expressed as means ± SD of triplicate experiments. ** p < 0.01 were compared with LPS-alone group.
Figure 5
Figure 5
Anti-inflammatory and protective effects of PP7 in LPS-induced inflammation in zebrafish embryo. Zebrafish embryos at 8 h post-fertilization (hpf) were pretreated with PP7 and exposed to LPS. (A) The NO generation in zebrafish larvae was measured by image analysis and fluorescence microscope after staining with 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM-DA). Scale bars represent 200 μm. (B) Quantitative analysis of fluorescence intensity of (A) using an image J program. Effects of PP7 on heart beat rate (C) and yolk sac edema size (D) of LPS treated zebrafish embryos. Data are represented as means ± SD from three independent experiments. n = 10/group, * p < 0.05, ** p < 0.01 as compared with LPS-treated alone.
Figure 6
Figure 6
PP7 affects CuSO4 induced inflammation in zebrafish. (A) BACmpx::GFP transgenic zebrafish larvae at 3 days post-fertilization (dpf) were treated with indicated concentrations of PP7 for 1 h prior to CuSO4 treatment (20 μM CuSO4 for 1 h) and were monitored for neutrophils present at the myoseptum. Scale bars represent 200 μm. (B) Quantification of numbers of neutrophils recruited to the lateral line after CuSO4 treatment. Data are represented as means ± SD from three independent experiments. n = 10/group, ** p < 0.01 as compared with CuSO4-treated alone.

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References

    1. Reuter S., Gupta S.C., Chaturvedi M.M., Aggarwal B.B. Oxidative stress, inflammation, and cancer: How are they linked? Free Radic. Biol. Med. 2010;49:1603–1616. doi: 10.1016/j.freeradbiomed.2010.09.006. - DOI - PMC - PubMed
    1. Bruscia E.M., Bonfield T.L. Cystic Fibrosis Lung Immunity: The Role of the Macrophage. J. Innate Immun. 2016;8:550–563. doi: 10.1159/000446825. - DOI - PMC - PubMed
    1. Decano J.L., Mattson P.C., Aikawa M. Macrophages in Vascular Inflammation: Origins and Functions. Curr. Atheroscler. Rep. 2016;18:1–7. doi: 10.1007/s11883-016-0585-2. - DOI - PubMed
    1. Gopinath V.K., Musa M., Samsudin A.R., Sosroseno W. Role of interleukin-1beta and tumour necrosis factor-alpha on hydroxyapatite-induced phagocytosis by murine macrophages (RAW264.7 cells) Br. J. Biomed. Sci. 2006;63:176–178. doi: 10.1080/09674845.2006.11978094. - DOI - PubMed
    1. Moncada S. Nitric oxide: Discovery and impact on clinical medicine. J. R. Soc. Med. 1999;92:164–169. doi: 10.1177/014107689909200402. - DOI - PMC - PubMed

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