J Vet Sci.  2016 Sep;17(3):315-321. 10.4142/jvs.2016.17.3.315.

Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells

• Bernardo Reyes AW
• Simborio H
• Hop HT
• Arayan LT
• Min WG
• Lee HJ
• Rhee MH
• Chang HH
• Kim S

• KMID: 2413130
• DOI: http://doi.org/10.4142/jvs.2016.17.3.315

Abstract

Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

Keyword

Brucella abortus; Korean red ginseng; macrophage; phagocytosis; red ginseng acidic polysaccharide

MeSH Terms

Animals
Brucella abortus/*drug effects
Brucellosis/microbiology/*prevention & control/veterinary
Macrophages/drug effects
Mice
Panax/*chemistry
Phagocytosis/drug effects
Polysaccharides/*pharmacology
RAW 264.7 Cells
Signal Transduction/drug effects
Polysaccharides

Figure

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  Fig. 1 Effect of red ginseng acidic polysaccharide (RGAP) on invasion and intracellular growth of Brucella (B.) abortus. (A) Bacterial internalization efficiency. (B) Bacterial intracellular growth efficiency. Data represent the means ± SD of duplicate samples in at least three independent experiments. Statistically significant differences relative to the untreated control are indicated by asterisks (*p < 0.05; **p < 0.01; ***p < 0.001).

• 

  Fig. 2 Effect of RGAP on the adherence of B. abortus to macrophages. Fluorescence images were collected using a microscope equipped with a camera. One hundred macrophages were selected at random, and the bacteria that adhered to the cells were counted. Data represent the mean ± SD of triplicate samples in at least three independent experiments. Statistically significant differences relative to the untreated control are indicated by asterisks (***p < 0.001).

• 

  Fig. 3 Fluorescence-activated cell sorting (FACS) analysis of the effects of RGAP on phagocytosis of B. abortus by F-actin polymerization modulation. (A) F-actin polymerization and bacterial co-localization (scale bars = 5 µm). (B) FACS analysis for F-actin content. (C) Intensification of F-actin polymerization. The data shown are representative of at least three independent experiments. Statistically significant differences relative to untreated control cells are indicated by an asterisk (*p < 0.05). DIC, differential interference contrast.

• 

  Fig. 4 Effect of RGAP on activation of intracellular signaling for the phagocytosis of B. abortus. Immunoblot analyses of total RAW 264.7 cell lysates pre-treated with RGAP were assessed using phospho-specific ERK1/2, JNK and p38α antibodies at the indicated times. Images shown are representative of at least three independent experiments.

• 

  Fig. 5 Effect of RGAP on intracellular trafficking of B. abortus. One hundred bacteria within macrophages were randomly selected, and the extent of LAMP-1 acquisition by the bacteria was determined. Data represent the means ± SD of triplicate samples. Statistically significant differences relative to the untreated control are indicated by asterisks (***p < 0.001).

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