Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 1:12:753599.
doi: 10.3389/fphar.2021.753599. eCollection 2021.

Achyranthes bidentata Polysaccharide Activates Nuclear Factor-Kappa B and Promotes Cytokine Production in J774A.1 Cells Through TLR4/MyD88 Signaling Pathway

Sairong Fan  1   2 , Yanxing Wang  1   2 , Yue Zhang  1   2 , Yamin Wu  1   2 , Xiaoming Chen  1   2
Affiliations

Achyranthes bidentata Polysaccharide Activates Nuclear Factor-Kappa B and Promotes Cytokine Production in J774A.1 Cells Through TLR4/MyD88 Signaling Pathway

Sairong Fan et al. Front Pharmacol. .

Abstract

Achyranthes bidentata Blume, a traditional Chinese medicine, is widely acknowledged for its function of invigorating the liver and kidneys and as a stranguria-relieving diuretic and used in the treatment of edema, gonorrhea, and other diseases. Polysaccharide (ABPS), isolated from Achyranthes bidentata Blume, has been demonstrated to have multiple biological activities including immunomodulatory effects. However, the mechanisms underlying the effects of ABPS have not been fully investigated. The present study is conducted to explore the underlying mechanism of immunomodulatory activities of ABPS. Results showed that ABPS significantly increased the secretion of IL-1β and TNF-α in J744 A.1 cells. Nitric oxide (NO) also significantly increased after ABPS treatment. The special antibodies (Toll-like receptor 4 (TLR4) antibody and CD14/TLR4 antibody) significantly decreased the activation, while the Toll-like receptor 2 (TLR2) antibody could not abolish this activation. Meanwhile, pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB, remarkably inhibited the secretion of IL-1β and TNF-α induced by ABPS in J744 A.1 cells. Western blotting (WB) and confocal laser scanning microscopy (CLSM) showed that ABPS promoted NF-κB translocation into the nucleus. Furthermore, the mRNA and protein expression of TLR4 and MyD88 were significantly increased after ABPS treatment. Taken together, these findings suggested that the immunomodulatory mechanism of ABPS was associated with the secretion of cytokines by stimulating the NF-κB pathway through TLR4/MyD88 signaling.

Keywords: Achyranthes bidentata; NF-kappa B; Toll-like receptors (TLRs); immunomodulatory; polysaccharides.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effects of ABPS on the secretion of cytokines and NO in J774 A.1 cells. The cells were treated with ABPS (0, 50, 200, 500, and 1,000 μg/ml, respectively) or LPS (5 μg/ml) for 24 h. Cell culture medium was collected, and the secretion levels of cytokines (A) IL-1β, (B) TNF-α, and (C) NO were detected. NC: normal control; ABPS50: 50 μg/ml ABPS treated; ABPS200: 200 μg/ml ABPS treated; ABPS500: 500 μg/ml ABPS treated; ABPS1000: 1,000 μg/ml ABPS treated; and LPS: 5 μg/ml LPS treated (positive control). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC. The values are presented as means ± SD.
FIGURE 2
FIGURE 2
Effects of ABPS on the mRNA expression of IL-1β and TNF-α in J774 A.1 cells. The cells were treated with ABPS (0, 50, 200, 500, and 1,000 μg/ml, respectively) or LPS (5 μg/ml) for 24 h. Total RNA was isolated, and the mRNA expression levels of (A) IL-1β and (B) TNF-α were determined. NC: normal control; ABPS50: 50 μg/ml ABPS treated; ABPS200: 200 μg/ml ABPS treated; ABPS500: 500 μg/ml ABPS treated; ABPS1000: 1,000 μg/ml ABPS treated; and LPS: 5 μg/ml LPS treated (positive control). * p < 0.05, ** p < 0.01, vs. NC. The values are presented as means ± SD.
FIGURE 3
FIGURE 3
ABPS activation of macrophages depends on TLR4. (A) IL-1β and (B) TNF-α. The cells were pretreated with or without 20 μg/ml anti-TLR2 antibody for 1 h and then treated with ABPS (0, 50, 200, 500, and 1,000 μg/ml, respectively) or LPS (5 μg/ml) for 24 h. Cell culture medium was collected, and the secretion levels of cytokines were detected. (C) IL-1β and (D) TNF-α. Cells were pretreated with or without 20 μg/ml anti-TLR4 antibody. (E) IL-1β and (F) TNF-α. Cells were pretreated with or without 20 μg/ml anti-CD14/TLR4 antibody. NC: normal control; ABPS50: 50 μg/ml ABPS treated; ABPS200: 200 μg/ml ABPS treated; ABPS500: 500 μg/ml ABPS treated; ABPS1000: 1,000 μg/ml ABPS treated; and LPS: 5 μg/ml LPS treated (positive control). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control (without antibody treatment group). The values are presented as means ± SD.
FIGURE 4
FIGURE 4
ABPS increased the expression of TLR4 and MyD88 in J774 A.1 cells. (A) The mRNA expression of TLR2, TLR4, TRAM, and MyD88. (B) The protein expression of TLR2, TLR4, TRAM, and MyD88. The blot shown is representative of one of the three similar experiments. Cells were treated 500 μg/ml ABPS for 24 h. NC: normal control; ABPS500: 500 μg/ml ABPS treated. * p < 0.05, ** p < 0.01 vs. NC. The values are presented as means ± SD.
FIGURE 5
FIGURE 5
Effect of ABPS on the expression and nuclear translocation of NF-κB in J774 A.1 cells. (A) The expression of NF-κB in J774 A.1. Cells were treated with ABPS (0, 50, 200, 500, and 1,000 μg/ml, respectively) or LPS (5 μg/ml) for 24 h. Nuclear extracts were prepared and determined by western blot. NC: normal control; ABPS50: 50 μg/ml ABPS treated; ABPS200: 200 μg/ml ABPS treated; ABPS500: 500 μg/ml ABPS treated; ABPS1000: 1,000 μg/ml ABPS treated; and LPS: 5 μg/ml LPS treated (positive control). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC. The values are presented as means ± SD. (B) and (C) ABPS promoted nuclear translocation of NF-κB. Cells were stained with FITC-tagged antibody (B1: control; C1: 500 μg/ml ABPS) or propidium iodide (PI) (B2: control; C2: 500 μg/ml ABPS). Micrographs (B3: control; C3: 500 μg/ml ABPS) represent merged images obtained through the red and green fluorescence channels.
FIGURE 6
FIGURE 6
Effect of NF-κB inhibitor (PDTC) on ABPS induced the secretion of IL-1β and TNF-α. The cells were pretreated with or without 100 μg/ml anti-TLR2 antibody for 2 h and then treated with ABPS (0, 50, 200, 500, and 1,000 μg/ml, respectively) or LPS (5 μg/ml) for 24 h. Cell culture medium was collected, and the secretion levels of cytokines (A) IL-1β and (B) TNF-α were detected. NC: normal control; ABPS50: 50 μg/ml ABPS treated; ABPS200: 200 μg/ml ABPS treated; ABPS500: 500 μg/ml ABPS treated; ABPS1000: 1,000 μg/ml ABPS treated; and LPS: 5 μg/ml LPS treated (positive control). ** p < 0.01, *** p < 0.001 vs. control (without antibody treatment group). The values are presented as means ± SD.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Ahmed H., Khan M. A., Kahlert U. D., Niemelä M., Hänggi D., Chaudhry S. R., et al. (2021). Role of Adaptor Protein Myeloid Differentiation 88 (MyD88) in post-subarachnoid Hemorrhage Inflammation: A Systematic Review. Ijms 22, 4185. 10.3390/ijms22084185 - DOI - PMC - PubMed
    1. Allam H. A., Abd El-Salam M., Mohammad A., Aboulkhair A. G. (2015). Clinical Value of Toll like Receptor 4 and CD14 in Children with Acute Lower Respiratory Tract Infection. Int. Biol. Biomed. J. Summer. 1 (3), 90–97.
    1. Ao X., Kim I. H. (2020). Effects of Achyranthes Bidentata Polysaccharides on Performance, Immunity, Antioxidant Capacity, and Meat Quality in Pekin Ducks. Poult. Sci. 99 (10), 4884–4891. 10.1016/j.psj.2020.06.026 - DOI - PMC - PubMed
    1. Chen L., Ge M. D., Zhu Y. J., Song Y., Cheung P. C. K., Zhang B. B., et al. (2019). Structure, Bioactivity and Applications of Natural Hyperbranched Polysaccharides. Carbohydr. Polym. 223, 115076. 10.1016/j.carbpol.2019.115076 - DOI - PubMed
    1. Chen X., Yu G., Fan S., Bian M., Ma H., Lu J., et al. (2014). Sargassum Fusiforme Polysaccharide Activates Nuclear Factor Kappa-B (NF-Κb) and Induces Cytokine Production via Toll-like Receptors. Carbohydr. Polym. 105, 113–120. 10.1016/j.carbpol.2014.01.056 - DOI - PubMed