Effect of Orally Administered Atractylodes macrocephala Koidz Water Extract on Macrophage and T Cell Inflammatory Response in Mice

Abstract

The rhizome of Atractylodes macrocephala Koidz (AM) is a constituent of various Qi booster compound prescriptions. We evaluated inflammatory responses in macrophages and T cells isolated from mice following oral administration of AM water extract (AME). Peritoneal exudate cells were isolated from thioglycollate-injected mice and alterations in scavenger receptors were examined. Peritoneal macrophages were stimulated with lipopolysaccharide (LPS). Serum cytokine responses to intraperitoneal LPS injection were also evaluated. Splenocytes were isolated and their composition and functional responses were measured. The content of atractylenolide I and atractylenolide III, known anti-inflammatory ingredients, in AME was 0.0338 mg/g extract and 0.565 mg/g extract, respectively. AME increased the number of SRA(+)CD11b(+) cells in response to thioglycollate. Peritoneal macrophages isolated from the AME group showed no changes in inflammatory markers such as tumor necrosis factor- (TNF-) α, interleukin- (IL-) 6, inducible nitric oxide synthase, and cyclooxygenase-2 but exhibited a decrease in CD86 expression. Interestingly, AME decreased the serum levels of TNF-α and IL-6 upon intraperitoneal injection of LPS. Regarding the adaptive immune system, AME increased the CD4(+) T cell population and major histocompatibility complex class II molecule expression in the spleen, and cultured splenocytes from the AME group showed increased production of IL-4 concurrent with decreased interferon-γ production during T cell activation. AME promoted the replenishment of peritoneal macrophages during the inflammatory response but its anti-inflammatory activity did not appear to be mediated by the modulation of macrophage activity. AME also altered the immune status of CD4 T cells, promoting the Th2 response.

Figure 1

HPLC chromatograms of Atractylodes macrocephala water extract (AME). (a, b) Standard markers. (c, d) AME.

Figure 2

Scavenger receptors expressed by peritoneal exudate cells after oral administration of AME. Mice were orally given AME (500 or 2500 mg/kg) for 10 days. Peritoneal exudate cells were isolated from thioglycollate-injected mice and double-stained with FITC-conjugated anti-SRA and PE-conjugated anti-CD11b Abs (a), FITC-conjugated anti-CD11b and PE-conjugated anti-CD36 Abs (b), or FITC-conjugated anti-CD11b and PE-conjugated anti-LOX-1 Abs (c). Cells were analyzed using flow cytometry and representative dot plots are shown. (d) Bars represent mean ± SEM (n=6). ∗ P <0.05, ∗∗∗ P<0.005 versus control.

Figure 3

Effect of AME on the surface expression of costimulatory molecules in LPS-stimulated macrophages. Peritoneal macrophages isolated from the control and AME groups were stimulated with 100 ng/ml LPS for 24 h and then stained with PE-conjugated anti-CD86 antibody. (a) Representative histograms are shown. (b) Bars represent mean ± SEM (n=6). (-): without LPS, (+): LPS treatment. MFI: mean fluorescence intensity. # P<0.005 versus control (-), ∗ P<0.05 versus control (+).

Figure 4

Effect of AME on inflammatory cytokines and enzymes in LPS-stimulated macrophages. Macrophages isolated from control or AME group (2500 mg/kg) were stimulated with LPS (100 ng/ml) for 24 h. (a) The levels of tumor necrosis factor- (TNF-) α and interleukin- (IL-) 6 in supernatant were determined by ELISA. (b) Quantitative PCR was used to measure the expression of iNOS and COX-2 genes. Target gene expression was normalized to GAPDH expression. Data represent mean ± SEM (n=6). (-): without LPS, (+): LPS treatment. # P<0.005 versus control (-), ∗ P<0.05 versus control (+).

Figure 5

Effects of oral administration of AME on serum inflammatory responses following intraperitoneal injection of LPS. AME (2500 mg/kg) was orally administered to mice for 10 days. Serum was obtained 1 h after intraperitoneal injection of LPS (1.3 mg/kg) and the serum levels of TNF-α and IL-6 were determined by ELISA. Data represent mean ± SEM (n=10). (-): without LPS, (+): LPS treatment. # P<0.001 versus control (-), ∗ P <0.05, ∗∗∗ P <0.001 versus control (+).

Figure 6

Effects of oral administration of AME on composition of the adaptive immune system in the spleen. Splenocytes were isolated from control or AME groups and double-stained with FITC-conjugated anti-CD4 antibody and PE-conjugated anti-CD8 antibody (a), FITC-conjugated anti-CD19 antibody (b), or FITC-conjugated anti-MHC II antibody (c) and evaluated using flow cytometry. (a-c) Representative dot blots or histograms are shown. (d-e) Bars represent mean±SEM (n=6). ∗∗∗ P <0.001 versus control.

Figure 7

Effects of AME on the proliferation and cytokine secretion of activated splenic T cells. Splenocytes isolated from control and AME groups were cultured and stimulated with anti-CD3 antibody (2 μg/ml) for 48 h. (a) The proliferative response of splenocytes was determined using the MTS assay. (b) Cytokine secretion at 48 h of stimulation was measured by ELISA. Bars represent the mean±SEM (n=6). (-): without anti-CD3 antibody, (+): anti-CD3 antibody treatment. # P <0.001 versus control (-), ∗ P <0.05 versus control (+).