br Results br Chemical properties of APS br APS
3.1. Chemical properties of APS
APS was white powder in extrinsic feature as shown in Fig. 2a. SEM observation found that most of the APS presented spherical-like shape with the mean size of 191.4 ± 51.84 nm, and the size of smallest particle of APS was 58.3 nm (Fig. 2b). At a higher magnification, the surface of APS was relatively rough with small protrusions (Fig. 2c). Meanwhile, the EDS analysis indicated the main elements of APS were carbon (72.56%) and oxygen (27.44%), and the undetected nitrogen and phosphorus also explained the preferable purity without mixing protein and nucleic acid (Fig. 2d).
Chemical composition analysis showed that APS contained 89.75% total carbohydrate and a small proportion of uronic acid (9.3%). As shown in Fig. 2e, the UV Methoctramine spectra of APS indicated no ab-sorption at 280 nm or 260 nm, which implied the absence of protein or nucleic acid. The IR spectrum of APS (Fig. 2f) exhibited absorption bands at 3400, 2925, 1647 and 1371 cm−1, attributing to OeH stretching vibration, CeH stretching vibration, C]O stretching
vibration and CeH bending vibration, respectively. The four bands above were characteristic absorption bands of polysaccharide. The three bands at 1010–1155 cm−1 indicated the pyran configurations of polysaccharides. The absorption peaks at 1081 and 1022 cm−1 were attributed to the glycosidic linkage CeOeH and CeOeC stretching vibration. The bands at 848 cm−1 and 930 cm−1 were characteristic of α-1,6 glucan .
3.2. Macrophage activation of APS
Following 24 h incubation with APS, the production of NO was gradually up-regulated with the increasing doses of APS (Fig. 3a). Significant enhancement of NO generation was identified after ex-posure to APS at the concentration of 200–1000 μg/mL compared with that of untreated cells (P < 0.01). However, NO production stimulated by APS was markedly lower than that in the presence of LPS (P < 0.05). For the expression of TNF-α, ELISA results indicated that all the APS-treated groups presented a significant increase of TNF-α levels in a dose-dependent manner as compared with that of control group (P < 0.001, Fig. 3b). Nevertheless, significant superiority of TNF-α secretion was noticed in LPS group compared with that of APS at the concentration of 1000 μg/mL (P < 0.05). On this basis, we speculated that APS may indirectly exert cytotoxicity against cancer cells though the macrophage activation with the release of TNF-α and NO.
To preliminarily elucidate the action mechanism of APS to macro-phages, we localized the site of their interaction by labeling APS with FITC. The obtained FITC-labeled APS was light yellow powder, and exhibited strong green fluorescence at 488 nm excitation. The major site of interaction could be noticed on cell membrane rather than pe-netrate into cells. FITC-APS specifically bound to RAW264.7 macro-phages in a time-dependent manner (20 min, 1 h, and 6 h), and the binding was blocked by unlabeled APS, which suggested that mem-brane receptors in macrophages appeared to saturate (Fig. 3c). It ex-plained the opposite decrease of NO and TNF-α levels over 1000 μg/mL. Dramatic changes of cellular morphology with long protrusions and
Fig. 3. APS activated macrophages. (a) and (b) indicated the eﬀects of APS on NO and TNF-α production in RAW264.7 murine macrophage cells. The data represent the means ± standard deviation; n = 3; *P < 0.05, **P < 0.01; # P < 0.05, APS (1000 μg/mL) vs. the LPS group. (c) Fluorescence microphotographs of RAW264.7 cells after incubation with FITC-labeled APS for 20 min, 1 h, and 6 h, as well as incubation with APS (1000 μg/mL) for 1 h followed by exposure to FITC-labeled APS for 5 h. Scale bar = 30 μm. (a–b) Macroscopic morphology of FITC-labeled APS. (d) Cellular morphology of RAW264.7 at P5 passage with/without the intervention of APS under phase contrast microscopy and SEM. The blue circle and arrows indicated the long protrusions. Scale bar = 3 μm. (e–f) The eﬀect of anti-TLR4 antibody on the production of APS-induced NO and TNF-α. *** P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
pseudopodia were identified after exposure to APS compared with those of untreated RAW 264.7 cells (Fig. 3d).
Moreover, treatment of RAW264.7 cells withTLR4 antibodies for 2 h before adding APS to the culture resulted in the down-regulation rather than complete suppression of NO and TNF-a production than that in corresponding samples treated with APS alone (Fig. 3e–f), which de-monstrated that binding of TLR4 receptor with APS was partially as-sociated with the activation of macrophages and following cytokine production.