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However, the antioxidant and immunostimulatory-effects are-
natural activities of polysaccharides [18–22], and previously our
laboratory focused on the TLH-3 of antioxidant and anti-aging func-
tion, thus the immunostimulatory effect of TLH-3 has been rarely
investigated up to now. Therefore, in the present study, we inves-
tigated the immunstimulatory effect of TLH-3 on RAW264.7 cells,
which is commonly accepted as a tool to investigate the molecular
mechanisms of macrophages about regulating immunity [23]. Our
studies indicated the immunostimulatory of TLH-3, which activat-
ing the IB-␣-NF-B pathway via TLR-4 in RAW264.7 cells, then
inducing phagocytes is and secreting pro-inflammatory cytokines
including TNF-␣, IL-6 and NO. This study suggested that TLH-3
potentially could be used as immunomodulatory agent.
cells/well) were pre-incubated in E-Plate8 at 37 ◦C in 5% CO2 incu-
bators on RTCATP Analyzer. After 24 h, the various concentrations
of TLH-3 were added into each well according to the procedure and
settings in RTCA Control Unit before the experiment started. After
24 h incubated, the assay of RAW264.7 proliferation was acquired
and processed using RTCA Control Unit and Graph Pad Prism 6
software.
2.5. Measurement of NO production
NO production was monitored by assessment of nitrite accumu-
lation as previous study [24,25]. RAW264.7 cells (1 × 105 cells/well)
were pre-incubated in 12-well plates for 24 h at 37 ◦C in 5%
CO2incubator and then were incubated with TLH-3 (25–200 g/ml)
or LPS (1 g/ml) for 24 h. One hundred microliter of culture super-
natants was mixed with an equal volume of 10% Griess-reagent
Sodium nitrite (NaNO2) was used to generate a standard curve,
and nitrite production was determined by measuring absorbance
at 540 nm.
2. Materials and methods
2.1. Materials
TLH-3
polysaccharide,
which
is
made
up
of
1,3-linked--d-glucopyranosyl branched at C-6 and 1,3-linked-
-d-galactopyranosyl [15], was separated and purified according
to our research group previous report [16]. DMEM (Dulbecco?s
modified eagle medium) high glucose medium was acquired from
Wisten Biotechnology (WISTEN Co. Ltd., Nanjing), fetal bovine
serum(FBS), penicillin and streptomycin were purchased from
GIBCO (life technology USA).3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide(MTT),Lipopolysaccharides (LPS) and
neutral red were purchased from Sigma Chemical Co. (SaintLouis,
USA).Mouse IL-6 ELISA kit and TNF-␣ ELISA kit were purchased
from eBioscience (San Diego, CA, USA), NO assay kit, cellular NF-B
translocation kit were supplied by Beyotime Institute of Biotech-
nology (Shanghai, China), lipopolysaccharide (LPS, Escherichia coli
0111:B4), Polymyxin B(PMB) (Sigma?Aldrich, St. Luis, MO, USA),
anti-TLR4-antibody (ab13556), anti-IB-␣ antibody (ab32518),
anti-NF-B −antibody (ab32536), anti--actin antibody (ab59381)
were purchased from Abcam (Cambridge, UK), HRP-labeled goat
anti-rabbit IgG and HRP-labeled goat anti-Mouse IgG antibody
were purchased from Protein techBiotechnology (Proteintech
Group, Inc, Wuhan). All other reagents were of the highest grade
commercially available.
2.6. Assay for phagocytic activity
The phagocytic ability of macrophage was measured by neu-
tral red uptake. After cells (1 × 105 cells/well) cultured in 12-well
with TLH-3 (25–200 g/ml) or LPS (1 g/ml)(Sigma) for 24 h, 100 l
neutral red solutions (dissolved in PBS with the concentration of
0.075%) was added and incubated for 30 min. The supernatant was
discarded and the cells in 12-well plates were washed with PBS
thrice to remove the neutral red that was not phagocytized by
RAW264.7 cells. Then cell lysate (50% ethanol and 1% acetic acid
at the ratio of 1:1, 100 l/well) was added to lyse cells at 4 ◦C for
2 h. After cells were incubated overnight at room temperature, the
optical density was measured by a micro-plate reader at 540 nm
(Biotek, USA).
2.7. ELISA for quantitative analysis of cytokines and antibody
inhibition experiments
RAW 264.7 cells (1 × 105 cells/well) in 12-well were pre-treated
with 20 g/ml of TLR4mAb for 30 min, and then incubated with
TLH-3 (25–200 g/ml) or LPS (1 g/ml) was used as positive control
for 24 h. The supernatant TNF-␣ and IL-6 levels were determined
using ELISA kit. The specific method was carried out according to
the ELISA kit manual. The TNF-␣ and IL-6 concentration was esti-
mated from a reference to a standard curve of murine recombinant
TNF-␣ and IL-6.
2.2. Cell culture
RAW264.7 was purchased from Institute of Cell Biology, Chinese
Academy of Sciences (Shanghai, China). The cells were main-
tained in DMEM medium supplemented with 100 U/ml penicillin,
100 g/ml streptomycin, and 10% heat-inactivated FBS at 37?C in an
incubator with a humidified atmosphere and 5% CO2.
2.8. Morphologic observations and immunofluorescence of
nuclear translocation of NF-ꢀB
2.3. Assay for cell viability
RAW 264.7 cells were grown on glass bottom cell culture dishes
and treated with TLH-3 (200 g/ml) or LPS (1 g/ml) for 24 h. The
morphological change was observed under a phase contrast micro-
scope (Olympus, Japan). For analysis of nuclear translocation of
NF-B, cells were washed with PBS and stained with cellular NF-
B translocation kit according to the manufacturer’s instructions
after removing supernatant. Briefly, cells were fixed and incubated
with a blocking buffer for 1 h to suppress non-specific binding.
Cells were then incubated with the primary NF-B p65 antibody
at 4?C overnight, followed by incubation with a Cy3-conjugated
secondary antibody at room temperature for another 1 h. DAPI as
nuclear staining was used after 5 min incubation with cells. Sam-
ples were visualized under laser-scanning confocal microscopy
(Olympus FV1000, Japan). Cells were luminescently imaged on
Olympus FV1000 upright confocal-laser scanning microscope with
60X and 100X oil-immersion lenses. For DAPI (500 nM), excitation
The effect of TLH-3 on the viability of RAW264.7 cells was deter-
mined by MTT method. Adherent RAW 264.7 cells were treated
with increasing concentrations of TLH-3 (25–800 g/ml) in the
growth medium at 37 ◦C in 96-well plates. After 24 h incubation,
MTT (5 mg/ml) was added to each well and incubated for additional
4 h. The supernatant was then removed and 100 l of DMSO was
added. The cell culture plate was shaken for 10 min until no partic-
ulate matter was visible. Absorbance in each well was measured at
570 nm using a micro-plate reader (Biotek, USA).
2.4. Cell proliferation assay
The proliferation of RAW264.7 was measured using xCELLi-
gence RTCA TP (ACEA Biosciences, Inc). RAW264.7 cells (5 × 104
Zhang, Mingzhu
