Biomedical Engineering Reference
In-Depth Information
of these cells by LPS has been linked to the pathogenesis of a variety of diseases. These
imply the possibility that in case of an abnormal condition, LPS has also been known to
induce endotoxemia and acute renal failure. However, the exact mechanism of LPS-induced
toxicity remains unclear.
3.4.1.2 Characterization
Measurement of nitrite : An aliquot of the incubation supernatant (100 μL) was transferred
to 96-well plates and nitrite was determined spectrophotometrically using Griess reagent
(0.8% sulfanilamide and 0.75% N -(naphthylethylene) diamine in 0.5 N HCl) by mixing 100 μL
of the Griess reagent. After 15 min incubation at room temperature, the nitrite concentrations
were measured at 540 nm using a microplate reader. Sodium nitrate (0.5-100 μM) was used
as nitrite standards and nitrite was linear over this concentration range.
Reverse transcriptase polymerase chain reaction ( RT-PCR ) RAW264.7 cells were treated with
1 μg/mL LPS for 6 or 12 h. The cells were harvested and total RNA was isolated by Trizol
according to the manufacturer's instructions. For amplification of IL-6 and TNF-α, the
following primers were used: TNF-α forward primer was 5′CCC AAA TGG CCT CCC
TCT C3′, reverse primer was 5′CAA ATC GGC TGA CGG TGT GTC C3′; IL-6 primer sense
was 5′ATG AAG TTC CTC TGC AAG AGA CT3′,antisense was 5′CAC TAG GTT TGC CGA
GTA GAT CTC3′. For PCR amplification, the following conditions were used: 94°C for
30 s (denaturation), 55°C for 1 min (annealing), and 72°C for 1 min (extension) for one
cycle and 72°C for 1 min for 30 cycles. The amplified PCR products were separated with
2% agarose gel, and then stained with ethidium bromide.
Western blotting : Proteins (20 μg) in incubation media were separated by 15% SDS-PAGE
and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked
with 5% skim milk in TPBS for 1 h at room temperature and incubated with anti-mouse
TNF-α antibody or anti-mouse IL-6 antibody for 3 h at room temperature or overnight at
4°C. After washing in TPBS three times, the blot was incubated with secondary antibody
(horseradish peroxidase-conjugated anti-goat antiserum) for 1 h at room temperature. The
antibody-specific proteins were detected by using West-ZOL (plus).
3.4.1.3 Function of Chitosan in Antiinflammatory Activity
Upon stimulation with increasing concentrations of chitosan, LPS-stimulated TNF-α
secretion was significantly recovered within the incubation media of RAW264.7 cells. The
effect of chitosan on LPS-stimulated TNF-α secretion was similar in 6 h and 12 h incuba-
tion media. The effective concentration range of chitosan was 0.05-0.5 wt% and, at higher
concentrations, TNF-α secretion plateaued. Also, above 1% concentration, chitosan was
toxic in vitro . Consistently, RT-PCR with mRNA of TNF-α and Western blot with anti-
TNF-α antiserum showed that the amount of TNF-α secretion in the incubation
media recovered with the concentration of chitosan. These results suggest that chitosan
may have an antiinflammatory effect in LPS-stimulated inflammation and may affect
LPS-stimulated TNF-α secretion within 6 h.
Upon stimulation with increasing concentrations of chitosan, LPS-stimulated IL-6
secretion was also significantly recovered within the incubation media of RAW264.7
cells. However, the effect of chitosan on LPS-stimulated IL-6 secretion was different in
6 h and 12 h incubation media. In 0.05% concentration of chitosan, the effect of chitosan
on IL-6 secretion was late, compared with TNF-α secretion. Consistently, RT-PCR with
mRNA of IL-6 and Western blot with anti-IL-6 antiserum showed that the amount of
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