68 5 ART for Anti-inflammation
By the enzyme-linked immunosorbent assay, we detected the antibody against
3-nitrotyrosine (3NT) that reflects the extent of ONOO−-mediated nitration of pro-
teins, and we also detected the antibody against cyclic citrulline peptide (CPP) that
indicates the occurrence and progression of RA. The results indicated that either
combination with l-NMMA or not, CFA/CII-CFA elevates the anti-3-nitrotyrosine
antibody levels, suggesting CFA/CII-CFA induces the production of both NO and
O 2 − by activating the immune responses, and thereby accelerates the reaction of
NO with O 2 − to give rise to ONOO−, which can directly enhance protein nitra-
tion. In contrast, SNP alone or in combination with CFA/CII-CFA leads to a lower
anti-3NT antibody level. This is likely because SNP only releases NO but does
not generate O 2 −. Intriguingly, a reverse correlation of anti-3NT antibody with the
anti-CCP antibody was noticed in mice after CFA/CII-CFA injection, suggesting
that the 3-nitrosyl group on the tyrosine residue is most likely from ARG of pro-
teins because the denitration of l-arginine might result in conversion to citrulline,
which induces the anti-CCP antibody due to absence in native proteins.
5.3.2.4 Modulation of Immune Responses and Hypoxia
Consequences in CIAA/LIAA Mice by ART, RAP,
and/or BLA
To test if administration of ART or RAP before or during CIAA modeling sup-
presses the immune responses and mitigate the hypoxia consequences, we
designed a pretreatment regimen by injecting mice with 60 μg/ml ART or 30 μg/
ml RAP on four hours prior to immunization and continuous injection in twice
a day for three days. As results, proinflammatory cytokines were found to be
remarkably downregulated. For example, ART and RAP decrease TNFα for 1.24-
and 1.11-folds, respectively, compared to 2.24-folds in untreated CIAA mice. RAP
seems to have a more potent immunosuppressive effect than ART because the
expression levels of proinflammatory cytokines are lower in the RAP group than
those in the ART group. In LIAA mice, ART (60 μg/ml) can decrease TNFα con-
tent from 1.691 ± 0.07 pg/ml (control mice) to 1.462 ± 0.07 pg/ml, although it is
lower than that after treatment by 0.5 mg/ml l-NMMA (1.079 ± 0.15 pg/ml).
To observe the effects of different administration procedures on disease
improvement in modeling mice, we determined the serum NO levels before or after
injection with ART or RAP in a preinjection group (four hours before CFA immu-
nization) and a postinjection group (three days after CFA immunization). While
pretreatment by ART (60 μg/ml) or RAP (30 μg/ml) for three days decreases the
NO level to that comparative to the control, posttreatment by RAP (50 μg/ml) for
five days causes a dramatic decline of the NO level. For LIAA mice, ART (60 μg/
ml) can decline the NO level from 8 μM (control mice) to 5 μM, albeit slightly
lower than that in l-NMMA (0.5 mg/ml)-treated LIAA mice (4 μM). Accordingly,
ART (60 μg/ml) or l-NMMA (0.5 mg/ml) can downregulate the expression level
of iNOS, represented by the weaker immunohistochemical staining from the gray
scale values, 92.67 ± 4.75 (LIAA mice) to 8.54 ± 2.03 or 21.46 ± 4.30.