59Furthermore, we quantified the expression of angiogenesis-responsible and
hypoxia-induced genes in CIA mice. In consequences, we noticed the upregula-
tion of HIF-1α and VEGF in the synovial tissue of mice after intra-articular CFA
injection. Also, the positive staining against HIF-1α or VEGF was remarkably
enhanced in the hypodermal tissue injected with 20 μg (100 μg/ml) SNP, account-
ing for 7-fold increases for HIF-1α staining and 4-fold increases in VEGF stain-
ing. From these results, it is clear that either endogenous or exogenous NO can
equally induce the synovial angiogenesis via upregulating HIF-1α and VEGF.
5.2.2.4 Replication of Inflammatory Synovitis by Administration
of SNP
To address NO as an initiating factor of inflammatory synovitis, we simply admin-
istered mice with SNP by intra-articular injection. Fascinatingly, a single injec-
tion with SNP can lead to the significant edema on the paw after only one day.
As expectation, coinjection with a mixture of SNP and CII-CFA induces a severe
inflammatory phenotype, confirming that NO legitimates a causative effector lead-
ing to inflammatory lesions. The exogenous NO-induced acute synovitis, there-
fore, represents a rapidly constructed early phase model of RA in mice.
As a critical hypoxic parameter, SpO 2 is very low (55–57 %) in mice after
injection with SNP regardless of treatment with or without CII-CFA. At the same
time, we also found that CII-CFA induces the moderate SpO 2 (62–66 %), which
are lower than control mice (82 %). It is worthy of noting that SpO 2 seems not to
correlate the severity of inflammation. For example, we saw that the inflammatory
signature is more severe in CII-CFA-injected mice with high SpO 2 (62 %) than
SNP-injected mice with lower SpO 2 (55–57 %). These results implied that CII-
CFA-triggered immune activation, in addition to NO, should be implicated in the
acceleration of inflammatory progression.
5.2.2.5 Abrogation of NO Production and Termination of Hypoxic
Induction Through Antibacteria and/or Inhibiting INOS
To validate bacterial infection as the origin of NO generation, we determined
the serum NO level in BIA mice after subcutaneous injection of CEF, ART, or
ART + CEF for three days (twice a day). As results, either treatment can consider-
ably decline the NO level lower than the control. Conceivably, ART declines the
NO level because it inhibits iNOS even though infection still exists. CEF allows
an equal NO level to the control because it kills bacteria and blocks infection. The
combined treatment leads to a lower NO level owing to the dual effects of CEF-
suppressed infection and ART-inhibited NO production. These results verified that
persistent gastrointestinal infection and potent NO burst can be abrogated by anti-
bacteria and/or iNOS inhibition.