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In rats, we found no difference in the magnitude of dose-dependent responses in the serum levels of proinflammatory cytokines IL-6 and TNF-α ( Figures 3 A and 3B). IL-6 was also significantly increased in the plasma samples after a 30-min exposure to 0.75 μg/kg, as was TNF-α concentrations ( Figure 3 C) in the C4b but not C3a strains. These results confirm a decrease in proinflammatory cytokine levels after 30-min exposure but do not confirm a suppressive effect on the production of inflammatory cytokine by this treatment. The results indicated that, in present study, the administration of M2a is able to protect against the development of severe inflammation.
(A) Serum levels of TNF and IL-6 in rats subjected to a 2 hr exposure saline or 1.0 M M2a and 1 mg/ kg intraperitoneally (i.p.). (Left) Serum concentrations of TNF-α in the C3a C4b group and (Right) in the C4b group that was previously treated with M2a prior to being exposed TNF-α. (B) Serum levels of IL-6 in the respective groups for 5, 7, 10, and 15 days by ELISA Western blot (p < 0.05, ∗ p +∗∗ 0.05 with Bonferroni's multiple comparisons test). (C) IL-6 levels were quantified by using ELISA and Western blot normalized to GAPDH levels (p < 0.05; ∗ p 0.05, +∗∗ < 0.05 with Bonferroni's multiple comparisons test). (D–J) The mRNA levels of T, IL-6, and IL-1α were determined in a 1∶5th ratio T cells isolated from the blood of a vehicle-treated group that received M4a, TNF-α, C3a, C4b, and TNF-α (n = 4, ∗ p < 0.05, ∗∗∗ with Bonferroni's multiple comparisons test). The results were expressed as fold change in mRNA levels compared to vehicle-treated group (n = 3–6 animals per genotype). Results are presented as mean ± SEM; ∗∗∗ p < 0.05, and ∗ 0.05 were significant.
Next, it was the goal to determine whether M2a protects against the development of a mouse model that is dependent upon T cells, as suggested by our previous finding that M2a attenuated an acute TNF-α-induced production of proinflammatory cytokines in wild-type mice [23]. this context, a group led by Atsuo Takahashi examined whether the administration of M2a at a dose 1.0 mg/kg induces suppressive effect on T-cell production by suppressing the T-cell-dependent of pro-inflammatory cytokines. Animals were subjected to an acute-phase response (a-P) test as described [19]. The results indicated that application of M2a was able to protect against the acute-phase response in serum levels of TNF-α ( Figure 4A ) and IL-1α ( Figure 4B ) and to decrease the duration of a-P that was evident in T cells isolated from the blood of mice treated with vehicle control ( Figure 4C ).
To determine whether the administration of M2a at lower doses would induce suppressive effects on proinflammatory cytokine production, we examined levels in C3a and C4b mice which the respective genotypes were randomly assigned to M2a-treated group and the vehicle-treated group. We observed significant protection on a-P levels after 1 week treatment with M2a, even in the groups where serum levels of proinflammatory cytokines were reduced (n = 4 for C3a C4b C3a, and n = 3 for C4b C3a). There were no significant effects on TNF-α, IL-6, IL-1a, or IL-1β when the serum levels of these inflammatory markers were measured over a longer duration. In addition, the number of T-cell-dependent IL-6, IL-1β, and IL-1a produced by the naïve mice.
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