Figure 1.
The chemical structure of inotilone (A) and the effects of inotilone on lipopolysaccharide (LPS)-induced cell viability (B), NO production (C), inhibition of iNOS and COX-2 protein expression (D), and MAPK (JNK, p38, and ERK) protein expression (E) were evaluated in RAW264.7 cells.
Cells were incubated for 24 h or 5, 10, 15, 30, and 60 mins with 100 ng/mL of LPS in the absence or presence of inotilone (0, 1.56, 3.12, 6.25, 12.5, and 25 µM). Inotilone was added 1 h before the incubation with LPS. Cell viability was performed by using MTT assay. Nitrite concentration in the medium was determined by using Griess reagent. Lysed cells were then prepared and subjected to Western blotting by using an antibody specific for iNOS, COX-2, and MAPK. β-actin was used as an internal control. The data were presented as mean ± S.D. for three different experiments performed in triplicate. **p<0.01 and ***p<0.001 were compared with LPS-alone group.
Figure 2.
Inotilone suppresses LPS-induced MMP-9 activities (A), MMP-9 protein (B), and NF-κB expressions (C) in RAW264.7 cells.
Cells were incubated for 24 h or 1 h with 100 ng/mL of LPS in the absence or the presence of inotilone (0, 6.25, 12.5, and 25 µM). Inotilone was added 1 h before the incubation with LPS. The conditioned media were collected MMP-9 activities determined by gelatin zymography. MMP-9 activities were quantified by densitometric analysis. Representative Western blot from two separate experiments was shown. MMP-9 and NF-κB levels were calculated with reference to a LPS-stimulated culture. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ###compared with sample of control group. **p<0.01 and ***p<0.001 were compared with LPS-alone group.
Figure 3.
Effects of inotilone and Indo on hind paw edema induced by Carr in mice (A), the tissue MDA concentration of foot in mice (B), Carr-induced NO (C), and TNF-α (D) concentrations of serum at the 5th
h in mice. Each value represents as mean ± S.E.M. ###p<0.001 as compared with the control group. *p<0.05, **p<0.01, and ***p<0.001 as compared with the Carr group (one-way ANOVA followed by Scheffe’s multiple range test).
Table 1.
Effects of inotilone and indomethacin (Indo) on changes in CAT, SOD and GPx activities was studied on Carr-induced mice paw edema (5th h).
Figure 4.
Inhibition of iNOS, COX-2 (A), MMP-9 (B), NF-κB (C), and MAPK (JNK, p38, and ERK) (D) protein expressions by inotilone induced by Carr of foot at the 5th
h in mice. Suspended tissue were then prepared and subjected to Western blotting using an antibody specific for iNOS and COX-2. β-actin was used as an internal control. A representative Western blot from two separate experiments is shown. Relative iNOS, COX-2, MMP-9, NF-κB, and MAPK (JNK, p38, and ERK) protein levels were calculated with reference to a Carr-injected mouse. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ###p<0.001 as compared with the control group. *p<0.05, **p<0.01 and ***p<0.001 as compared with the Carr group (one-way ANOVA followed by Scheffe’s multiple range test).
Figure 5.
Histological appearances of mouse hind footpads after subcutaneously injecting 0.9% saline (Control group) or Carr, and then stained with H&E stain, while others were processed for iNOS and COX-2 immunohistochmistry staining.
(A). Control mice: show the normal appearance of dermis and subdermis without any significant lesions, (F) iNOS and (J) COX-2 immunoreactive cells existed in the paws of normal mice; (B). Carr Only: Hemorrhage with moderately extravascular red blood cell and large amounts of inflammatory leucocytes, mainly neutrophils infiltrating the subdermis interstitial tissue. Moreover, the detail of the subdermis layer show enlargement of the interstitial space caused by the exudate fluid in the edema, (G) numerous iNOS and (K) COX-2 immunoreactive cells were observed in the brown site of paw tissue; (C). Carr + Indo 10 mg/kg (i.p.) (100×): there were obvious morphological alterations and improvements, (H) iNOS and (L) COX-2 immunoreactive cells; (D). Carr + inotilone: there were significant morphological alterations compared to the tissue with Carr treatment only. The lesions showed no hemorrhage and the number of neutrophils infiltrating the subdermis interstitial tissue was markedly reduced and also in (I) iNOS and (M) COX-2 immunoreactive cells in paws. Scale bar = 100 µm. There were markedly fewer inflammatory cells, and iNOS and COX-2 immunoreactive cells in the paws of Carr treated mice. The infiltrating cells were predominantly neutrophils (N; arrows). The brown staining indicated the interaction of primary and secondary antibodies and the presence of iNOS and COX-2.
Figure 6.
Proposed mechanism of inotilone inhibition of LPS-induced inflammation in RAW 264.7 cells.
Inotilone abrogates the phosphorylation of MAPKs/IKK and subsequently inactivates NF-κB, which may result from inotilone down-regulation of iNOS and COX-2. Arrows indicate the main inflammatory pathway activated by LPS stimulation. The prohibition signs indicate the inhibitory effects of inotilone. TLR4; Toll-like receptor 4.