Figure 1.
Chemical structures of thiosemicarbazone complexes.
(A) 2,3-dihydroxybenzaldehyde –N4-subsituted thiosemicarbazone, the backbone structure of the complexes. (B) Chemical structures of thiosemicarbazone complexes with N4 substituted with –H, -CH3, - C6H5 and -C2H5 groups. These complexes were regarded as ligands. (C) Chemical structures of ligands with additional triphenyl phosphine group as coligand.
Figure 2.
Crystal structures of two of the complexes.
(A) ORTEP diagram for complex [Ni(HL3)(PPh3)] (7). (B) ORTEP diagram for compound [Ni(HL4)(PPh3)] (8).
Table 1.
IR spectral assignments for the Ni(II) thiosemicarbazone ligands and their Ni(II)-phosphine complexes.
Table 2.
Selected bond lengths (Å) and angles (°) for complex 8.
Table 3.
Cell proliferation assay.
Figure 3.
Effect of N4-substituteted thiosemicarbazone complexes on LPS-induced NF-κB nuclear transloction in RAW264.7 macrophage cells.
Staining of Hoechst (nucleus) and NF-κB in RAW264.7 cells. Cells were pre-treated with 25 µg/ml of compounds for 4 h, followed by LPS stimulation for 1 h.
Figure 4.
Complex 5 blocks LPS-induced NF-κB nuclear translocation by inhibiting IκBα degradation.
(A) RAW264.7 cells pretreated with or without complex 5 for 4 h were treated with LPS for the indicated time points. Cytoplasmic and nuclear extracts were prepared and were subjected to Western blot analysis with IκBα, p65, HSP90 and PARP antibodies. (B) Relative density of IκBα and p65. Western blot signal intensities were quantified using ImageJ software. Densities were normalized to 0 min DMSO.
Figure 5.
Complex 5 blocks TNFα-induced NF-κB nuclear translocation.
Complex 5 inhibits TNFα -induced NF-B nuclear translocation. (A) NF-κB activation assay on TNFα treated HeLa S3 cells. Staining of Hoechst (nucleus) and NF-κB in HeLa S3 cells. Cells were pre-treated with 25 µg/ml of Ligand 1 and Complex 5 for 4 h, followed by TNFα stimulation for 1 h. (B) HeLa S3 cells pretreated with or without complex 5 for 4 h were incubated with TNFα for the indicated time points. Cytoplasmic and nuclear extracts were prepared and were subjected to western blot analysis with IκBα, p65, HSP90 and PARP antibodies. (C) Relative density of IκBα and p65. Western blot signal intensities were quantified using ImageJ software. Densities were normalized to 0 min DMSO. (D) HeLa S3 cells pretreated with or without complex 5 for 4 h were incubated with TNFα for the indicated time points. Whole cell extracts were prepared and were subjected to western blot analysis with IκBα, p-IκBα and PARP antibodies.
Figure 6.
Complex 5 inhibits NF-κB transactivation activity.
(A) RAW264.7 cells pretreated with or without complex 5 for 4 h were treated with LPS for 4 h. The expression of TNFα, IFNβ, IL6 and IP10 were measured by QPCR. (B) HeLa S3 cells pretreated with or without complex 5 for 4 h were stimulated with TNFα for 4 h. The expression of TNFα, IL8, CCL5, ICAM1, IL6 and A20 were measured by QPCR. (C) K562 or KCL22 cells pretreated with or without 50 µg/ml of complex 5 for 4 h were stimulated with 100 ng/ml TNFα for 2 h. The expression of IL8 and TNF were measured by QPCR. (D) 293T-luc reporter cells pretreated with or without various concentrations of complex 5 were stimulated with TNFα for 12 h. Cell lysates were prepared and the luciferase activities were measured. Error bars represent the variation range of duplicate experiments. Asterisks represents statistical significance by Student's t-test (*P<0.05).
Figure 7.
Complex 5 suppresses acute inflammatory responses in vivo.
Male C57BL/6 mice at age of 8-12 weeks old were non-injected or injected i.p. with 2.5 mg/kg or 5.0 mg/kg complex 5, whereas for positive controls, mice were injected i.p. with 2.0 mg/kg dexamethasone. After 1 h, acute inflammation was induced in the right hind paw of each mice by injecting 50 µl of carrageenan. The size of the paw were measured after (A) 1 h or, (B) 1, 3 and 5 h post carrageenan injection. Shown were mean ±SD (n = 4). Statistical analysis was performed with Student's t-test. At 1 h post-carrageenan induction, control versus 2.5 mg/kg complex 5 (P<0.05), control versus 5.0 mg/kg complex 5 (P<0.01), and control versus 2.0 mg/kg dexamethasone (P<0.01).
Table 4.
ORAC assay.
Figure 8.
Molecular docking of complex 5 to IKK complex.
(A) The binding of complex 5 in the kinase domain of IKKβ. The protein is represented by the protein surface and complex 5 in a stick model. (B) The overlay of complex 5 and inhibitor (compound 1 [30]). The inhibitor is represented by a balls and sticks model and complex 5 by a sticks model, in which the Nickel is colored in orange, nitrogen is colored in blue, oxygen is in red, carbon is in grey, sulphur is in green, hydrogen in white and phosphate is in purple. (C) The interaction between complex 5 and the binding site. One hydrogen bond is formed between complex 5 and TYR98, which is indicated by a green dotted line.