Figures index : The Anti-Inflammatory and Immunomodulatory Activity of Thymulin Peptide is NF-B-Dependent and Involves the Downregulation of IB- : Science and Education Publishing

Figure 1. The expression of various NF-κB subunits in the hippocampus in response to ICV ET. Varying concentrations of thymulin (0.1 – 5μg; ICV) were introduced stereotypically 30 minutes before ICV injection of ET (1μg; 45 minutes). ET (Lane-3) upregulated the nuclear translocation of NF-κB₁ (p50), NF-κB₂ (p52), RelA (p65), RelB (p68) and c-Rel (p75) in the hippocampus (HC). The protein expression of NF-κB₁ (p50) and NF-κB₂ (p52) was mild as compared with other NF-κBsubunits, with prominent expression of p65. Thymulin (5 μg; ICV) alone (Lane-2) or control (Lane-1) that received no injections showed faint constitutive or no expression, respectively, as compared with ET. The inductive effect of ET is ameliorated and attenuated with thymulin, particularly at concentrations ≥ 1 μg (Lanes-4, 5 and 6), with prominent effects on p52, p65 and p75 subunits. β-Actin standard was used as an internal reference for semi-quantitative loading in parallel lanes for each variable. n = 3 – 5, which represents the number of independent experiments in separate animals

Figure 2. The effect of ET and thymulin pretreatment on IκB-α. Regulated by an upstream kinase (IKK), IκB-α is phosphorylated prior to the nuclear translocation of NF-κB. ET induced the phosphorylation of IκB-α, thereby tagging it for proteasome degradation, hence its cytosolic concentration was decreased (Lane-3). Thymulin, in a dose-dependent manner, reduced the phosphorylation of IκB-α, with maximal inhibitory effect at ICV concentration of 5μg (Lanes-4, 5 and 6). Thymulin restored IκB-α cytosolic accumulation, in a dose-dependent manner, consistent with the downregulation of its phosphorylation (Lanes-4, 5 and 6; Figure 2). Neither the control (Lane-1) nor thymulin alone (Lane-2) showed any effect on the phosphorylation of IκB-α, but there is constitutive expression as compared with ET alone. β-Actin standard was used as an internal reference for semi-quantitative loading in parallel lanes for each variable. n = 3 – 5, which represents the number of independent experiments in separate animals

Figure 3. The modulation of the expression of various NF-κB subunits in the hippocampus in response to ICV ET/PAT. (A), (B) Varying concentrations of PAT (1 – 25μg; ICV) were introduced stereotaxically 30 minutes before ICV injection of ET (1μg; 45 minutes). ET (Lanes-1 and 2) upregulated the nuclear translocation of NF-κB₁ (p50), RelA (p65), RelB (p68), c-Rel (p75) and, to a lesser extent, NF-κB₂ (p52) in the hippocampus (HC). The protein expression of NF-κB₂ (p52) and RelB (p68) was mild as compared with other NF-κBsubunits, with prominent expression of p65. PAT (1 – 25 μg; ICV) alone (Lanes-3-5) showed faint constitutive or no expression of NF-κB₁ (p50), NF-κB₂ (p52) and RelB (p68), as compared with ET. The inductive effect of ET is ameliorated and attenuated with PAT, particularly at concentrations ≥ 1μg, with prominent effects on p65 subunit. β-Actin standard was used as an internal reference for semi-quantitative loading in parallel lanes for each variable. n = 3 – 5, which represents the number of independent experiments in separate animals

Figure 4. The effect of ET and PAT pretreatment on IκB-α. Regulated by an upstream kinase (IKK), IκB-α is phosphorylated prior to the nuclear translocation of NF-κB. ET induced the phosphorylation of IκB-α, thereby tagging it for proteasome degradation, hence its cytosolic concentration was decreased. (A), (B) PAT induced the cytosolic accumulation of IκB-α. PAT-mediated restoration of IκB-α cytosolic accumulation is, to an extent, dose-dependent. β-Actin standard was used as an internal reference for semi-quantitative loading in parallel lanes for each variable. n = 3 – 5, which represents the number of independent experiments in separate animals

Figure 5. The effect of ET and PAT pretreatment on IκB-α phosphorylation. (A), (B) PAT reduced, in a dose-dependent manner, the phosphorylation of IκB-α, with maximal inhibitory effect at ICV concentration of 5 μg. This is consistent with restoration of IκB-α cytosolic accumulation (as shown in Figure 4). Neither PAT (1μg) nor PAT (25μg) alone showed any effect on the phosphorylation of IκB-α, but there is constitutive expression as compared with ET alone. β-Actin standard was used as an internal reference for semi-quantitative loading in parallel lanes for each variable (the β-actin for Lanes-1 and 2 [ICV ET] has not been reproduced in this experiment; however, it was shown in Figure 1 and Figure 2). n = 3 – 5, which represents the number of independent experiments in separate animals

Figure 6. DNA-binding activity of NF-κB in response to ET/PAT. PAT reduced (Lanes-4-6), in a dose-dependent manner, ET-mediated NF-κB activation (Lane-3) in the hippocampus, as compared with saline (Control; Lane-1) or PAT alone (Lane-2), n = 3 – 5, which represents the number of independent experiments in separate animals

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