IKK/NF-κB Inactivation by Salidroside via Targeting TNF-α for the Treatment of LPS-Induced Colitis

Figures

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  Figure 1. Sal alleviates inflammation on LPS-Induced mice. (A). Modeling and treatment cycle. (B). The body weight of various treatment groups. PBS vs. Sal, ns: no significance; PBS vs. LPS, ** p < 0.01; LPS vs. LPS + Sal, * p < 0.05. (C). Photographs of colons in various treatment groups. (D). The length of colons in various treatment groups. PBS vs. Sal, * p < 0.05; PBS vs. LPS, * p < 0.05; LPS vs. LPS + Sal, * p < 0.05. (E). Colon sections were stained with H&E and histological score. PBS vs. Sal, ns: no significance; PBS vs. LPS, **** p < 0.001; LPS vs. LPS + Sal, *** p < 0.005. (F). Colon sections were stained with AB-PAS and the number of goblet cells. PBS vs. Sal, ns: no significance; PBS vs. LPS, **** p < 0.001; LPS vs. LPS + Sal, * p < 0.05. (G). The mRNA levels of TNF-α, IL-1β and IL 6. PBS vs. Sal, ns: no significance; PBS vs. LPS, **** p < 0.001; LPS vs. LPS + Sal, *** p < 0.005 or **** p < 0.001. (H). Representative immunohistochemistry images of colon sections stained for the macrophage marker F4/80, the pan-T cell marker CD3, and the helper T cell marker CD4. Sal treatment reduced the infiltration of these immune cells induced by LPS. Scale bar, 100 μm. PBS vs. Sal, ns: no…

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  Figure 2. Effects of Sal on NCM460 and RAW264.7 cells induced by LPS. (A). Cell viability of different doses of Sal, LPS and Dex in NCM 460 cells. Different concentration of Sal, LPS or Dex vs. control, ns: no significance; * p < 0.05; ** p < 0.01; **** p < 0.001. (B). Measurement of proinflammatory (TNF-α, IL-1β and IL-6) factors produced from different treatment in NCM 460 cells by ELISA assay. PBS vs. 200 μM Sal or 2 μg/mL Dex, ns: no significance; PBS vs. 2 μg/mL LPS, **** p < 0.001; LPS vs. LPS + Sal, **** p < 0.001. LPS vs. LPS + Dex, **** p < 0.001. (C). The mRNA levels of TNF-α, IL-1β and IL-6 with different treated in NCM 460 cells. PBS vs. Sal or Dex, ns: no significance, PBS vs. LPS, * p < 0.05, *** p < 0.005, **** p < 0.001; LPS vs. LPS + Sal, * p < 0.05, *** p < 0.005, **** p < 0.001. LPS vs. LPS + Dex, **** p < 0.001. (D). Cell viability of different doses of Sal, LPS and Dex in RAW264.7 cells. Different concentration of Sal, LPS or Dex vs. control, ns: no significance; * p < 0.05. (E). Measurement of proinflammatory (TNF-α, IL-1β and IL-6) factors produced from different treatment in RAW264.7 cells by ELISA assay. PBS vs. Sal, ** p < 0.05; PBS vs. Dex, ns: no significance;…

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  Figure 3. Salidroside ameliorates cell inflammation by inhibiting the IKK/NF-κB signaling pathway. (A). Representative blot images of IKKα + IKKβ, p-NF-κB, IκBα (Ser36), p-IκBα (Ser36) and IL-1β in NCM 460 cells. (B). Quantitative analysis of IKKα + IKKβ, p-NF-κB, IκBα (Ser36), p-IκBα (Ser36) and IL-1β in NCM 460 cells. NC vs. Sal, ns, no significance, * p < 0.05, ** p < 0.01; NC vs. Dex, ns, no significance, * p < 0.05, ** p < 0.01; NC vs. LPS, ns, no significance, * p < 0.05, ** p < 0.01; LPS vs. LPS +Sal, ns, no significance, * p < 0.05, ** p < 0.01; LPS vs. LPS +Dex, ns, no significance, * p < 0.05, ** p < 0.01. (C). Representative blot images of IKKα + IKKβ, p-NF-κB, IκBα (Ser36), p-IκBα (Ser36) and IL-1β in RAW264.7 cells. (D). Quantitative analysis of IKKα + IKKβ, p-NF-κB, IκBα (Ser36), p-IκBα (Ser36) and IL-1β in RAW264.7 cells. The values represent the mean ± S.D. NC vs. Sal, ns, no significance, ** p < 0.01; NC vs. Dex, ns, no significance, * p < 0.05, ** p < 0.01; NC vs. LPS, ** p < 0.01, *** p < 0.005, **** p < 0.001; LPS vs. LPS +Sal, * p < 0.05, ** p < 0.01, *** p < 0.005; LPS vs. LPS + Dex, ns, no significance, * p < 0.05.

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  Figure 4. Sal ameliorates colitis by inhibiting the TNF-α signaling pathway. (A). A Venn diagram depicting the overlap between candidates and predicated targets of Sal in the context of IBD. (B). The PPI network of targeted proteins was generated via the STRING database and Cytoscape. (C). A bar graph illustrating the results of GO enrichment analysis, including biological process (BP), cell component (CC) terms, molecular function (MF). (D). The top 12 pathways related to the effect of Sal against UC.

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  Figure 5. Arg179, Lys188 and Tyr191 played roles in the binding of Sal with TNF-α. (A). Molecular docking of Sal with TNF-α. (B). Co-immunoprecipitation (Co-IP) and CETSA of TNF-α protein stability in NCM 460 cells treated with Sal. (C). Sal abolished the anti-inflammatory (TNF-α, COX-2) in 1 μg/mL TNF-α-induced NCM 460 cells. (D). Quantitative analysis of TNF-α, COX-2 in NCM 460 cells. NC vs. TNF-α, ** p < 0.01, *** p < 0.005; TNF-α vs. TNF-α + Sal, *** p < 0.005. (E). Sal abolished the anti-inflammatory (TNF-α, COX-2) in 1 μg/mL TNF-α-induced RAW264.7 cells. (F). Quantitative analysis of TNF-α, COX-2 in RAW264.7 cells. NC vs. TNF-α, * p < 0.05; TNF-α vs. TNF-α + Sal, * p < 0.05. (G). Partial protein sequence alignment of wild-type (WT) and mutant (Arg179Ala, Lys188Ala, Tyr191Ala) TNF-α. Mutated residues are highlighted in red. Western blot analysis of TNF-α protein expression in NCM 460 cells transfected with the empty pcDNA3.1 vector, pcDNA3.1-TNF-α WT, or the respective pcDNA3.1-TNF-α mutants (Arg179Ala, Lys188Ala, Tyr191Ala). β-actin served as a loading control. (H). Arg179Ala, Lys188Ala, Tyr191Ala abolished the binding of salidroside with TNF-α. The values represent the mean ± S.D.…