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Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction

  • Authors:
    • Elisa Porchietto
    • Eleonora Aimaretti
    • Giacomo Einaudi
    • Gustavo Ferreira Alves
    • Debora Collotta
    • Enrica Marzani
    • Leonardo Camillò
    • Chiara Rubeo
    • Raffaella Mastrocola
    • Natasha Irrera
    • Manuela Aragno
    • Carlo Cifani
    • Massimo Collino
  • View Affiliations / Copyright

    Affiliations: Pharmacology Unit, School of Pharmacy, University of Camerino, I‑62032 Camerino, Italy, Department of Neurosciences ‘Rita Levi Montalcini’, University of Turin, I‑10125 Turin, Italy, Department of Clinical and Biological Sciences, University of Turin, I‑10125 Turin, Italy, Department of Clinical and Experimental Medicine, University of Messina, I‑98125 Messina, Italy
    Copyright: © Porchietto et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 175
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    Published online on: August 26, 2025
       https://doi.org/10.3892/ijmm.2025.5616
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Abstract

Kinases are activators of well‑known inflammatory cascades implicated in metabolic disorders, and abnormal activation of casein kinase II (CK2) is associated with several inflammatory disorders. However, thus far, its role in the low‑grade chronic inflammatory response known as ‘metaflammation’, which is a hallmark of obesity and type 2 diabetes, has not yet been elucidated. The present study aimed to evaluate the role of CK2 in diet‑induced metaflammation and the effects of the CK2 inhibitor 4,5,6,7‑tetrabromobenzotriazole (TBB) on a murine model fed a high‑fat‑high‑sugar (HFHS) diet. C57BL/6JOlaHsd mice were fed a standard diet (n=12) or HFHS diet (n=24) for 12 weeks. A subgroup of the HFHS group received TBB (2.5 mg/kg/day, orally, n=12) for the last 8 weeks. Subsequently, plasma and liver samples were harvested for ex vivo biomolecular analyses (immunohistochemistry, western blotting, multiplex assay to determine the plasma levels of pro‑inflammatory cytokines, reverse transcription‑quantitative PCR and enzymatic assays) Statistical significance was determined using one‑way ANOVA with post‑hoc analysis (P<0.05). The results revealed that HFHS feeding induced glucose and lipid intolerance, elevated circulating pro‑inflammatory cytokines and increased hepatic neutrophil infiltration. By contrast, TBB treatment improved glucose and lipid homeostasis, and reduced systemic inflammation without altering body weight. Notably, TBB attenuated hepatic inflammation, reduced neutrophil recruitment and suppressed HFHS‑induced CK2α hyperactivation. This was accompanied by modulation of key inflammatory pathways, including NFκB/nucleotide‑binding domain, leucine‑rich‑containing family, pyrin domain‑containing‑3 and AMPK signaling. In conclusion, the present study demonstrated the beneficial effects of pharmacological inhibition of CK2 in a murine model of diet‑induced metabolic dysfunction, identifying CK2 as a potential target for dampening metaflammation. The efficacy of TBB in relieving hepatic inflammation was mainly due to the interference with selective inflammatory pathways.
View Figures

Figure 1

Effects of hypercaloric diet
consumption and casein kinase II inhibition on hepatic inflammatory
status. (A) ALT and (B) AST were measured in plasma samples using
commercially available kits. Data are presented as the mean ± SEM
(n=12 mice/group). Statistical analysis was performed by one-way
ANOVA followed by Bonferroni's post hoc test for ALT, whereas AST
was analyzed by Kruskal-Wallis followed by Dunn's post hoc test.
(C) H&E staining was performed on hepatic cryopreserved tissues
(n=5 mice/group). Representative photomicrographs of were recorded
with a Zeiss Airyscan confocal microscope at (magnification, ×10
and ×20). (D) Relative area of inflammatory cells. Data are
presented as the mean ± SEM (n=5 mice/group). Statistical analysis
was performed by one-way ANOVA followed by Bonferroni's post hoc
test. *P<0.05 vs. SD; •P<0.05 vs. HFHS.
ALT, alanine aminotransferase; AST, aspartate aminotransferase;
H&E. hematoxylin and eosin; HFHS, high-fat high-sugar; SD,
standard diet; TBB, 4,5,6,7-tetrabromobenzotriazole.

Figure 2

Assessment of ICAM-1 expression and
neutrophilic recruitment at the hepatic level among the
experimental groups. (A) Western blot analysis of ICAM-1 normalized
to β-actin. Densitometric analysis of the bands is expressed as
relative O.D. Data are presented as the mean ± SEM (n=4-5
mice/group). (B) Representative photomicrographs of MPO
immunostaining were recorded (n=5 mice/group). Stained tissues were
viewed under a Zeiss Airyscan confocal microscope (magnification,
×10 and 20×). (C) Mean positive MPO area on ×20 magnification
images (n=3-4 mice/group). Data are presented as the mean ± SEM
(n=3/4 mice per group). (D) MPO activity was measured using an
in vitro assay. Data are presented as the mean ± SEM (n=10
mice/group). Statistical analysis was performed by one-way ANOVA
followed by Bonferroni's post hoc test. *P<0.05 vs.
SD; •P<0.05 vs. HFHS. HFHS, high-fat high-sugar;
ICAM-1, intercellular adhesion molecule 1; MPO, myeloperoxidase;
O.D., optical density; SD, standard diet; TBB,
4,5,6,7-tetrabromobenzotriazole.

Figure 3

Effects of hypercaloric diet and
pharmacological inhibition of casein kinase II on NFκB and NLRP3
inflammasome cascades, as well as AMPK phosphorylation in the
liver. Western blot analysis of (A) IκBα phosphorylated on
Ser32/36 normalized to IκBα; (B) NFκB p65 subunit
expression at nuclear and cytosolic levels; (C) NLRP3 inflammasome
expression; (D) cleaved caspase-1 and total caspase-1 expression;
and (E) AMPK phosphorylated on Thr172 and total AMPK.
Densitometric analysis of the bands is presented as relative O.D.
Data are presented as the mean ± SEM (n=4-5 mice/group).
Statistical analysis was performed by one-way ANOVA followed by
Bonferroni's post hoc test. *P<0.05 vs. SD;
•P<0.05 vs. HFHS. HFHS, high-fat high-sugar;
inhibitor of κB α; NLRP3, nucleotide-binding domain,
leucine-rich-containing family, pyrin domain-containing-3; O.D.,
optical density; PCNA, proliferating cell nuclear antigen; SD,
standard diet; TBB, 4,5,6,7-tetrabromobenzotriazole.

Figure 4

Effects of HFHS chronic consumption
and TBB administration on CK2 activity and expression. Western blot
analysis of CK2α phosphorylation on Tyr255 normalized to
CK2α. Densitometric analysis of the bands is presented as relative
O.D. Data are presented as the mean ± SEM (n=4-5 mice/group).
Statistical analysis was performed by one-way ANOVA followed by
Bonferroni's post hoc test. *P<0.05 vs. SD;
•P<0.05 vs. HFHS. CK2, casein kinase II; O.D.,
optical density; HFHS, high-fat high-sugar; SD, standard diet; TBB,
4,5,6,7-tetrabromobenzotriazole.

Figure 5

Assessment of hepatic
pro-inflammatory and lipogenic transcript levels following dietary
challenges and casein kinase II inhibition. Quantitative PCR was
performed for the following genes: (A) TNF-α, (B) IL-1β, (C) IL-6,
(D) FASN and (E) SREBP1c. Relative gene expression was obtained
after normalization to GAPDH or 18S genes, using the formula
2−ΔΔCq and fold change was determined by comparison to
the SD group. Data are presented as the mean ± SEM (n=5-6
mice/group). Statistical analysis was performed by Kruskal-Wallis
followed by Dunn's post hoc test for TNF-α and IL-6, whereas IL-1β,
FASN and SREBP1c were analyzed by one-way ANOVA followed by
Bonferroni's post hoc test, *P<0.05 vs. SD;
•P<0.05 vs. HFHS. FASN, fatty acid nuclear synthase;
HFHS, high-fat high-sugar; SD, standard diet; SREBP1c, sterol
regulatory element-binding protein 1c; TBB,
4,5,6,7-tetrabromobenzotriazole.

Figure 6

Effects of HFHS consumption and
casein kinase II inhibition on systemic cytokine release. Plasma
levels of pro-inflammatory cytokines (A) TNF-α, (B) IL-1β, (C)
IL-6, (D) IFN-γ and (E) IL-17A were determined using Luminex
suspension bead-based multiplexed Bio-Plex Pro™ Mouse Cytokine Th17
Panel A 6-Plex assay. Data are presented as the mean ± SEM (n=6-8
mice/group). TNF-α and IL-1β were analyzed by Kruskal-Wallis
followed by Dunn's post hoc test, whereas IL-6, IFN-γ and IL-17A
were analyzed by one-way ANOVA followed by Bonferroni's post hoc
test, *P<0.05 vs. SD; •P<0.05 vs. HFHS.
HFHS, high-fat high-sugar; SD, standard diet; TBB,
4,5,6,7-tetrabromobenzotriazole.
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Copy and paste a formatted citation
Spandidos Publications style
Porchietto E, Aimaretti E, Einaudi G, Alves GF, Collotta D, Marzani E, Camillò L, Rubeo C, Mastrocola R, Irrera N, Irrera N, et al: Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction. Int J Mol Med 56: 175, 2025.
APA
Porchietto, E., Aimaretti, E., Einaudi, G., Alves, G.F., Collotta, D., Marzani, E. ... Collino, M. (2025). Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction. International Journal of Molecular Medicine, 56, 175. https://doi.org/10.3892/ijmm.2025.5616
MLA
Porchietto, E., Aimaretti, E., Einaudi, G., Alves, G. F., Collotta, D., Marzani, E., Camillò, L., Rubeo, C., Mastrocola, R., Irrera, N., Aragno, M., Cifani, C., Collino, M."Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction". International Journal of Molecular Medicine 56.5 (2025): 175.
Chicago
Porchietto, E., Aimaretti, E., Einaudi, G., Alves, G. F., Collotta, D., Marzani, E., Camillò, L., Rubeo, C., Mastrocola, R., Irrera, N., Aragno, M., Cifani, C., Collino, M."Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction". International Journal of Molecular Medicine 56, no. 5 (2025): 175. https://doi.org/10.3892/ijmm.2025.5616
Copy and paste a formatted citation
x
Spandidos Publications style
Porchietto E, Aimaretti E, Einaudi G, Alves GF, Collotta D, Marzani E, Camillò L, Rubeo C, Mastrocola R, Irrera N, Irrera N, et al: Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction. Int J Mol Med 56: 175, 2025.
APA
Porchietto, E., Aimaretti, E., Einaudi, G., Alves, G.F., Collotta, D., Marzani, E. ... Collino, M. (2025). Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction. International Journal of Molecular Medicine, 56, 175. https://doi.org/10.3892/ijmm.2025.5616
MLA
Porchietto, E., Aimaretti, E., Einaudi, G., Alves, G. F., Collotta, D., Marzani, E., Camillò, L., Rubeo, C., Mastrocola, R., Irrera, N., Aragno, M., Cifani, C., Collino, M."Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction". International Journal of Molecular Medicine 56.5 (2025): 175.
Chicago
Porchietto, E., Aimaretti, E., Einaudi, G., Alves, G. F., Collotta, D., Marzani, E., Camillò, L., Rubeo, C., Mastrocola, R., Irrera, N., Aragno, M., Cifani, C., Collino, M."Pharmacological inhibition of casein kinase II attenuates metaflammation in a murine model of diet‑induced metabolic dysfunction". International Journal of Molecular Medicine 56, no. 5 (2025): 175. https://doi.org/10.3892/ijmm.2025.5616
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