Fig 2: Irisin levels in mice. Mean FNDC5/irisin levels (37kDA band) in the hippocampus of htau (purple) and control C57BL/6 J (grey) mice as a function of treatment (striped bars = r‐irisin treated mice; solid bars = vehicle‐treated mice). (A) Female brain FNDC5/irisin levels did not differ as a function of treatment or strain. (B) Male brain FNDC5/irisin levels did not differ as a function of treatment, but vehicle‐treated male htau mice exhibited significantly higher FNDC5/irisin levels than vehicle‐treated C57 males. (C) Dot density scatterplots show individual composite measurements summed across 20kDA and 37kDA bands for FNDC5/irisin protein in the hippocampus, brainstem, hypothalamus and prefrontal cortex for female and male htau and C57 mice. These summed composite values were normalised across all brain regions for depiction. (D&E) Serum levels of irisin are elevated in female and male mice treated with r‐irisin regardless of strain. * indicates significant treatment group differences, significance = p < 0.05. n = 16 htau mice (4/sex/treatment group); 14 C57 mice (3–4/sex/treatment group)

Fig 3: TNFα as a surrogate marker of inflammation in brain and serum of mice. Mean TNFα chemiluminescence in brain and serum of htau (purple) and control C57BL/6 J (grey) mice as a function of treatment (striped bars = r‐irisin treated mice; solid bars = vehicle‐treated mice). (A) Treatment with r‐irisin significantly reduced elevated levels of pro‐inflammatory cytokine TNFα in hippocampus of female htau mice. TNFα in C57 control mice did not vary as a function of treatment. (B) Brainstem and prefrontal cortex ptau levels appeared to be reduced in irisin‐treated female htau mice, but this was not statistically significant. Scatterplots depict strong correlation (indicated by Pearson's r value, p < 0.05) between hippocampal TNFα and ptau measurements for individual female htau mice. (C) Mean serum measurements of TNFα were significantly reduced in irisin‐treated female htau mice. (D) Irisin‐treated male htau mice showed significantly enhanced hippocampal inflammation compared to vehicle‐treated htau mice. (E) TNFα also appeared to be elevated in irisin‐treated male htau prefrontal cortex data but this was not statistically significant. Scatterplots illustrate the lack of correlation between hippocampal TNFα and ptau measurements for individual male htau mice (p = ns). (F) Mean serum measurements of TNFα did not differ as a function of irisin treatment in male htau mice, however they trended towards an irisin‐induced increase. Results are calibrated by total protein loaded per sample. Error bars show s.e.m.; * indicates significant difference between treatment groups. # indicates significant strain difference between htau and C57 mice of same treatment group. p < 0.05 in all significant comparisons. n = 16 htau mice (4/sex/treatment group); 14 C57 mice (3‐4/sex/treatment group)

Fig 4: Schematic model of the mechanism by which irisin suppresses nicotine-mediated migration, proliferation, cell cycle arrest, and cell senescence in endothelial cells. [Irisin’s protein data bank (PDB) ID: 4LSD (42, 43)].

Fig 5: Irisin inhibits nicotine-mediated endothelial cell migration and proliferation. (A) Wound healing assay under nicotine, irisin and nicotine + irisin interventions performed at 0, 6 and 12 h during phase II intervention, n = 3. (B) Migration rate among groups at 6 and 12 h during phase II administration and 2 phases treatment pattern illustration. (C) CCK-8 cell proliferation test performed at 6–24 h during phase II intervention, n = 4. (D) Immunoblotting bands of PI3K, AKT, p-AKT (S473), GAPDH, and β-actin. (E) Quantification of PI3K, AKT, and p-AKT. All results were normalized to the expression level of GAPDH or β-actin and presented as the fold change of the Control group, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are mean ± SD; ns, no significance.