Phospho-BTK (Ser179) Antibody - #AF8361

Figure 7. Running exercise reverses KLK8 upregulation and inactivates Met/Src/Btk/NF-κB signaling pathways, thereby attenuating microglia activation and neuroinflammation in the hippocampus of STZ-induced diabetic mice. Control or STZ-induced diabetic mice were subjected to moderate intensity treadmill training for 5 weeks. A, The levels of KLK8 mRNA and protein were measured in the hippocampus of the STZ-induced diabetic mice subjected to sedentary conditions or running exercise by qRT-PCR (F1,24 (running exercise) = 57.427, p < 0.001; F1,24 (treatment) = 74.348, p < 0.001; F1,24 (running exercise×treatment) = 37.177, p < 0.001) and western blotting F1,24 (running exercise) = 157.255, p < 0.001; F1,24 (treatment) = 195.184, p < 0.001; F1,24 (running exercise×treatment) = 78.996, p < 0.001), respectively. Representative protein bands were presented on the top of the histograms. B, Hippocampal sections were stained with fluorophore-labeled antibodies against phosphorylated Met (p-Met, red). DAPI staining was used to detect nuclei (blue). Scale bar = 50 μm. C, Hippocampal sections were stained with fluorophore-labeled antibodies against phosphorylated Btk (p-Btk, red). DAPI staining was used to detect nuclei (blue). Scale bar = 50 μm. D, Hippocampal sections were stained with fluorophore-labeled antibodies against the microglial cell marker Iba1 (red) and p-p65 (green). DAPI staining was used to detect nuclei (blue). The merge image represents double positive staining for Iba1 and p-p65. Areas in white boxes were shown enlarged. Scale bar = 50 μm. E, Immunofluorescent staining showed Iba1 (red) expression in the CA1, CA2/3, and DG subregions of the hippocampus. Nuclei were counterstained with DAPI (blue). Scale bar = 100 μm. The quantification of Iba1+ cell numbers in each subfield of the hippocampus were presented in the right panels (CA1: F1,24 (running exercise) = 30.348, p < 0.001; F1,24 (treatment) = 37.189, p < 0.001; F1,24 (running exercise×treatment) = 26.515, p < 0.001. CA2/3: F1,24 (running exercise) = 17.621, p < 0.001; F1,24 (treatment) = 32.211, p < 0.001; F1,24 (running exercise×treatment) = 15.279, p < 0.001. DG: F1,24 (running exercise) = 33.171, p < 0.001; F1,24 (treatment) = 32.722, p < 0.001; F1,24 (running exercise×treatment) = 17.916, p < 0.001). F, The mRNA expression levels of Iba1 in the hippocampus were detected by qRT-PCR (F1,24 (running exercise) = 9.947, p = 0.004; F1,24 (treatment) = 15.644, p < 0.001; F1,24 (running exercise×treatment) = 8.656, p < 0.007). G, The mRNA expression levels of TNF-α, IL-6, CCL2, and iNOS in the hippocampus were detected by qRT-PCR (TNF-α: F1,24 (running exercise) = 85.403, p < 0.001; F1,24 (treatment) = 28.816, p < 0.001; F1,24 (running exercise×treatment) = 36.099, p < 0.001. IL-6: F1,24 (running exercise) =17.182, p < 0.001; F1,24 (treatment) = 33.374, p < 0.001; F1,24 (running exercise×treatment) = 14.296, p < 0.001. CCL2: F1,24 (running exercise) = 24.974, p < 0.001; F1,24 (treatment) = 10.641, p = 0.003; F1,24 (running exercise×treatment) = 8.261, p = 0.008. iNOS: F1,24 (running exercise) = 71.497, p < 0.001; F1,24 (treatment) = 15.102, p < 0.001; F1,24 (running exercise×treatment) = 27.053, p < 0.001). Data were presented as means ± SEM (n = 7, two-way ANOVA). ** p < 0.01.