3.3. Effect of α-Klotho on LPS-induced NF-κB activation in astrocytes primary mouse glia culture
To evaluate the influence of α-Klotho on NF-κB signaling we performed experiments in astrocytes primary culture based on previous evidence that secrected neuronal α-Klotho modulates astrocytic metabolic activity (Mazucanti, Kawamoto et al., 2019). For immunofluorescence experiments, we used staining of RelA (p65), which is a NF-kB subunit that is activated during LPS-induced inflammation (Fig. 4). Pre-treatment of cells with concentrations of 1 nM α-Klotho for 24h, followed by challenge with LPS at 4 hours (Fig. 4A,B) confirmed increased RelA nuclear translocation by LPS vs control. RelA nuclear translocation induced by LPS was completely revert by recombinant α-Klotho treatment (Fig. 4A,B). For a quantitative assessment, cells extract from cytosolic and nuclear fractions were used to evaluate the RelA (p65) content in each compartment by Western Blotting (Fig.4C,D). Results confirmed immunofluorescence data as the RelA (p65) subunit translocation subunit induced by LPS is inhibited by recombinant α-Klotho (Fig.4C,D). Finally, these groups were submitted to an EMSA assay to more precisely detect whether the NF-kB that translocated to the nucleus was active and bound to its specific sequence in DNA (Fig. 4E,F). EMSA data confirmed both previous data as the binding of the nuclear extract to the 32P-labeled probe was much higher in LPS-treated astrocytes and pre-treatment with α-Klotho was able to inhibit the activation of this transcription factor. A super-shift assay was also performed (Fig. 4G) to clarify that NF-kB subunits are involved in this activation. Data confirmed that RelA (p65) and p50 subunits are involved, which typically occurs following activation by LPS (Glezer, Munhoz et al., 2003) .