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) .