3.2. Effect of α-Klotho on LPS-induced cytokines secretion in primary mouse glia culture
After analyzing cell viability and toxicity of different concentrations of α-Klotho in primary glia culture, time-course and concentration of α-Klotho effects on LPS - induced TNF-α secretion was evaluated. This cytokine was chosen based on previous studies in glial cells showing an increase of this cytokine after LPS treatment (Yshii, Denadai-Souza et al., 2015). Thus, concentrations ranging from 0.1 – 2 nM of α-Klotho and different time points of treatment (1,4 and 24 hours) were used followed by a LPS challenge (1 µg /mL for 8 hours) and TNF-α levels were determined by ELISA. Data showed that LPS induced increase in TNF-α levels compared to the control group and this effect was blocked by α-Klotho protein pretreatment at concentrations of 2 nM for 1 hour, and 1-2 nM for 4 and 24 hours (Fig. 2A, 2B, and 2C).
Based on these data, the concentration of 1 nM for 24 hours was chosen for evaluating the influence of α-Klotho on LPS (1 µg / mL for 8 hours) induced change in proinflammatory cytokines. In addition to TNF-α, LPS increased the production and secretion of other pro-inflammatory cytokines, such as IL-1β, IL6, and IFN-γ (Fig 3A, 3B, and 3C). However, α-Klotho treatment (1 nM for 24 hours) only reversed the LPS -induced increase of IL-6 levels (Fig. 3B) but not of IL-1β and IFN-γ (Fig. 3A and 3C).