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  • Astrocytes play a key role in


    Astrocytes play a key role in the brain, as these cells are involved in fluid, ion, pH, and neurotransmitter homeostasis, synapse function, energy and metabolism and blood-brain barrier (BBB) maintenance (Sofroniew and Vinters, 2010). PA is able to activate different damaging responses in astrocytes, such as inflammation (Gupta et al., 2012), de novo ceramide synthesis (Patil et al., 2007) and endoplasmic reticulum stress (Ortiz-Rodriguez et al., 2018). Moreover, PA has a significant effect on mitochondrial functionality since this saturated fatty Aliskiren is able to dampen mitochondrial membrane potential (Gonzalez-Giraldo et al., 2017; Wong et al., 2014). Additionally, there is evidence that PA actions on astrocytes can involve epigenetic mechanisms, such as DNA methylation (Su et al., 2015) and microRNAs (Geekiyanage and Chan, 2011). Previous studies from our group showed that tibolone has protective effects against cell damage induced by PA on an astrocytic cell model (Gonzalez-Giraldo et al., 2017). In order to explore the possible mechanisms of actions of tibolone on astrocytic cells, in the present study, we aimed to determine which ERs are involved in the protective effects of this compound and how tibolone can modulate gene expression in cells treated with PA.
    Materials and methods
    Discussion In the present study, we have assessed which ER was involved in the protective effects of tibolone on astrocytic cells treated with PA. Then, we evaluated whether tibolone may modulate the expression of genes related to inflammation, astrocytic markers, apoptosis, steroid receptors, telomere complex and DNA methylation. Our findings indicate that although both agonists of either ERα or ERβ mimic the protective actions of tibolone against PA, only the ERβ antagonist was able to block the protective effect of tibolone, particularly, on the mitochondrial membrane potential analysis. It is noteworthy that tibolone reduces inflammatory gene expression and affects epigenetic and telomere pathways, but does not ameliorate the negative effects of PA on astrocyte genes (Fig. 7). High concentrations of PA induce several impairments in astrocytes; for example, PA reduces cell viability, increases inflammatory signals and affects glucose uptake and lactate release (Gupta et al., 2012; Patil et al., 2007). Interestingly, a previous study has demonstrated that astrocytes from different brain areas react differentially to PA stimulation (Oliveira et al., 2018). Moreover, PA upregulates BACE1 and amyloid beta (Aβ) protein production in neurons, with a direct involvement of astrocytes (Patil et al., 2007). Based on these previous findings, PA has been implicated in many pathological states such as neurodegenerative diseases (Hussain et al., 2013). Therapeutic strategies have been evaluated to reverse detrimental effects of PA in astrocyte cells, including the activation of estrogen receptors by estradiol (Frago et al., 2017; Morselli et al., 2014). Estradiol may reduce the expression of inflammatory markers and astrocyte activation via ERα (Morselli et al., 2014), but failed to prevent cellular apoptosis in hypothalamic astrocytes (Frago et al., 2017). In contrast, in the present study, we found that ERα agonist (PPT), ERβ agonist (DPN) and estradiol protected T98G cells from the deleterious effects induced by PA on cell viability, mitochondrial membrane potential and IL-6 cytokine expression (Fig. 7). Overall, these results may suggest that therapeutic strategies targeting estrogen receptors can be useful for neurological diseases. Here, we found that ERβ is involved in the protective action of tibolone on T98G cells exposed to PA (Fig. 4B). Although our results show that activation of the ERα leads to a more efficacious response than the activation of the ERβ, these differences did not reach significance (Fig. 2C). These differences in response could be explained by alterations in the pharmacodynamics of each estrogenic compound, induced by changes in both receptor density and/or affinity (Salahudeen and Nishtala, 2017). In addition, in contrast to other studies using animal models (Crespo-Castrillo et al., 2018) or cells exposed to other stress conditions (Avila-Rodriguez et al., 2016), in the current study, higher concentrations of tibolone, estradiol and estrogen receptor agonists were used. This is due to the fact that physiological concentrations of these compounds did not have any effect on cell viability or mitochondrial membrane potential in our experiments (Gonzalez-Giraldo et al., 2017). Nevertheless, it is important to note that previous studies have also found that pharmacological concentrations (μM) of tibolone and estradiol have beneficial effects (Dodel et al., 1999; Maran et al., 2006). Indeed, effects on the regulation of gene expression by high concentrations of tibolone have been quite similar to those exerted by physiological concentrations of estradiol (Maran et al., 2006). Notably, the affinity of estrogenic compounds with estrogen receptors can be affected by sulfation mechanisms, which have been associated with the inactivation of estradiol (Falany and Falany, 1997) and tibolone (Falany et al., 2004). Therefore, high concentrations could be required to induce a response through ERs (Falany and Falany, 1997).