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Davis et al. Neuroimmunol Neuroinflammation 2020;7:300-10 I http://dx.doi.org/10.20517/2347-8659.2020.19 Page 305

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Figure 3. Effects of chronic β-FNA on viability of NHA. Cells were cultured in growth medium containing 3 µmol/L β-FNA for 24 h; the
medium was then replaced with serum-free medium containing β-FNA for an additional 48 h. IL-1β (3 ng/mL) was added to cultures for
the final 24 h. Cell viability was assessed using the MTT assay. Data are presented as mean ± SEM (n = 8). ANOVA did not reveal any
significant differences. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SEM: standard error of the mean; β-FNA:
beta-funaltrexamine; NHA: normal human astrocytes; IL-1β: interleukin-1β

Effects of chronic β-FNA on NHA viability
Cell viability was not significantly (P = 0.34) affected by chronic exposure to β-FNA alone or in
combination with IL-1β, as revealed by ANOVA [Figure 3].

Effects of chronic β-FNA on NF-κB activation in NHA
One-way ANOVA and pairwise comparison by a Fisher’s LSD test revealed that chronic exposure to β-FNA
significantly (P < 0.05) inhibited IL-1β-induced phosphorylation of nuclear NF-κB p65 in NHA [Figure 4].
The expression of total nuclear NF-κB p65 in NHA was not significantly (P = 0.2) affected by IL-1β or
β-FNA alone, or the combination of chronic β-FNA plus stimulation with IL-1β [Figure 4].

Effects of acute β-FNA on IL-1β-induced chemokine/cytokine expression in C20 microglial cells
Two-way ANOVA revealed that IL-1β significantly increased CXCL10 (F = 51.4; P < 0.0001), CCL2 (F 1,66
1,79
= 0.71; P < 0.0001), and IL-6 (F = 55.6; P < 0.0001) levels in C20 microglial cells [Figure 5]. Acute (24 h)
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exposure to β-FNA did not significantly affect expression of CXCL10 (F = 0.19; P = 0.9), CCL2 (F =
3,79
3,66
0.71; P = 0.54), or IL-6 (F = 0.065; P = 0.98) in C20 microglial cells. Furthermore, there was no significant
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interaction between stimulation and β-FNA dose for CXCL10, CCL2, or IL-6 (F = 0.24; P = 0.86; F =
3,66
3,79
0.60; P = 0.62; F = 0.07; P = 0.98, respectively.
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Effects of acute β-FNA on C20 microglial cell viability
Two-way ANOVA indicated that the viability of C20 microglial cells was not significantly affected by IL-1β
(F = 0.025; P = 0.87), yet there was a significant main effect of β-FNA (F = 4.99; P < 0.005) [Figure 5].
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1,86
Additionally, there was no significant interaction between stimulation and β-FNA (F = 0.26; P = 0.85).
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DISCUSSION
Astrocytes and microglia are instrumental in neuroinflammation and both cytokines and chemokines are
among the inflammatory molecules released by these cells during neuroinflammation [34-36] . Hence, there is
substantial interest in targeting neuroinflammation as a therapeutic strategy for selected brain disorders.
The therapeutic potential of β-FNA is of particular interest to our group, and the results of this study are in