Abstract
Objective: This study was aimed to evaluate the role of genistein or 4',5,7-trihydroxyisoflavone as a phytoestrogen in the treatment of estrogen deficiency-induced neuroinflammatory. The specific objectives of this study were to determine the anti-neuroinflammatory effect of genistein through measurement of MHC II and Arg1 expressions on microglia HMC3 cell line, as well as to prove that the effect occurs in an ER-dependent manner, through the measurement of free-ERβ expression.
 Methods: The cells were cultured in 24-well microplates, induced with 10 ng IFN-γ, and incubated for 24 h to activate the cell to M1 phenotype which has pro-inflammatory characteristics. Genistein with a concentration of 50 μM was added to the cells. The expression of MHC II, Arg1, and free-ERβ as markers was tested through an immunocytochemistry method and measured using the CLSM instrument. In silico approach was also conducted to determine the interaction between genistein and ERβ, compared to 17β-estradiol. Genistein structure was prepared with Avogadro 1.0.1, and molecular docking was done using PyRx 0.8 software. Biovia Discovery Studio Visualizer 2016 was used to visualize the structure of genistein against 3OLS protein. The physicochemical characteristics of genistein were analyzed using the SwissADME web tool.
 Results: Genistein can decrease MHC II expression and increase Arg1 expression in microglia HMC3 cells compared to negative controls (p<0.005), with expression value of 472.577±26.701 AU and 114.299±6.578 AU. But, genistein cannot decrease the free-ERβ expression in cells (p<0.005). The results of in silico analysis showed that genistein is an ERβ agonist.
 Conclusion: Genistein shows anti-neuroinflammatory effects by decreasing the MHC II expression and increasing Arg1 expression in the microglia HMC3 cell line. However, this effect does not occur through the binding of genistein to ERβ, but it is likely to occur through the binding of genistein with other types of ER.
Highlights
The global prevalence of neurodegenerative diseases has been increasing in recent decades [1]
Visualization of marker expression in microglia human microglia clone 3 (HMC3) cells can be seen in fig. 2 and fig. 3, while the results of the analysis of marker expression are as shown in table 1
The decrease in major histocompatibility complex II (MHC II) expression was indicated by the decrease in the fluorescence intensity of the microglia HMC3 cell line
Summary
The global prevalence of neurodegenerative diseases has been increasing in recent decades [1]. One of the main causes of neurodegenerative diseases is neuroinflammatory. Estrogen deficiency triggers postmenopausal women to be prone to neuroinflammatory, which can causes some neurodegenerative disorders [2,3,4]. There is an increase of the number of microglia cells that are activated in the M1 polarity state which has pro-inflammatory characteristics due to estrogen deficiency. The increasing of activated microglia cells causes an increase in inflammatory cytokines, such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-α (TNF-α), and nitric oxide (NO), as well as major histocompatibility complex II (MHC II) [5, 6]. Continuous M1 polarity state activation of microglia cells can cause prolonged inflammation and induces hippocampal neuron cell death leading to decreased cognitive function [7,8,9]
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