Half maximal inhibitory concentration (IC50) values are presented below. constituent of [27] and SG-Tang [25], glycyrrhizic acid has been used to treat inflammatory diseases [29]. VB-037 is usually a quinoline compound protecting neurons against A aggregates-induced cytotoxicity through reduction of P38- and JNK-mediated inflammation [30]. In addition, anandamide transport inhibitor AM404 (C26H37NO2) displays anti-inflammatory and neuroprotection effects on N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity FLJ45651 [31]. In the present study, we examined the anti-inflammatory effects of AM404, VB-037, glycyrrhizic acid, SG-Tang, and on BV-2 microglia and inducible A53T SNCA-GFP-expressing SH-SY5Y cells. We also explored if these CHMs or compounds exert their effects through mitigating the NLRP1/3, caspase 1, IL-1, IL-6, and associated IB/P65, JNK/JUN, P38/STAT1, and JAK2/STAT3 pathways. 2. Results 2.1. -Synuclein Induced Microglial Activation in Mouse BV-2 Cells -Synuclein has been known to induce neuroinflammation and cell death [32]. In the brain, activated microglia release proinflammatory mediators in response to neuroinflammation [33]. To investigate -synuclein induced inflammation in mouse BV-2 microglial cells, -synuclein fibrils prepared from the = CPI 4203 0.006?0.002) and increased expression of the microglia marker Iba1 (192C219%, = 0.041?0.030) were observed after LPS or -synuclein stimulation (Figure 1c,d). Exposure of BV-2 cells to -synuclein (2.5 M) also resulted in increased expression of CD68 (252%, = 0.004) and major histcompatibility complex class II (MHCII) (198%, = 0.002) (Physique 1e). -Synuclein fibrils at 2.5 M was selected to provide inflammatory stimulus to BV-2 microglia for the following experiments. Open in a separate window Physique 1 -Synuclein induced inflammation in mouse BV-2 microglial cells. (a) Experimental flow chart. BV-2 cells were plated in 1% fetal bovine serum (FBS) on day 1. On day 2, the cells were treated with lipopolysaccharide (LPS) (1 g/mL) or -synuclein fibrils (-Syn, 2.5C5 M) to induce inflammation. On day 3, the cells were examined for microglial activation by morphology, NO release in cell culture medium and Iba1 Western blotting. (b) Morphology, (c) NO production (= 3), and (d) Iba1 level (= 3) of inactive and LPS or -synuclein fibrils activated BV-2 cells. For normalization, the relative Iba1 level in inactive cells was set as 100%. (e) Immunocytochemistry of CD68 and CPI 4203 major histcompatibility complex class II (MHCII) (red) and fluorescent intensity with or without -synuclein (2.5 M) activation. Nuclei were counter stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). For normalization, the relative fluorescent intensity in inactive cells was set as 100% (= 3). values: comparisons between inactive and activated cells (*: 0.05 and **: 0.01). (Two-tailed Students test). Initial blot images can be found in the Supplementary Materials. 2.2. Anti-Inflammatory Potentials of Test Compounds and Herbs in BV-2 Microglia Three compounds (AM404, VB-037 CPI 4203 and glycyrrhetic acid; Physique 2a) and two herbs (extract and formulated SG-Tang) were tested. Ammonium glycyrrhizinate, a common active constituent in both and SG-Tang, is usually a glycyrrhizic acid salt. It is hydrolyzed by intestinal flora to glycyrrhetic acid. The amounts of ammonium glycyrrhizinate in these two herbs were 2.23% (26.6 mM) in extract [27] and 2.43% (14.52 mM) in formulated SG-Tang [25]. As shown in Physique 2b, AM404, VB-037, glycyrrhetic acid, and SG-Tang had half maximal inhibitory concentration (IC50) for BV-2 viability of 100 M, 71 M, 100 M, 500 g/mL and 500 g/mL, respectively, in BV-2 cells. The anti-inflammatory effects of these compounds and herbs on -synuclein fibrils (2.5 M)-activated BV-2.