Effects of Strigolactones on NLRP3 Activation, Nitrosative Stress, and Antioxidant Mox Phenotype: In Vitro and In Silico Evidence

Phytohormones have significant roles in redox metabolism, inflammatory responses, and cellular survival mechanisms within the microenvironment of the mammalian brain. Herein, we identified the mammalian molecular targets of three representative strigolactone (SL) analogues structurally derived from apocarotenoids and the functional equivalent of plant hormones. All tested SL analogues have an inhibitory effect on NLRP3 inflammasome-mediated IL-1β release in murine microglial cells. However, IND and EGO10 became prominent among them due to their high potency at low micromolar doses. All SL analogues dose-dependently suppressed the release and expression of proinflammatory factors. For EGO10 and IND, IC50 values for iNOS-associated NO secretion were as low as 1.72 and 1.02 μM, respectively. In silico analyses revealed that (S)-EGO10 interacted with iNOS, NLRP3, and Keap1 ligands with the highest binding affinities among all stereoisomeric SL analogues. Although all compounds were effective in microglial Mox phenotype polarization, 4-Br-debranone exhibited a differential pattern for upregulating Nrf2-driven downstream enzymes.


MTT cell viability assay
Cells were seeded into 24-well plates at a concentration of 4x10 5 cells/well and incubated for 24 hours.After the cells were treated and incubated for 24 hours under appropriate conditions, the medium of each sample was aspirated gently.The 20 µL MTT at 0.1 mg/mL concentration was simultaneously added to each well and incubated for two hours at 37 ° C, 5% CO 2 , and 75% humidity conditions.The medium was aspirated, and the formazan formed by living cells was dissolved with 200 µL of DMSO for 10 minutes.Cell survival rates were quantified at 570 nm and 620 nm (background) using a Tecan Infinite M200 PRO microplate reader.

NO release measurement and IC 50 determination
NO release levels were determined by analyzing the media collected from the inflammation model of SIM-A9 microglia cells according to the Griess method as previously described. 1fferent doses of SL analogs were administered to SIM-A9 cells together with 1 µg/mL of LPS and incubated for 24 hours.After 24 hours, 100 µL medium for each sample and sodium nitrite (NaNO 2 ) standards were mixed with Griess reagent at equal amounts and incubated in a 96-well plate for 10 minutes at room temperature and in a dark environment.The absorbance of color that formed after incubation was quantified at 520 nm using the microplate reader.The calculated NO concentrations were normalized by cell viability percentages.IC 50 and ANOVA analyses were performed with the GraphPad Prism 8 program.

TNF-α and IL-1β ELISA assays
First of all, SIM-A9 cells were seeded at a density of 4x10 5 cells/well and incubated for 24 hours.Subsequently, media was exchanged with DMEM media without serum, and selected doses of SL analogs with LPS were applied to the SIM-A9 cells.After 24 hours of incubation, the collected media were analyzed using Invitrogen mouse IL-1β and TNF-α ELISA kits (ThermoFisher Invitrogen, Cat.No. BMS6002 and BMS607-3) according to the protocols provided by the manufacturer.The concentrations of IL-1β and TNF-α secreted into the medium were normalized to the total concentration of cellular protein measured by the BCA assay.IL-1β ELISA assay was also carried out for the samples of LPS+ATP/nigericin-induced SIM-A9 cells which were treated with the aforementioned doses of SL analogs.

NLRP3 inflammasome activation model in SIM-A9 cells
SIM-A9 cells were treated firstly with LPS in the presence or absence of SL analogs at selected concentrations and 5 µM of sulforaphane (Cayman Chemical, USA, item no 10496) as a positive control or DMSO as vehicle control 2,3 for 24 hours.After LPS priming, the treatment process followed with 1 mM of adenosine 5′-triphosphate (ATP) (Sigma Aldrich, Cat.No. A6419) or 10 µM of nigericin (Tocris Bioscience, Cat No. 4312) for 40 minutes to switch on the NLRP3 inflammasome mechanism. 4,5The supernatants were collected to evaluate the effects of SL analogs against NLRP3-mediated IL-1β release in microglia cells by ELISA assay.

Gene expression analysis
SIM-A9 cells were treated with certain doses of SL analogs and LPS.After 12 hours, the total RNA of each sample was collected from the cell pellet by the NORGEN Total RNA Purification Plus kit according to the protocols provided by the manufacturer.The cDNA synthesis was actualized using the ABI-High-Capacity cDNA reverse transcriptase kit, according to the manufacturer's instructions.Relative gene expression levels of samples were analyzed by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) that was performed by using cDNAs and TaqMan probes specific for iNOS, IL-1β, COX-2, TNF-α, NQO1, HO-1, Gclc, Srxn1, and Nrf2 genes.

Molecular docking simulations
Molecular docking simulations were performed in AutoDock 4.2.6 software. 6SL analogs were designed using ChemDraw Structure software (RRID: SCR 016768).3D structures of 1400W (PubChem CID: 1433), sulforaphane (PubChem CID: 5350), and EGCG (PubChem CID: 65064) molecules were downloaded from PubChem. 78][9] 3D structure of NLRP3 (PDB ID: 3qf2), 10 KEAP1 (PDB ID: 2flu), and iNOS (PDB ID: 3nw2) 11 were downloaded from the RCSB Protein data bank (rcsb.org). 12Water molecules, inhibitors, and ions were removed from the structure of proteins.Polar hydrogens and Kollman charges were added to the atoms of the proteins and Gastiger charges were added to ligands prior to docking studies.Grid boxes were generated according to the active site of the proteins.
The dimensions for boxes were 40 x 40 x 40 Å for NLRP3, 40 x 50 x 60 Å for KEAP1, and 82 x 80 x 126 Å for iNOS.Fifty Lamarckian genetic algorithm runs with 300 population sizes were carried out for docking studies.The best docking pose for each complex was chosen according to the docking affinity score (more negative value meaning better binding).Amino

Statistical analysis
All data were analyzed statistically using One Way ANOVA with Dunnett's post hoc test and a 95% confidence interval by GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA) (*p < 0.02, **p< 0.005, ***p < 0.001).The mean of each column was compared with the mean of a control column, which is represented as C2 and corresponds to the SIM-A9 cells induced by LPS or LPS+ATP/NIG depending on the evaluation of the inflammatory or NLRP3 inflammasome activation conditions.All results are presented as the individual values of three separate experiments and bars plotted for the mean ± SEM of each group.