Mpp dihydrochloride

Chlorogenic acid, rutin, 3,4‐dicaffeoylquinic acid (DA), isorhamnetin 3‐O‐glucoside (IG), calenduloside E (CE), curcumin, nomifensine and MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) were purchased from Shanghai Yuanye Biotechnology Co. Ltd. MPP (dihydrochloride) was purchased from Sigma‐Aldrich. Acetonitrile and MS‐grade water were acquired from Tedia Company Inc. and Watsons Ltd., respectively. All other chemicals were of analytical grade. SH‐SY5Y cells were obtained from the American Type Culture Collection (ATCC). Fresh flower heads of C. officinalis were obtained from the Anguo medicine market, Hebei Province. One gram of plant material was extracted with 8 mL of 70% ethanol for 1 h using ultrasound, and, subsequently, the solution was concentrated to acquire the residue (111.4 mg).

Agathisflavone (FAB) was extracted from Poincianella pyramidalis (Tul.) as previously described [17 (link)], stored at 100 mM in dimethyl sulfoxide (DMSO, Sigma Aldrich, Saint Luis, MO, USA, 472301), and kept out of light at −20 °C until use. For the experiments, FAB was diluted in culture medium to make a final concentration of 0.1 or 1 µM in the neuron–glial cocultures; control cultures were treated with DMSO, the vehicle of dilution of FAB. To induce neuroinflammation, at 26 DIV, cocultures were treated for 24 h with LPS (1 µg/mL; Sigma Aldrich, Saint Luis, MO, USA, L2880) or IL-1β (10 ng/mL; R&D Systems, Minneapolis, MN, USA, 501-RL-010); then, the medium was removed and replaced with medium containing just agathisflavone (0.1 or 1 µM) or vehicle, and cultures were analyzed after 24 h. To assess whether the effects of agathisflavone were mediated through ERs, neuron–glial cocultures were treated with the selective ER-α antagonist MPP dihydrochloride at 10 nM (1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride; Sigma Aldrich, Saint Luis, MO, USA, M7068) or the selective ER-β antagonist PHTPP at 1 μM (4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol; Tocris, Bristol, UK, #2662); control cultures were treated with DMSO vehicle.