Fisetin

Rabbit anti-p-Akt (Ser473), anti-p-Akt (Thr308), anti-Akt, anti-PDK1, anti-p-PDK1, anti-PI3K p85, anti-p-PI3K p85, and anti-ERK1/2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-CLDN2, rabbit anti-CLDN2, mouse anti-ZO-1, and rabbit anti-ZO-1 antibodies were from Thermo Fisher Scientific (Rockford, IL, USA). Mouse anti-p-Stat3 (Y705) and anti-Stat3 antibodies were from BD Biosciences (Franklin Lakes, NJ, USA). Goat anti-β-actin and rabbit anti-p-ERK1/2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fisetin, GEF, and LY-294002 were obtained from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide (DMSO). Control cells were treated with DMSO as a vehicle. The concentration of DMSO in the control and drug-treated cells was 0.1%. CDDP and DXR were from Fujifilm Wako Pure Chemical Industries (Osaka, Japan). DOC and hypoxia probe solution (LOX-1) were from Tokyo Chemical Industry (Tokyo, Japan) and Medical and Biological Laboratory (Tokyo, Japan), respectively. All other reagents were of the highest purity available.

Fisetin was procured from Cayman Chemical Co. (Ann Arbor, MI, United States). PTZ and sodium valproate (VPA) were obtained from Sigma Aldrich (St. Louis, MO, United States). Fisetin was suspended in 0.1% carboxymethylcellulose (CMC) prepared in double-distilled water. PTZ was solubilized in double-distilled water. Fisetin was administered at the doses that have been reported to inhibit the onset and progression of seizures in experimental animals in previous studies (41 (link), 42 (link)). For acute studies, Fisetin was administered in doses of 5, 10, and 20 mg/kg body weight each day by oral (p.o.) route for a week. For the chronic study, the same doses of Fisetin were administered daily for 5 weeks. PTZ was administered in the dose of 25 mg/kg on alternate days by intraperitoneal (i.p.) route. The volume of 10 ml/kg was kept constant for all the administered doses.