Investigating neuroprotective agents for the development of new stroke therapies have frequently been unsuccessful (Grupke et al., 2014). Because of this, quite a few researchers have already been exploring potentially active plantderived agents, hoping to meet this unmet require and uncover a diseasemodifying drug (Zhang et al., 2014). Regular Chinese medicine focuses around the all round regulation from the pathophysiological condition with the complete body, a trait that tends to make these compounds specifically promising in the remedy of complex illnesses (Ghosh et al., 2014). TMP is amongst the primarily biologically active constituents derived in the traditional Chinese medicine Chuanxiong (Ligusticum wallichii Franchat) and has been widely employed to treat cardioand cerebrovascular diseases in clinic (Liu et al., 2003; Xue et al., 2011). In our prior study, a TMPderived Dihydroactinidiolide Purity & Documentation compound 22a was developed to combine caffeic acid (Touaibia et al., 2011) (one more all-natural compound with versatile pharmacological activities) and also a nitrone group (Floyd et al., 2013) (a robust free radicaltrapping agent) with TMP. We discovered that compound 22aexhibited strong ROS scavenging activity and exerted protective effects in models of ischemic stroke in vivo (Chen et al., 2017). Inside the present study, the neuroprotective effects of compound 22a against glutamateinduced excitotoxicity on major culture of rat CGNs, as well as the underlying mechanisms of action, have been additional investigated.Supplies AND Solutions Chemical compounds and ReagentsAll media and supplements employed for cell cultures have been purchased from Gibco (Carlsbad, CA, United states), unless otherwise noted. ATP assay kit was obtained from Beyotime (Beyotime, China). LY294002 and Aktiv was obtained from SigmaAldrich (St. Louis, MO, Usa). PGC1 siRNA, scrambled siRNA and transfection reagent have been purchased from Santa Cruz Biotechnology (Santa Cruz, CA, Usa). RIPA lysis buffer, phenylmethanesulfonyl fluoride (PMSF), and halt phosphatase inhibitor cocktail were bought from Pierce Biotechnology (Rockford, IL, United states). Antibodies against phosphoSer473 Akt, phosphoSer9 GSK3, Bcl2 and Bax had been obtained from Cell Signaling Technologies (Beverly, MA, United Role Inhibitors Reagents states of america). Antibodies against actin, PGC1, Nrf2 and HO1 have been obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United states of america). All other reagents have been from SigmaAldrich (St. Louis, MO, United states of america) except where stated otherwise.Principal Cell CulturesEightdayold SpragueDawley rats have been obtained from the Experimental Animal Center of Sun Yatsen University. The detailed methodology made use of to separate the CGNs is described in our previous publication (Chen H.Y. et al., 2015). Briefly, neurons had been seeded at a density of 1.0.five 105 cellswell in basal modified Eagle’s medium supplemented with ten fetal bovine serum, 25 mM KCl, two mM glutaMax and penicillin (100 UmL)streptomycin (one hundred mL). The cultures had been grown at 37 C for 24 h. The growth of nonneuronal cells was limited by adding cytosine arabinoside (ten ). Applying this protocol, 9599 from the cultured cells had been granule neurons. All experiments were performed in CGNs at eight days in vitro (DIV). All experiments have been carried out in accordance with all the suggestions in the Experimental Animal Care and Use Committee of Jinan University. The experimental protocols had been authorized by the Ethics Committee for Animal Experiments of Jinan University.MTT Reduction AssayThe tetrazolium salt 3(four,5dimethylthiazol2yl)two,5diphenyltetrazolium bromide dye (.