Neuroprotective Mechanism of TC-G 1008

The expression levels of GPR39, SIRT1, PGC-1α and Nrf2 were measured at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days post-HIE by Western blot following the manufacturer’s recommendations [34 (link), 35 (link)]. To analyze whether GPR39 receptor and SIRT1/PGC-1α/Nrf2 pathway were involved in the neuroprotective effects of TC-G 1008, the expression levels of GPR39, SIRT1, PGC-1α and Nrf2, and pivotal inflammatory cytokines IL-6, IL-1β, TNF-α were assessed via Western blot. RIPA lysis buffer (Santa Cruz Biotechnology, USA) was used to obtain whole cell lysates. Primary antibodies used were rabbit anti-GPR39 (1:500, Bioss), mouse anti-SIRT1 (1:2000, Abcam), rabbit anti-PGC-1α (1:1000, Abcam), rabbit anti-Nrf2 (1:1000, Abcam), rabbit anti-interleukin (IL)-1β (1:1000, Abcam), rabbit anti-interleukin (IL)-6 (1:1000, Abcam), mouse anti-TNF-α(1:500, Abcam) and mouse anti-β-actin(1:3000, Santa Cruz). The next day, the anti-rabbit (or anti-mouse) secondary antibodies (1:3000, Santa Cruz Biotechnology, USA) were incubated at room temperature for 1–2 h. The gray values were quantified and analyzed by Image J software (NIH).