Protective effect of Toll-like receptor 4 inhibitor TAK-242 on β-amyloid peptide25-35 inducing PC12 cytotoxicity

Abstract

Objective To study the role of TAK-242, a Toll-like receptor 4 (TLR4) specific inhibitor, in β-amyloid peptide (Aβ)25-35 inducing PC12 cytotoxicity and its potential mechanism. Methods PC12 cells were cultured with different concentrations of Aβ25-35 (0, 10, 20 and 30 μmol/L) for 24 h, and then, the cell survival rate was detected by CCK-8 kit to choose the specific concentration of Aβ25-35 to establish cell AD models. The survival rate of Aβ25-35 inducing PC12 cells was further detected one h after TAK-242 intervention. The PC12 cells were divided into four groups: control group, Aβ treatment group, Aβ+TAK-242 pretreatment group and TAK-242 group. The apoptosis of cells was observed with Hoechst 33258 kit. The secretions of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) were detected with ELISA. The protein expression levels of TLR4, myeloid differentiation factor 88 (MyD88), IκB kinase complexus α/β (IKKα/β) and nuclear factor (NF)-κB were detected by Western blotting. Results The cell survival rate decreased gradually with the increase of Aβ25-35 concentrations after PC12 cells cultured with Aβ25-35 for 1 h. Twenty μmol/L Aβ25-35 was used to establish the AD models, with which the cell survival rate was closely half of the control group. As compared with Aβ treatment group, Aβ+TAK-242 pretreatment group had significantly increased cell survival rate and significantly decreased apoptosis (P<0.05). The secretions of IL-1β and TNF-α in Aβ treatment group were significantly increased than those in the control group (P<0.05), and Aβ+ TAK-242 pretreatment group had significantly decreased secretions of IL-1β and TNF-α (P<0.05). As compared with those in the control group, the protein expressions of TLR4, MyD88, IKKα/β and NF-κB in the Aβ treatment group were significantly increased (P<0.05); as compared with Aβ treatment group, the protein expressions of TLR4, MyD88, IKKα/β and NF-κB in the Aβ + TAK-242 pretreatment group were degraded obviously, with significant differences (P<0.05). Conclusions Aβ25-35 could reduce the cell survival rate and apoptosis in PC12 cells by up-regulating the expressions of TLR4/MyD88 signal pathway related proteins and increasing the secretions of IL-1β and TNF-α, and the phenomenon is concentration-dependent. TAK-242 could resist Aβ25-35-induced PC12 cytotoxicity through down-regulating the TLR4/MyD88 signal pathway related proteins levels and decreasing the secretions of TNF-α and IL-1β. Key words: Toll-like receptor 4; TAK-242; Myeloid differentiation factor 88; Aβ25-35