The Effects Of TLR4 On The Injury Of HT22 Cell Induced By Ox-LDL

[Backgroud and Objective] Neuronal loss is associated with neurodegeneration such as Alzheimer's disease(AD).Oxidized LDL(ox-LDL)is capable of eliciting neuronal death,which contributes to the development of neurodegerative diseases.However,the precise mechanisms are still not fully understood.In present study,to explore the potential role and mechanisms of TLR4-PI3K/AKT/ NF-?B signaling in the injury of HT22 cell induced by ox-LDL,Toll-like receptor 4(TLR4)inhibitor TAK-242,nuclear factor-kappa B(NF-?B)inhibitor pyrrolidine dithiocarbamate(PDTC)and PI3 K inhibitor LY294002 were used to suppress TLR4-PI3K/AKT/NF-?B signaling pathway.[Methods] To investigate the role of ox-LDL on the HT22 cell injury the HT22 cells activity and apoptosis were detected by CCK8 assay and by flow cytometer with Annexin-V/PI double stain after treatment with different concentration of ox-LDL(12.5 ?g/mL,25 ?g/mL,50 ?g/mL and 100 ?g/mL)for 24 h,respectively.Furthermore,morphological alterations of HT22 neuron cells were analyzed by light microscope and TEM(transmission electron microscope,TEM).To explore ox-LDL on the activation of TLR4-PI3K/AKT/NF-?B signaling pathway,the expressions of TLR4,p-AKT,p-NF-?B p65,Bcl-2,Bax and IL-1? were measured by Western blotting method.To study the effects and mechanisms of TLR4-PI3K/AKT/NF-?B pathway in ox-LDL induced injury of HT22 cell,the cultured TH22 cells were pre-treated with TLR4 inhibitor TAK-242,or pre-treated with NF-?B inhibitor PDTC,or pre-treated with PI3 K inhibitor LY294002 for 0.5 h,respectively,and then all cells were co-incubated with ox-LDL at concentration of 100 ?g/mL for another 24 h.The HT22 cell activity and apoptosis were detected by CCK8 assay and by flow cytometer with Annexin-V/PI double stain,respectively.The expressions of TLR4,p-AKT,p-NF-?B p65,Bcl-2,Bax and IL-1? were measured by Western blotting method.[Results] CCK-8 analysis indicated that ox-LDL remarkably decreased HT22 cells activity in a dose-dependent manner at the range of 12.5-100 ?g/mL and in a time-dependent manner at the range of 4-24 h.The percentage of apoptotic HT22 cells was significantly increased in ox-LDL group as compared with that in the control group,.Apoptotic cells and morphologic alterations were observed both under light microscope(the lowered refractivity of neuronal somata;the diminished connections between dendrites and axons)and TEM.Furthermore,Western blotting results showed that the protein levels of TLR4,p-AKT,p-NF-?B p65 levels IL-? and Bax significantly elevated,while the expression of Bcl-2 was down-regulated by ox-LDL in TH22 cells.Flow cytometer analysis demonstrated that pre-treated with TLR4 inhibitor TAK-242,NF-?B inhibitor PDTC and PI3 K inhibitor LY294002 obviously decreased the apoptosis rate of HT22 cells induced by 100?g/mL ox-LDL.Western blotting results indicated that TLR4,p-NF-?B p65,p-AKT,IL-? and Bax protein levels were markedly lowered,while the expression of Bcl-2 significantly elevated by pro-treatment with TAK-242,PDTC and LY294002 as compared with ox-LDL group,respectively.[Conclusion] The present study indicates that PI3K/AKT-NF-?B signaling pathway mediated by TLR4 contributes to the apoptotic processes of the HT22 hippocampal neurons induced by ox-LDL,and inhibition of this signaling pathway decreases HT22 cells apoptosis.