Impacts Of Mild Hypothermia On LPS-mediated TLR4/NF-κB Signaling Pathway In Microglia

Backgroung: Sepsis is a systemic inflammatory response syndrome caused by infection. Microglia is immunocompetent cells of the nervous system, the main expression of toll-like4 receptor. The TLR4 exist on the cell membrane, can specifically recognize LPS, trigger SIRS. Hypothermia therapy in the treatment of various diseases of the nervous system in the occurrence and development have obvious advantages, and was included in the 2010 United States guidelines for cpr. But the mechanism of hypothermia on sepsis course of cerebral protection is not clear. We conducted this study, to understand whether mild hypothermia by inhibiting inflammatory factors influencing TLR4/NF-k B signaling pathway in microglia release, the protective effect to the nervous system.Objective: To investigate the impacts of mild hypothermia on LPS-mediated TLR4/NF-κB signal pathway in microglia.Methods: The microglia cells infected with LPS in vitro,cells and cellular supeinatants were harvested at the selected time points after LPS infection(0h、2h、6h、12h、24h,respectively).The uninfected microglia cells were as normal controls. The expression of TLR4, NF-κB gene m RNA were evaluated semiquantitatively by RT-PCR. The expression levels of TLR4, NF-κB protein were detected by Western blot. The concentration of TNF alpha and IL-10 in cell supernatant was detected by ELISA. The MTT assays were used to detect the cell proliferation.Results:(1)37℃-LPS: The levels of TNF alpha and IL-10 increased gradually. The LPS infection could markedly up-regulate the expression of TLR4, NF-κB m RNA in a time-dependent manner. The cell supernatant concentration of TNF-α and IL-10 increased gradually. The expression levels of TLR4, NF-κB protein gone up. It was found that LPS infection could up-regulate the growth inhibition of cell in a time-dependent manner.(2) 33℃-LPS: The levels of TNF alpha and IL-10 were lower than in 37℃-LPS infected cells. The expression of TLR4, NF-κB m RNA could up-regulate markedly in a time-dependent manner and these were lower than the group of infection(TNF-α:2hh: P< 0.01;6h:P<0.01;12h: p<0.0001;24h: P<0.0001,IL-10:2h: P2<0.05; 6h:P<0.005;12h: P<0.0001, 24h: P<0.0001). The cell supernatant concentration of TNF-α and IL-10 were lower than in in 37℃-LPS infected cells. The expression levels of TLR4、NF-κB protein was lower than that in the infected cells(TLR4 m RNA:0h: P=0.4496;2h: P<0.0001, P2< 0.01;6h:P<0.0001,12h: P<0.0001,24h: P24<0.005;TLR4protein:0h: P=0.0763, 2h: P< 0.01;6h: P<0.01;12h:P<0.01;24h:P<0.0.01;NF-κB m RNA:0h:P=0.9048;2h:P=0.0001;6h:P=0.001;12h:P=0.0001;24h:P=0.0001;NF-κBprotein:0h:P=0.1277, 2h:P< 0.01;6h: P<0.05;12h: P<0.01; 24h:P<0.001); At different time points were statistically significant.(3) The control group:There is no statistical significance between normothermic and mild hypothermic control group of inflammatory cytokines TNF-α and IL-10 content at different time points; cell Toll4, NF kappa B gene transcription level and protein content between different time points not statistically significant, and there is no time dependence, and LPS group have statistical significance.Conclusions:LPS infected microglia cells in vitro can induce oxidative stress, LPS infection could up-regulate the expression of TLR4 and its major downstream signal molecules. In mild hypothermia condition, the expression of TLR4 and its downstream molecules, although the performance of inflammatory factors at different time points to continue to rise, but at the same time than the normal LPS group at each time point is low, with statistical significance. The above data show that microglial inflammatory reaction mediated by LPS, mild hypothermia can reduce inflammation by downregulation of TLR4/NF-KB signal molecules.