JBM  Vol.7 No.2 , February 2019
Impacts of Mild Hypothermia on LPS-Mediated TLR4/NF-κB Signaling Pathway in Microglia
Abstract: Background: Existing studies have found that some inflammatory factors cause brain cell damage through the TLR4/NF-κB pathway, and that mild hypothermia has a protective effect on nerve cells. It is not clear whether the mild hypothermic brain protection is achieved through the TLR4/NF-κB pathway in microglia. Objective: To investigate the impacts of mild hypothermia on lipopolysaccharide (LPS)-mediated TLR4/NF-κB signaling pathway in microglia. Method: The cultured microglia cells in vitro were divided into the NS group and the LPS group at 33?C and 37?C, respectively; quantitative RT-PCR was performed to detect the expressions of TLR4 and NF-κB mRNA in the microglia, Western blot was used to detect the expressions of TLR4 and NF-κB protein in the microglia, and ELISA was performed to detect the levels of tumor necrosis factor α (TNF-α) and interleukin-10 (IL-10) in the culture medium. Results: Under the LPS stimulation, the mRNA and protein expressions of TLR4 and NF-κB at different time points had significant changes between the normothermia group and the mild hypothermia group, in which the expressions in the former group were firstly increased and then decreased, while those in the latter showed a continuous increasing trend (P < 0.01); and the expressions of TNF-α in all the groups presented the trend of first-increasing then-decreasing, while IL-10 exhibited one slow linear increasing trend (P < 0.01). Conclusions: Mild hypothermia could inhibit the mRNA and protein expressions of LPS-mediated TLR4/NF-κB signaling pathway in the microglia, and inhibit the production and release of downstream inflammatory cytokines (TNF-α and IL-10).
Cite this paper: Liu, L. , Li, X. , Wang, Y. , Cao, F. , Zhang, S. , Zhan, Z. , Meng, Y. and Xie, Q. (2019) Impacts of Mild Hypothermia on LPS-Mediated TLR4/NF-κB Signaling Pathway in Microglia. Journal of Biosciences and Medicines, 7, 86-97. doi: 10.4236/jbm.2019.72008.

[1]   Chelazzi, C., Consales, G. and De Gaudio, A.R. (2008) Sepsis Associated Encephalopathy. Current Anaesthesia & Critical Care, 19, 15-21.

[2]   Gilmore, E.J., Gaspard, N., Choi, H.A., Cohen, E., Burkart, K.M., Chong, D.H., Claassen, J. and Hirsch, L.J. (2015) Acute Brain Failure in Severe Sepsis: A Prospective Study in the Medical Intensive Care Unit Utilizing Continuous EEG Monitoring. Intensive Care Medicine, 41, 686-694.

[3]   Lu, Y.C., Yeh, W.C. and Ohashi, P.S. (2008) LPS/TLR4 Signal Transduction Pathway. Cytokine, 42, 145-151.

[4]   Aravalli, R.N., Peterson, P.K. and Lokensgard, J.R. (2007) Toll-Like Receptors in Defense and Damage of the Central Nervous System. Journal of Neuroimmune Pharmacology, 2, 297-312.

[5]   Ishizuka, F., Shimazawa, M., Inoue, Y., et al. (2013) Toll-Like Receptor 4 Mediates Retinal Ischemia/Reperfusion Injury through Nuclear Factor-KappaB and Spleen Tyrosine Kinase Activation. Investigative Ophthalmology & Visual Science, 54, 5807-5816.

[6]   Song, J., Oh, Y. and Lee, J.E. (2015) MiR-Let7A Modulates Autophagy Induction in LPS-Activated Microglia. Experimental Neurobiology, 24, 117-125.

[7]   Franco, R. and Fernandez-Suarez, D. (2015) Alternatively Activated Microglia and Macrophages in the Central Nervous System. Progress in Neurobiology, 131, 65-86.

[8]   Park, S.Y., Kim, Y.H. and Park, G. (2015) Cucurbitacins Attenuate Microglial Activation and Protect from Neuroinflammatory Injury through Nrf2/AREactivation and STAT/NF-κB Inhibition. Neuroscience Letters, 609, 129-136.

[9]   Block, M.L. and Hong, J.S. (2005) Microglia and Inflammation-Mediated Neurodegeneration: Multiple Triggers with a Common Mechanism. Progress in Neurobi-ology, 76, 77-98.

[10]   Streit, W.J., Conde, J.R., Fendrick, S.E., et al. (2005) Role of Microglia in the Central Nervous System’s Immune Response. Neurological Research, 27, 685-691.

[11]   Gilgun-Sherki, Y., Rosenbaum, Z., Melamed, E., et al. (2002) Antioxidant Therapy in Acute Central Nervous System Injury: Current State. Pharmacological Reviews, 54, 271-284.

[12]   Wu, M.H., Huang, C.C., Chio, C.C., Tsai, K.J., Chang, C.P., Lin, N.K. and Lin, M.T. (2016) Inhibition of Peripheral TNF-α and Downregulation of Microglial Activation by Alpha-Lipoic Acid and Etanercept Protect Rat Brain against Ischemic Stroke. Molecular Neurobiology, 53, 4961-4971.

[13]   Fan, B., Dun, S.H., Gu, J.Q., Guo, Y. and Ikuyama, S. (2015) Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation. PLoS ONE, 10, e0137837.

[14]   Kang, B.K., Kim, M.K., Kim, S.Y., Lee, S.J., Choi, Y.W., Choi, B.T. and Shin, H.K. (2015) Anti-Neuroinflammatory Effects of Uncaria sinensis in LPS-Stimulated BV2 Microglia Cells and Focal Cerebral Ischemic Mice. The American Journal of Chinese Medicine, 43, 1099-1115.

[15]   Matsui, T., Yoshida, Y., Yanagihara, M., et al. (2014) Hypothermia at 35 Degrees C Reduces the Time-Dependent Microglial Production of Pro-Inflammatory and Anti-Inflammatory Factors That Mediate Neuronal Cell Death. Neurocritical Care, 20, 301-310.

[16]   Kalita, J., Bastia, J., Bhoi, S.K., et al. (2015) Systemic Inflammatory Response Syndrome Predicts Severity of Stroke and Outcome. Journal of Stroke and Cerebrovascular Diseases, 24, 1640-1648.

[17]   Kim, F., Nichol, G., Maynard, C., Hallstrom, A., Kudenchuk, P.J., Rea, T., Copass, M.K., Carlbom, D., Deem, S., Longstreth, W.T., Olsufka, M. and Cobb, L.A. (2014) Effect of Prehospital Induction of Mild Hypothermia on Survival and Neurological Status among Adults with Cardiac Arrestra Randomized Clinical Trial. JAMA, 311, 45-52.

[18]   Gancia, P. and Pomero, G. (2012) Therapeutic Hypothermia in the Prevention of Hypoxic-Ischaemic Encephalopathy; New Categories to Be Enrolled. The Journal of Matemal-Fetal & Neonatal Medicine, 25, 94-96.

[19]   Nakamura, Y., Si, Q.S. and Kataoka, K. (1999) Lipopolysaccharide-Induced Microglial Activation in Culture: Temporal Profiles of Morphological Change and Release of Cytokines and Nitric Oxide. Neuroscience Research, 35, 95-100.

[20]   Bhatia, H.S., Candelario-Jalil, E., de Oliveira, A.C., Olajide, O.A., Martínez-Sánchez, G. and Fiebich, B.L. (2008) Mangiferin Inhibits Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Activated Ratmicroglial Cells. Archives of Biochemistry and Biophysics, 477, 253-258.

[21]   Iwashyna, T.J., Ely, E.W., Smith, D.M. and Langa, K.M. (2010) Long-Term Cognitive Impairment and Functional Disability among Survivors of Severe Sepsis. JAMA, 304, 1787-1794.

[22]   Shah, F.A., Pike, F., Alvarez, K., Angus, D., Newman, A.B., Lopez, O., Tate, J., Kapur, V., Wilsdon, A., Krishnan, J.A., Hansel, N., Au, D., Avdalovic, M., Fan, V.S., Barr, R.G. and Yende, S. (2013) Bidi-rectional Relationship between Cognitive Function and Pneumonia. American Journal of Respiratory and Critical Care Medicine, 188, 586-592.

[23]   Widmann, C.N. and Heneka, M.T. (2014) Long-Term Cerebral Consequences of Sepsis. The Lancet Neurology, 13, 630-636.

[24]   Tang, Z.H., Hu, J.T., Lu, Z.C., et al. (2014) Effect of Mild Hypothermia on the Expression of Toll-Like Receptor 2 in Lung Tissues with Experimental Acute Lung Injury. Heart, Lung and Circulation, 23, 1202-1207.

[25]   Hiller, S., DeKroon, R., Xu, L., et al. (2014) Alpha-Lipoic Acid Protects Mitochondrial Enzymes and Attenuates Lipopolysaccharide-Induced Hypothermia in Mice. Free Radical Biology & Medicine, 71, 362-367.

[26]   Hakim, T.S., Pedoto, A., Nandi, J., Bosco, G., Rubini, A., Mangar, D., et al. (2014) Hypothermia Attenuates NO Production in Anesthetized Rats with Endotoxemia. Naunyn-Schmiedeberg's Archives of Pharmacology, 387, 659-665.

[27]   Wang, S., Tang, Z., Hu, J., Jiang, L. and Yin, X. (2014) Impact of Hypothermia on Toll-Like Receptor 4 mRNA Transcription and Inflammatory Balance of Macrophage Induced by Lipopolysac-charide. Chinese Critical Care Medicine, 26, 84-88.

[28]   Altinsoy, C., Tuzun, F., Duman, N., Sever, A.H., Dilek, M., Ozbal, S., et al. (2014) Effect of Induced Hypothermia on Lipopolysaccharide-Induced Lung Injury in Neonatal Rats. The Journal of Maternal-Fetal & Neonatal Medicine, 27, 421-429.

[29]   Schwarzl, M., Seiler, S., Wallner, M., von Lewinski, D., Huber, S., Maechler, H., et al. (2013) Mild Hypothermia Attenuates Circulatory and Pulmonary Dysfunction during Experimental Endotoxemia. Critical Care Medicine, 41, e401-e410.

[30]   Chang, Y.T., Wann, S.R., Tsai, J.S., Kao, C.H., Lee, P.T., Huang, N.C., et al. (2013) The Role of Autonomic Nervous System Function in Hypothermia-Mediated Sepsis Protection. The American Journal of Emergency Medicine, 31, 375-380.

[31]   Kumar, A., Bhatia, H.S., de Oliveira, A.C. and Fiebich, B.L. (2015) microRNA-26a Modulates Inflammatory Response Induced by Toll-Like Receptor 4 Stimulation in Microglia. Journal of Neurochemistry, 135, 1189-1202.

[32]   Wang, H.Y., Wang, H., Wang, J.H., Wang, Q., Ma, Q.F. and Chen, Y.Y. (2015) Protocatechuic Acid Inhibits Inflammatory Responses in LPS-Stimulated BV2 Microglia via NF-κB and MAPKs Signaling Pathways. Neurochemical Research, 40, 1655-1660.

[33]   Kim, P.K. and Deutschman, C.S. (2000) Inflammatory Responses and Mediators. Surgical Clinics of North America, 80, 885-894.

[34]   Yang, G.Y., Schielke, G.P., Gong, C., Mao, Y., Ge, H.L., Liu, X.H. and Betz, A.L. (1999) Expression of Tumor Necrosis Factor-Alpha and Intercellular Adhesion Molecule-1 after Focal Cerebral Ischemia in Interleukin-1beta Converting Enzyme Deficient Mice. Journal of Cerebral Blood Flow & Metabolism, 19, 1109-1117.

[35]   Zou, J.Y. and Crews, F.T. (2005) TNF Alpha Potentiates Glutamate Neurotoxicity by Inhibiting Glutamate Uptake in Organotypic Brain Slice Cultures: Neuroprotection by NF Kappa B In-hibition. Brain Research, 1034, 11-24.

[36]   Tambuyzer, B.R., Ponsaerts, P. and Nouwen, E.J. (2009) Microglia: Gatekeepers of Central Nervous System Immunology. Journal of Leukocyte Biology, 85, 352-370.

[37]   Streit, W.J. (2002) Microglia as Neuroprotective, Immunocompetent Cells of the CNS. Glia, 40, 133-139.

[38]   Dai, X.J., Li, N., Yu, L., et al. (2015) Activation of BV2 Microglia by Lipopolysaccharide Triggers an Inflammatory Reaction in PC12 Cell Apoptosis through a Toll-Like Receptor 4-Dependent Pathway. Cell Stress Chaperones, 20, 321-331.

[39]   Huang, R.L., Yuan, Y., Zou, G.M., et al. (2014) LPS-Stimulated Inflammatory Environment Inhibits BMP-2-Induced Osteoblastic Dif-ferentiation through Crosstalk between TLR4/MyD88/NF-kappaB and BMP/Smad Signaling. Stem Cells and Development, 23, 277-289.

[40]   Guo, L., Li, S., Zhao, Y., et al. (2015) Silencing Angiopoietin-Like Protein 4 (ANGPTL4) Protects against Lipopolysaccharide-Induced Acute Lung Injury via Regulating SIRT1/NF-kB Pathway. Journal of Cellular Physiology, 230, 2390-2402.

[41]   Zhu, J.P., Wu, K., Li, J.Y., et al. (2015) Cryptoporus volvatus Polysaccharides Attenuate LPS-Induced Expression of Pro-Inflammatory Factors via the TLR2 Signaling Pathway in Human Alveolar Epithelial Cells. Pharmaceutical Biology, 54, 1-7.

[42]   Zhao, H., Cheng, L., Liu, Y., et al. (2014) Mechanisms of Anti-Inflammatory Property of Conserved Dopamine Neurotrophic Factor: Inhibition of JNK Signaling in Lipopolysaccha-ride-Induced Microglia. Journal of Molecular Neuroscience, 52, 186-192.

[43]   Badshah, H., Ali, T., Rehman, S.U., Amin, F.U., Ullah, F., Kim, T.H. and Kim, M.O. (2016) Protective Effect of Lupeol against Lipopolysaccharide-Induced Neuroinflammation via the p38/c-Jun N-Terminal Kinase Pathway in the Adult Mouse Brain. Journal of NeuroImmune Pharmacology, 11, 48-60.

[44]   Ma, B., Yu, J., Xie, C., Sun, L., Lin, S., Ding, J., Luo, J. and Cai, H. (2015) Toll-Like Receptors Promote Mitochondrial Translocation of Nuclear Transcription Factor Nuclear Factor of Activated T-Cells in Prolonged Microglial Activation. Journal of Neuroscience, 35, 10799-10814.