EETs Inhibit Induction Of Toll-like Receptor4Nuclear Factor Kappa B Pathway In Adipocytes And Macrophages Induced By Saturated Fatty Acids

Research Backgrounds and Aims： Arachidonic acid (AA) is the most widelydistributed polyunsaturated fatty acids in human body and is one of the most abundantsubstances. It is well known that the AA is metabolized by CYP epoxygenases to fourbiological active substances, respectively,5,6-EET,8,9-EET,11,12-EET and14,15-EET,which play a vital biological role in depreesuization, anti-inflammation, anti-apotosis,anti-mycardialdamage and anti-atherosclerosis.Metabolic syndrome is a complex disease arising from the interaction between thelifestyle and the emerging sudden risk factors, including central(intra-abdominal) obesity,dyslipidemia, hyperglycaemia and hypertension, interested in the more than three riskfactors can be diagnosed as metabolic syndrome. Central obesity, as assessed by waistcircumference, was agreed as an essential diagnostic criterion because of the strength ofthe evidence linking waist circumference and other metabolic co-morbidities and thelikehood than central obesity is an early step in the aetiological cascade leading to full MS.obesity is regarded as a chronic and low-grade inflammatory disease. Type2diabetesmellitus, insulin resistance and the metabolic syndrome are characterized by increased levels of systemic fatty acids, by an increased lipolysis of visceral adipose tissue. FFAsrepresent an important energy source mobilized from triglycerides stored in the adiposetissue, particularly during periods of starvation, but recent evidence has suggested thepathophysiologic roles other than the supply of nutrients in times of fasting or increasedenergy demand. There are a couple of reports demonstrating that exogenousadministration of saturated fatty acids (FAs) exerts the proinflammatory effects in certaincells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that play acritical role in the innate immune system by activating pro-inflammatory signalingpathways in response to microbial pathogens. TLR4, the best-characterized TLR, binds toLPS of gram-negative bacterial cell walls and to non-bacterial molecules, such as saturatedfatty acids. The activation of TLR4upregulates intracellular inflammatory pathways relatedto the induction of insulin resistance, such as JNK or NF-κ B. TLR4has been proposed tobe a molecular link between lipids, inflammation and insulin resistance. It has beensuggested that a paracrine loop between adipocytes and macrophages establishes a viciouscircle that aggravates the inflammatory changes in AT. This paracrine loop involves FFAsand TNF-α.Enlarged adipocytes release in excess saturated FAs that activatemacrophages via TLR4signaling. As a result, macrophages secrete the pro-inflammatoryadipokine TNF-α, which in turn acts on TNFR1and induces inflammatory changes inhypertrophied adipocytes through activation of the NF-κ B pathway and as well asenhanced FFA release.In this study, we assume that exogenous EETs improve obesity by inhibiting theinflammation induced by FFA. This study is aim to investigate whether EETs inhibite theinflammation in3T3-L1and RAW264.6cells induced by FFA and its possible molecularmechanism, as well as further clarifing whether EETs can improve inflammation of adiposetissue and attenuate the progress of the metabolic syndrome..Methods: Under the guidance3T3-L1preadipocytes are induced to differentiate intomature adipocytes, which indentified by Oil red staning. FFA plays a role for the experimental group in cells, with or without EETs and CLI-095, The cells were collected byReal-time PCR detection of TNF-α IL-6, MCP-1mRNA expression levels; With WesternBlot detection groups pIKBα and NF-kappa B protein expression levels; Cells with highlevels of expression of TLR4, with or without EETs, the cells were collected by Real-timePCR detection of TNF-α, IL-6, MCP-1mRNA expression levels; With Western Blotdetection groups pIKBα and NF-kappa B protein expression levels; SPSS13.0statisticalsoftware. Integrated to determine exogenous EETs whether the impact of the FFA-inducedcell inflammatory response and its possible molecular mechanism.Results: For3T3-L1cells and RAW264.7cells comparing with the FFA group, themRNA levels of MCP-1, IL-6and TNF-αof the8,9-EET group decreased（P＜0.05），the proteins levels of pIKBα and nuclear NF-kappa B decreased（P＜0.05）,and thislowering effect disappears while TLR4inhibitors function. For3T3-L1cells andRAW264.7cells, Comparing with the overexpression of TLR4group, the mRNA levels ofMCP-1, IL-6and TNF-α and the proteins levels of pIKBα and nuclear NF-kappa Bdecreased in TLR4+14,14-EET group（P＜0.05）.Conclusion:1. Exogenous EETs suppressed the increased secretion of MCP-1,IL-6,andTNF-a stimulated by FFA in3T3-L1and raw264.7cells.2. Exogenous EETs caninhibit the activation of the inflammatory signaling pathways stimulated by FFA in3T3-L1and raw264.7cells, mechanism of action is likely to be through Toll-like receptor4/NF-κBpathway.