The Experimental Study On The Mechanisms Of Atorvastatin Suppressing Nuclear Factor Kappa B Activation

阿托伐他汀；；血管内皮细胞；；动脉粥样硬化；；炎症；；核因子κB；；核因子κB抑制因子α；；磷酸化核因子κB抑制因子α；；泛素-蛋白连接酶亚单位；；泛素偶连酶5；；Toll样受体4；；核因子κB抑制因子激酶β；；蛋白激酶Cθ The Experimental Study on the Mechanisms of Atorvastatin Suppressing Nuclear Factor Kappa B ActivationObjectiveArteriosclerosis has became the main reason of death in our country as common pathological foundation of serious diseases such as coronary artery disease and stroke. So, arteriosclerosis is focused and emphasized in studies constantly. Arteriosclerosis caused by multiple factor, but its mechanism has not been clarified precisely yet. Immune reaction theory become a focal poin increasingly among theories trying to explain the reason of arteriosclerosis. The theory presume the arteriosclerosis as a immune reaction result after artery stimulated by lower intensity inflammation for a long time. Inflammation play a important role in the process of arteriosclerosis whether in the initiation period, the development period or the acute period of plaque disruption. Inflammation enhance the development of atherosclerotic lesion through the pathphysiologic procedure as follow: promoting adhesion molecule expression and chemical chemotatic factor production; then inducing inflammatory cell as leucocyte to adhere blood vessel endothelium and to invade artery wall; activating macrophage or vascular endothelial cell or vascular smooth muscle cell to secrete fibroblast growth factor including growth factor peptid; facilitating smooth muscle cell proliferation and extracellular matrix composition. Besides, evidence-based medicine has proved that inflammation can be detected in every phases of arteriosclerosis, furthermore, markers of inflammation in serum increase significantly when the plaque are instable and have close relationship with prognosis. Nuclear factor kappa B (NF-κB) play a center role in regulating many inflammatory factor transcription. NF-κB has been proved present in atheromatous plaque of mankind, but absent in the blood vessel without plaque. In the atheromasia and fibrotic endomembrane and tunica media, NF-κB activation can be find. Endothelial cell, smooth muscle cell, macrophage are involved. Studies have discovered that the activation of NF-κB is essential in vascular smooth muscle cell migrating down to the endomembrane. In order to relieve the injure and to block the following pathological changes, how to suppress the activation of NF-κB become a tendency in the study of arteriosclerosis prevention and cure.The excellent effect of statins on anti-arteriosclerosis and coronary artery disease treatment have been proved by evidence-based medicine. Beside regulating lipid level in blood, statins have many other effects including anti-inflammatory and NF-κB activation suppression. The mechanism of the additional effects have not been illuminated clearly, so, our research focus on it. Almost all compound of NF-κB are regulated in a similar way: in normal condition, compound of NF-κB combined with inhibitor of nuclear factorκBα(IκBα) remain in quiescent stage. After stimulated by different factor, IκBαcan be phosphorylated by signals from upstream, then, phosphorylated IκBαlinked with ubiquitin by specified ubiquitin joining enzyme. Finally, the compound degradate after discerned by 26s proteasomes. The released NF-κB enter cell nucleus to activate the transcription of target gene. The procedure is the classical pathway of NF-κB activation. In the procedure, the phosphorylation and degradation of IκBαare important. It is the IκB kinases(IKKs) who have the function. The mechanism of IKK activation by upstream signals still remains unclear.Because liposoluble statins can diminish NF-κB activation caused by almost all factors which enhance arteriosclerosis, we presume that lipsoluble statins work on the classical pathway of NF-κB activation. We design experimens to test the following hypothesis: 1. atorvastatin can influence NF-κB activation and IκBαphosphorylation. 2. atorvastatin can lighten IκBαdegradation by influencing related enzyme of ubiquitin system. 3. atorvastatin can influence the function of important kinase and receptor expression in upstream signal system.Methods1. Cultivation and passage of human vascular endothelial cells.2. The cultured ECV304 cells in vitro were divided into 5 groups, the control group(incubated with serum-free medium for 24h), the LPS group(incubated with serum-free medium for 24h), the group of low density atorvastatin (incubated with serum-free medium and atorvastatin at a does of 1×10^-7 molL^-1 for 24h), the group of middle density atorvastatin(incubated with serum-free medium and atorvastatin at a does of 1×10^-6 molL^-1 for 24h), the group of high atorvastatin(incubated with serum-free medium and atorvastatin at a does of 1×10^-5 molL^-1 for 24h). All groups except the control group were stimulated with 10 mgL^-1 LPS for 0.5h. Immumofluorescence method are used to detect the change of the p65 location in cells. The p65 mRNA and IκBαmRNA were examined by RT-PCR. The protein of p65, IκBαand p-IκBαwere tested by Western blot.3. The experiment groups adopt two systems. The groups in the first system are the same as those above. In the second system, cells incubated with serum-free medium and atorvastatin at a does of 1×10^-7 molL^-1 for Oh, 1h, 6h, 12h, 24h respectively, then stimulated with 10 mgL^-1 LPS for 0.5h. The E3RSIκB mRNA and UBC5 mRNA were examined by RT-PCR and protein tested by Western blot.4. The cells were divided into 5 groups as those in part 2. TLR4 were tested by flow cytometry. Protein of PKCθ, p-IκKβare tested by Western blot.Results1. Atorvastatin can block the p65 that is the subunit of NF-κB move to the cell nucleus in a dose-dependent way.2. Atorvastatin can lighten the dramatic degradation of IκBαinduced by LPS in a dose-dependent way. IκBαmRNA increase in groups have no statistical significance except the high atorvastatin group.3.Atorvastatin can decrease the intensified phosphorylation stimulated by LPS of IκBαin a dose-dependent way.4. Atorvastatin downregulate the increased mRNA and protein expression of E3RSIκB and UBC5 mRNA stimulated by LPS of IκBαin a time-dependent way. The difference among mRNA and protein expression in different atorvastatin groups have statistical significance.5. Atorvastatin can decrease the increased protein expression of TLR4 and p-IκKβstimulated by LPS in a dose-dependent way; Neither atorvastatin nor LPS have influence on the expression of PKCθ.Conclusion1. By blocking the degradation of IκBα, atorvastatin downregulate the increased activation of NF-κB induced by LPS.2. Atorvastatin lighten the reduced IκBαlevel by blocking the phosphorylation of IκBαand degradation of p-IκBα.3. Atorvastatin downregulate the degradation of p-IκBαby reduce the expression of E3RSIκB and UBC5, this effect related to the incubation time of atorvastatin and the dose of atorvastatin.4. After stimulated by LPS, the increased expression of membrane receptor TLR4 and phosphorylation of upstream signal IκK can be downregulated by atorvastatin.